CN108865949B - Bacillus subtilis TKM-1 and application thereof - Google Patents

Abstract

The invention relates to bacillus subtilis TKM-1 and application thereof. The Bacillus subtilis TKM-1 has the preservation number of CGMCC NO. 14950. The invention relates to a bacillus subtilis TKM-1 with antagonistic action on pathogenic bacteria of a rot disease, which is obtained by screening a plant with the rot disease of an apple. The bacteria is prepared into a microbial inoculum or is subjected to field expansion culture, and the rot disease is prevented and controlled by spraying and smearing fruits and branches. Experiments prove that the method can effectively prevent and treat apple canker, has a degradation effect on canker barks which are already ill, is harmless to human and livestock, is environment-friendly, is not easy to generate drug resistance, can heal wounds quickly, has low scab recurrence rate, can effectively avoid a series of problems caused by excessive application of chemical pesticides, and has wide application prospect in agricultural production.

Description

Bacillus subtilis TKM-1 and application thereof

Technical Field

The invention belongs to the field of biological control, and particularly relates to bacillus subtilis TKM-1 and application thereof.

Background

According to the statistics of the Food and Agriculture Organization (FAO) of the United nations, the cultivation area of Chinese apples occupies the first place of the world from 1988, and the cultivation area and the total yield approximately account for 2/5 and 1/3 of the total area and the total yield of the world apples. With the increase of yield and the planting in successive years, various diseases of apples frequently occur, wherein apple canker is one of fungal diseases with wide disease and serious harm.

The apple rot disease is commonly called as foul skin disease, rotten skin disease and skin tainting disease, and the pathogenic bacteria in east Asia region is apple Humicola melanosporum (Valsa mali Miyabe et Yamada), which belongs to Ascomycotina fungi. In autumn, ascospores are colorless, and the asexual generation of the unit cell is apple stem canna putida (Cytospora mandshurica Miura), which belongs to fungi of Deuteromycotina. Conidiophores (small black dots) form under the bark, producing multiple conidiophores. Conidia are colorless, unicellular, sausage-shaped. The apple tree rot mainly attacks a plurality of parts of main branches, trunks, fruits and the like of the apple tree, so that the apple tree rot causes the defects of branches and trunks, weak tree vigor and low yield, and is an apple disease with wide distribution and serious harm. When the disease is serious, the branches are withered, the fruiting capacity is reduced, and even the whole plant is dead. Saplings and seedlings can also be damaged, but with less morbidity. At the early stage of the disease, the appearance is not easily recognized, and if the epidermis of the branches is opened, dark brown or reddish brown moist small spots or yellowish brown dry spots are visible, or the internal lesion is serious and the external lesion is still not recognized well. When the patient suffers from serious injury, the cortex is rotten and necrotic, and then the patient can sink down by pressing with fingers. The diseased skin is easy to peel off, the rotten skin layer is red brown, and the taste of vinasse is generated when the diseased skin is wet and rotten. In the later stage of disease, the affected part becomes dark brown due to dehydration and drying shrinkage, and produces dark brown speckles, namely conidiophore, on the epidermis, which becomes the source of secondary disease.

The fruit tree rot is a plant disease caused by fungi, usually, the fruit tree rot begins to occur after the fruit tree enters a fruiting period, the rot can increase year by year along with the increase of the tree age and the continuous improvement of the yield, the tree vigor can be weakened due to continuous low temperature and freezing damage, high temperature, drying, extensive management and the like in summer, and the rot becomes a disease which has the widest disease area and the most serious harm after the fruit tree enters a full fruit.

At present, the prevention and treatment of the rot disease of the fruit trees mainly comprises chemical prevention and treatment, the prevention and treatment cost is high and incomplete, the soil, water and atmosphere pollution can be caused by long-term repeated and large-scale use of chemical pesticides, the pesticide residue is increased, and the problem of food safety is increasingly serious; and the chemical pesticide can easily destroy the surrounding ecological environment while killing pathogenic bacteria.

Disclosure of Invention

The invention aims to provide a Bacillus subtilis TKM-1 with a remarkable control effect on apple rot.

The invention also aims to provide the application of the bacillus subtilis TKM-1 in preventing and treating plant diseases.

In order to realize the purpose of the invention, the inventor obtains a strain TKM-1 with remarkable control effect on apple rot by separating and purifying a scab sample by a conventional method from a certain apple plantation in a small temple ditch town of plain spring city in Hebei province in the process of separating the apple rot pathogen, and identifies the strain TKM-1 as Bacillus subtilis according to the bacteriological characteristics of the strain TKM-1 and the 16S rDNA sequencing result (SEQ ID NO: 1). The TKM-1 strain is deposited in China general microbiological culture Collection center, institute No. 3 of West Lu 1 of the sunward region of Beijing, institute No. 3 of microbiology of China academy of sciences, zip code 100101, preservation number CGMCC NO.14950, and preservation date of 2017, 11 months and 22 days.

The strain TKM-1 has strong capability of inhibiting the growth of pathogenic bacteria, and is proved to be non-toxic, harmless and hemolytic negative by non-toxicity tests, hemolytic tests and the like.

The invention also provides a pathogenic bacteria inhibitor, the active component of which is the bacillus subtilis TKM-1 and/or the metabolite thereof.

The pathogenic bacteria inhibitor has inhibitory effect on Fusarium (Fusarium Oxysporum) and Fusarium (Fusarium) fungi, such as Fusarium Oxysporum (Fusarium Oxysporum) which is a pathogenic bacterium of Banana. In addition, TKM-1 has biocontrol effect on most fungi.

The invention also provides a plant disease inhibitor, the active component of which is the bacillus subtilis TKM-1 and/or the metabolite thereof.

The plant diseases comprise apple rot, banana Panama disease and plant root rot.

Wherein the apple rot disease is caused by apple Humicola melanosporum.

The invention also provides any one of the following applications of the bacillus subtilis TKM-1:

1) the application of the bacillus subtilis TKM-1 in inhibiting pathogenic bacteria;

2) the application of the bacillus subtilis TKM-1 in preparation of a pathogenic bacterium inhibitor;

3) the application of the bacillus subtilis TKM-1 in plant disease inhibition;

4) application of bacillus subtilis TKM-1 in preparation of plant disease inhibitors.

1) -4), said pathogenic bacteria comprise pythium aphanidermatum, Fusarium (Fusarium) fungi; the plant diseases comprise apple rot, banana Panama disease and plant root rot.

Through a fermentation culture mode, the strain TKM-1 can be prepared into fermentation powder, fermentation liquor or bacteria powder rich in viable bacteria, enzymes and metabolites and field propagation products.

When the method is specifically applied, the fermentation liquid of the bacillus subtilis can be mixed with 0.6-2.0kg of brown sugar, 0.3-1.5kg of compound fertilizer, 500g of shrimp and crab powder and 200kg of water according to per 100mL of the fermentation liquid, cultured at the temperature of 25-30 ℃, stirred once every 1h until the viable count is 100 plus materials and 500 hundred million cfu/mL, and the microbial inoculum is diluted by 100 plus materials and sprayed on the diseased part by 1000 times.

The preparation method of the bacillus subtilis fermentation liquor comprises the following steps: inoculating the bacillus subtilis to an LB culture medium, performing fermentation culture at the temperature of 30-32 ℃ for 24-48h, and concentrating to obtain the bacillus subtilis.

Wherein the LB medium comprises the following components: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of glucose.

N: P in the compound fertilizer₂O₅:K₂The mass ratio of O is 15-17:15-17:15-17, preferably 15:15: 15.

Preferably, before spraying, the method further comprises the step of adding the soapberry extract with the final concentration of 0.0005% -0.001% into the diluted microbial inoculum.

The preparation method of the soapberry extracting solution comprises the following steps: oven drying and pulverizing soapberry seed coat, extracting with water, refluxing at 60 deg.C for 2 hr, extracting for 3 times, filtering, mixing the filtered extractive solutions, and concentrating to obtain concentrated soapberry extractive solution; wherein the mass ratio of the extracting solution to the raw materials is 4: 1.

The invention relates to a bacillus subtilis TKM-1 with antagonistic action on pathogenic bacteria of a rot disease, which is obtained by screening a plant with the rot disease of an apple. The bacteria is prepared into a microbial inoculum or is subjected to field expansion culture, and the rot disease is prevented and controlled by spraying and smearing fruits and branches. Experiments prove that the method can effectively prevent the rotten disease spots from expanding, promote the drying and healing of the diseased parts, reduce the disease spots, is harmless to human and livestock, environment-friendly, not easy to generate drug resistance, fast in wound healing and low in scab recurrence rate, can effectively avoid a series of problems caused by excessive application of chemical pesticides, and has wide application prospect in agricultural production.

Drawings

FIG. 1 shows the inhibitory effect of Bacillus subtilis TKM-1 on putrescence pathogenic bacteria; wherein A, B respectively refers to A is the inhibition zone of Bacillus subtilis TKM-1 to pathogenic bacteria, and B is blank control.

FIG. 2 shows the colony morphology of Bacillus subtilis TKM-1.

FIG. 3 is a microscopic picture of Bacillus subtilis TKM-1.

FIG. 4 shows the inhibition zone of Bacillus subtilis TKM-1 against pathogenic bacteria.

FIG. 5 shows the inhibition zone of the metabolite of Bacillus subtilis TKM-1 against pathogenic bacteria.

FIG. 6 is a microscopic picture of pathogenic bacteria inhibited by Bacillus subtilis TKM-1 and a control group; wherein A is a hypha picture of pathogenic bacteria of apple rot disease after being inhibited by bacillus subtilis TKM-1, and B is a hypha microscopic picture under normal and non-interference conditions.

FIG. 7 is a picture of a lesion after the bacillus subtilis TKM-1 cures apple rot.

FIG. 8 is a graph showing the dilution factor and surface tension change of the soapberry extract.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The percentages in the following examples are by weight unless otherwise specified.

Example 1 isolation and characterization of Bacillus subtilis TKM-1

1. The bacterial strain TKM-1 capable of preventing and treating apple rot is collected from a certain apple plantation in the town of the temple ditch in Pingquan, Hebei, and is obtained by separating a scab sample by a conventional method in the process of separating the apple rot pathogen. Selecting tissue blocks with scabs, disinfecting the surfaces of the tissue blocks by using alcohol and saline water, cleaning the tissue blocks for several times by using sterile water, placing the tissue bacterium liquid on the surface of a PDA (personal digital Assistant) plate after the water on the surface of the tissue blocks is dried, culturing the tissue bacterium liquid in an incubator at 28 ℃, observing once a day, and recording the surface change of the plate. During the growth process, fungal hyphae and bacteria appear simultaneously, bacterial colonies which inhibit the fungi and form inhibition rings are picked for purification culture, activated by LB culture medium and placed in a refrigerator at 4 ℃ for later use.

The preparation method of the PDA culture medium comprises the following steps: 200g of peeled potatoes are boiled in water and filtered, filtrate is remained, 20g of glucose, 1.5g of magnesium sulfate, 1100 mg of vitamin B and 3g of monopotassium phosphate are added into the potato filtrate, and the volume is fixed to 1L by using distilled water. The LB solid culture medium has the formula: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride, 20g/L of glucose and 20g of agar powder, and the volume is up to 1000 mL.

2. Screening of Bacillus subtilis TKM-1

Inoculating apple rot pathogen on a PDA (personal digital Assistant) plate, culturing at 28 deg.C for 3-5 days, and inoculating 2 fungus cakes (with diameter of 2-3mm) on the PDA plate, wherein the fungus cakes are uniformly distributed on two sides of the center point of the plate, and the three points are in a line; inoculating the purified strain culture solution with filter paper sheet in the cross vertical direction (viable count of 2 × 10)⁸-10×10⁸cfu/mL), blank as control for direct pathogen, 3 replicates per treatment, incubated at 28 ℃.

After fungus colonies on the blank control plate overgrow the plate, observing the size and the existence of a bacteriostatic zone, and taking the nearest distance (bacteriostatic transparent zone) between pathogenic bacteria and bacterial colonies as a bacteriostatic intensity mark, wherein the ratio of the distance to the radius of the control group colonies can be used as bacteriostatic rate reference.

The inhibition rate is (control colony radius-treated colony radius)/(control plate colony radius-cake radius) × 100%.

The test shows that: the bacillus subtilis TKM-1 has the best inhibition effect on apple rot pathogenic bacteria, the transparent area is the largest, and the inhibition rate can reach 76.1% (figure 1).

3. Identification of rot-resistant bacillus subtilis

FIG. 2 is a colony morphology diagram of the strain of the invention, Bacillus subtilis TKM-1. And (3) observing morphological characteristics of the bacteria under a microscope after gram staining of the colonies selected in the step 2, wherein the morphological diagram of the bacillus subtilis TKM-1 after gram staining is shown in a figure.

Through observation, the bacillus subtilis TKM-1 is a rod-shaped bacterium, gram staining is positive, a single colony is not round, milky white, creamy and opaque, the middle of the colony is raised and rough, and the edge of the colony is incomplete.

The most suitable culture medium of the strain TKM-1 is an LB-culture medium, the most suitable growth temperature is 28-37 ℃, and the most suitable growth pH is 5-9; is aerobic bacteria, and grows slowly under anaerobic condition; malonate and citrate can be used as carbon source; the hydrogen peroxide test of the strain is positive; the methyl red test shows positive reaction; positive in V-P test; the hemolytic test was negative.

Further carrying out 16S rDNA identification on the Bacillus subtilis TKM-1 (the sequencing result is shown in SEQ ID NO:1), and determining that the Bacillus subtilis is Bacillus subtilis.

Example 2 determination of inhibition of Bacillus subtilis against pathogenic bacteria of rot disease

The inhibitory effect of Bacillus subtilis TKM-1 on apple rot pathogen was determined by plate confrontation method, which was the same as step 2 in example 1. Pathogenic bacteria of apple rot disease the fungus Valsa mali of the genus Humicola is selected as a disease strain sample. The inhibition rate of the bacillus subtilis TKM-1 on apple rot disease is shown in table 1. The inhibition zone of Bacillus subtilis TKM-1 on pathogenic bacteria is shown in FIG. 4.

TABLE 1 inhibition of apple rot pathogen by three parallel tests

Example 3 determination of the inhibitory Rate of the Bacillus subtilis TKM-1 sterile filtrate on the pathogens of rotten diseases

Preparation of fermentation filtrate: inoculating Bacillus subtilis TKM-1 into 250mL triangular flask containing 100mL LB liquid medium, and shaking at 30 deg.C for 24-48h to OD₆₀₀The obtained fermentation broth was centrifuged at 10000r/min for 10min, and the supernatant was filtered through a 0.22 μm microporous membrane to obtain a sterile filtrate.

Two holes are reserved on a PDA plate by using an Oxford cup, the distance between the two holes and the inoculation mode of pathogenic bacteria are the same as the step 2 in the embodiment 1, 100 mu L of sterile filtrate is added into the holes, the filtrate is replaced by sterile water in a contrast test, the culture is carried out for 4 days at the temperature of 28 ℃, the bacteriostatic effect of the filtrate is observed, and the diameter of a bacterial colony is measured to calculate the bacteriostatic rate. Three sets of parallel experiments were set up and calculated in the same manner as in step 2 of example 1.

The result shows that the sterile fermentation filtrate of the bacillus subtilis TKM-1 has obvious inhibition effect on apple rot pathogenic bacteria. The antagonistic effect is shown in fig. 5, and the bacteriostatic rate reaches 64%.

Example 4 inhibitory Effect of Bacillus subtilis TKM-1 culture solution on pathogenic bacteria of putrefactive disease

Inoculating apple Humicola fungus Valsa mali on PDA plate, culturing at 28 deg.C for 3 days to obtain PDA culture of pathogenic bacteria, inoculating bacterial cake (diameter of bacterial cake is 2-3mm) on two sides of center of another PDA culture medium, and inoculating Bacillus subtilis TKM-1 culture solution (viable count > 6 × 10)⁸cfu/mL) were soaked in sterile filter paper sheets, two filter paper sheets were placed symmetrically in a cross-hatch orientation. After 3-5 days of culture, the hypha condition was observed by microscopic examination. The hyphal growth and microscopic picture are shown in FIG. 6.

Microscopic examination shows that the hyphae of the pathogen close to the sterile filter paper sheet are sparse and linear, have no nodular protrusions and have no spores, while the hyphae of the blank control grow vigorously and have many nodular protrusions and spores. The sterile filtrate plays a good role in inhibiting the propagation of pathogenic bacteria.

Example 5 comparison with methods for chemical control of apple rot

The culture solution of the TKM-1 strain is transversely compared with other common chemical control methods for apple canker, and the results are shown in Table 2.

TABLE 2

The results show that after the treatment by the TKM-1 culture solution, the apple rot disease spots are obviously reduced, and the recurrence rate is lowest compared with other treatment groups, and after the treatment of foliage spraying and root irrigation, the immunity of apple diseased plants is enhanced, and the growth vigor is good.

Example 6 field application example

Mixing the bacillus subtilis TKM-1 fermentation broth with 1kg of brown sugar, 1kg of compound fertilizer, 200g of shrimp and crab powder and 100kg of water according to per 100mL of fermentation broth, culturing at 25-30 ℃, stirring once every 1h until the viable count is 100 plus materials and 500 hundred million cfu/mL, and diluting the microbial inoculum by 1000 times to spray the microbial inoculum on the diseased part. The result shows that the scab of the rotten part of the apple is not enlarged any more, the surface layer is dried slowly, and the area of the scab tends to be smaller. The lesions after the decay disease is cured are shown in fig. 7.

The preparation method of the bacillus subtilis fermentation liquor comprises the following steps: inoculating the bacillus subtilis to an LB culture medium, performing fermentation culture at 32 ℃ for 36h, and concentrating to obtain the bacillus subtilis.

The LB medium comprises the following components: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of glucose.

The compound fertilizer is purchased from XK13-001-00005 of Shikefeng chemical Co Ltd (Lu) and N: P in the compound fertilizer₂O₅:K₂The mass ratio of O is 15:15: 15.

Example 7 method for improving the ability to transfer the Bacillus subtilis TKM-1 culture solution

The soapberry extract is a natural saponin extracted from the peel or the seed coat of a soapberry plant, the saponin is also a surfactant, and the soapberry extract and the TKM-1 culture solution are used together, so that the surface tension of the culture solution can be reduced, and the components in the culture solution can be diffused more quickly.

According to the embodiment 6, before spraying, the method further comprises the step of adding the soapberry extract with the final concentration of 0.0005% -0.001% into the diluted microbial inoculum.

The preparation method of the soapberry extracting solution comprises the following steps: oven drying and pulverizing soapberry seed coat, extracting with water, refluxing at 60 deg.C for 2 hr, extracting for 3 times, filtering, mixing the extractive solutions, and concentrating to obtain concentrated soapberry extractive solution. The mass ratio of the extracting solution to the raw materials is 4: 1.

The surface tension value of the soapberry extract was measured using a surface tension meter, the soapberry extract was diluted in a gradient of 5, 10, 20, 50, 100, 500, 1000, 5000 and 10000 times at concentrations of 0.2, 0.1, 0.05, 0.02, 0.01, 0.002, 0.001, 2E-4 and 1E-4, respectively, relative to the soapberry extract stock solution, and then a curve was drawn using the values obtained (FIG. 8).

As can be seen from FIG. 8, the higher the concentration of the soapberry extract, the smaller the surface tension, from 10 times to 10000 times, the surface tension increased from 30mN/m to about 55mN/m, and when the dilution factor was more than 10000 times, the surface tension tended to be stable.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing Taikemeigao technology Co., Ltd

<120> bacillus subtilis TKM-1 and application thereof

<130> KHP181113428.9

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ccccggctca accggggagg gtcattggaa actggggaac ttgagtgcag aagaggagag 660

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cgactctctg gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag 780

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tagtgctgca gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac 900

tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960

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Claims (3)

1. A method for preventing and treating apple rot is characterized in that a fermentation liquid of Bacillus subtilis tmm-1 with the preservation number of CGMCC NO.14950 is mixed with 0.6-2.0kg of brown sugar, 0.3-1.5kg of compound fertilizer, 500g of shrimp and crab powder and 200kg of water according to per 100mL of the fermentation liquid, cultured at the temperature of 25-30 ℃, stirred once every 1h until the viable count is 100 plus 500 hundred million cfu/mL, and the microbial inoculum is diluted by 100 plus 1000 times and sprayed on the diseased part.

2. The method of claim 1, wherein the fermentation broth is prepared by: inoculating bacillus subtilis to an LB culture medium, performing fermentation culture at the temperature of 30-32 ℃ for 24-48h, and concentrating to obtain the bacillus subtilis;

wherein the LB medium comprises the following components: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of glucose.

3. The method as claimed in claim 1, wherein the N: P in the compound fertilizer₂O₅:K₂Quality of OThe amount ratio is 15-17:15-17: 15-17.

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