CN108570432B

CN108570432B - Bacillus amyloliquefaciens and application thereof

Info

Publication number: CN108570432B
Application number: CN201810434828.6A
Authority: CN
Inventors: 王刚刚; 苟艳; 谢天; 蒲莉
Original assignee: Chengdu Institute of Biology of CAS
Current assignee: Chengdu Institute of Biology of CAS
Priority date: 2018-01-12
Filing date: 2018-05-09
Publication date: 2021-01-01
Anticipated expiration: 2038-05-09
Also published as: CN108034611A; CN108570432A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a bacillus amyloliquefaciens and application thereof. The technical scheme is as follows: screening and separating a Bacillus amyloliquefaciens strain MY with chitin degradation capacity from the soil planted with asparagus, wherein the Bacillus amyloliquefaciens strain MY is stored in the common microorganism center of China general microbiological culture Collection management Committee in 7 months and 26 days in 001,2017 years, the strain name is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MY001, and the preservation number is as follows: CGMCC NO. 14460. The strain can be used for inhibiting phomopsis asparagi, and the specific use method comprises the following steps: at a concentration of 1X 10⁷1% of colloidal chitin is added into cfu/mL bacillus amyloliquefaciens MY001 microbial inoculum to form a compound microbial inoculum, and the compound microbial inoculum has important significance for preventing and treating stem blight of asparagus officinalis L.

Description

Bacillus amyloliquefaciens and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a bacillus amyloliquefaciens and application thereof.

Background

The Asparagus stem blight (Asparagus stem bright) is also called as the 'cancer' of the Asparagus, the disease symptoms of the Asparagus are mainly concentrated on stems, lateral branches or leaves, the Asparagus stem blight has the characteristics of quick disease, easy infection and serious loss, once the Asparagus is infected, the Asparagus is easy to spread in a large area, the whole Asparagus plant is suddenly yellow and withered, and the Asparagus product can not be effectively produced. The main pathogenic bacterium is Phomopsis Asparagus (Phomopsis asparagi). At present, chemical pesticides such as carbendazim and chlorothalonil are mainly used for preventing and treating the stem blight of the asparagus, but long-term use of the chemical pesticides easily causes drug resistance of germs, and the stem blight of the asparagus cannot be continuously and effectively prevented and treated. Therefore, the development of effective biopesticides is a feasible method for preventing and treating the stem blight of asparagus.

Bacillus is widely distributed in nature and comprises Bacillus subtilis, Bacillus licheniformis, Bacillus cereus and the like, and the strains have strong resistance to external harmful factors. A plurality of bacillus species have the function of inhibiting the propagation and growth of crop pathogenic bacteria and are good biocontrol bacteria.

Bacillus amyloliquefaciens (B. amyloliquefaciens) belongs to gram-positive bacteria, and has been found to have broad-spectrum bacteriostasis on plant pathogenic bacteria, but the bacteriostasis effectiveness of the bacillus amyloliquefaciens is still extremely high in specificity, and specific biocontrol strains need to be screened for different pathogenic bacteria to achieve the optimal bacteriostasis effect. However, the research of applying the bacillus amyloliquefaciens to the prevention and the control of plant fungal diseases mostly focuses on crops such as strawberries, tomatoes, peaches, potatoes and the like, and the bacillus amyloliquefaciens for specifically preventing and controlling the stem blight of the asparagus and a specific prevention and control method are not reported.

Disclosure of Invention

The invention aims to provide a bacillus amyloliquefaciens and application thereof.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a strain of Bacillus amyloliquefaciens is preserved in China general microbiological culture Collection center in 2017, 7 and 26 months, wherein the strain is named as Bacillus amyloliquefaciens, Bacillus amyloliquefaciens and MY001, and the preservation number is as follows: CGMCC NO. 14460.

Correspondingly, the 16SrDNA sequencing sequence of the Bacillus amyloliquefaciens strain is shown as SEQ ID NO 1, the gyrA sequencing sequence of the Bacillus amyloliquefaciens strain is shown as SEQ ID NO 2, and the gyrB sequencing sequence of the Bacillus amyloliquefaciens strain is shown as SEQ ID NO 3.

Correspondingly, the bacillus amyloliquefaciens is applied to degrading chitin.

Correspondingly, the bacillus amyloliquefaciens is applied to the prevention and the treatment of plant fungal diseases.

Preferably, the bacillus amyloliquefaciens is used for controlling plant fungal diseases, and the fungi comprise: phomopsis asparagi, Pestalotiopsis sp, Curvularia sp.

Preferably, the bacillus amyloliquefaciens is applied to control of stem blight of asparagus.

Correspondingly, the preparation method of the bacterial preparation for preventing and treating the stem blight of the asparagus comprises the following steps: the bacillus amyloliquefaciens is prepared into the concentration of 1 multiplied by 10⁷cfu/mL of bacterial suspension, namely the bacterial preparation.

Preferably, the bacillus amyloliquefaciens is combined with chitin to control stem blight of asparagus.

Correspondingly, the compound bacterium preparation for preventing and treating the stem blight of the asparagus comprises the following steps: the bacillus amyloliquefaciens is prepared into the concentration of 1 multiplied by 10⁷Adding chitin into cfu/mL bacterial suspension, and mixing to obtain a composite bacterial preparation; in the compound bacterium preparation, the final concentration of chitin is 1%.

Preferably, the chitin is colloidal chitin.

The invention has the following beneficial effects:

1. a new bacillus amyloliquefaciens, MY001, with chitin degradation capability is screened and separated, the strain has excellent inhibiting effect on pathogenic bacteria of phomopsis asparagi and other fungi causing stem blight of asparagus, and has important practical significance for preventing and treating the stem blight of asparagus.

2. The bacillus amyloliquefaciens screened and separated by the method has good degradation effect on chitin, can be practically applied to the production of degrading the chitin, and has guiding significance for further follow-up research.

3. The inventor also surprisingly finds that the bacillus amyloliquefaciens and the chitin which are screened by the invention are used together, and have stronger inhibiting effect on pathogenic bacteria of stem blight of asparagus than the microbial inoculum which is used alone. An unknown synergistic effect occurs between bacillus amyloliquefaciens and chitin.

At present, although the application of oligosaccharide generated by degrading chitin to biological control has been reported, the invention combines bacillus amyloliquefaciens and chitin for the first time and jointly uses the bacillus amyloliquefaciens and the chitin for controlling plant fungal diseases.

This may be because of the following reasons:

(1) chitin-induced bacillus amyloliquefaciens strain MY001 secretes chitinase, chitin is degraded to generate oligosaccharides, and the oligosaccharides have strong antibacterial effect on phoma asparagi pathogenic bacteria.

(2) Chitinase secreted by the MY001 strain can also degrade and destroy the cell wall of pathogenic fungi, thereby playing a role in inhibiting pathogenic bacteria.

(3) When chitin exists, MY001 strain can be induced to secrete more chitinase, so that the bacteriostatic effect is enhanced through the (1), (2) or other unknown ways.

Due to the complex secretion of bacillus amyloliquefaciens, in addition to chitinase, other antibacterial components may exert bacteriostatic effects, such as lipopeptides, polyketides, siderophiles, and the like.

The invention provides a new idea for controlling pathogenic bacteria of crops by using chitin and bacillus amyloliquefaciens in a combined manner, and has important guiding significance for subsequent similar researches.

Drawings

FIG. 1 is a transparent hydrolysis ring formed by a target strain;

FIG. 2 is a phylogenetic tree comparing the 16SrDNA of a target strain and a Bacillus model strain;

FIG. 3 is a phylogenetic tree that performs multiple sequence alignments of the sequencing results with the gyrA genes of the model strains;

FIG. 4 is a phylogenetic tree that performs multiple sequence alignments of the sequencing results with the gyrB genes of the model strains;

FIG. 5 is a graph showing the effect of a target strain on the inhibition of Phomopsis asparagi;

FIG. 6 is a graph showing the effect of a target strain on the inhibition of a Pestalotiopsis strain;

FIG. 7 is a graph showing the effect of a target strain on the inhibition of Curvularia strains;

FIG. 8 is a graph showing the comparison of chitin degradation by target strains;

FIG. 9 is a graph showing the effect of a target strain in inhibiting Phomopsis asparagi when used in combination with chitin.

Detailed Description

The first embodiment is as follows: screening of strains

1. Collecting a soil sample: collecting soil samples from the Mianyang asparagus planting area.

2. Preparing a culture medium:

(1) chitosanase screening medium (g/L): colloidal chitosan 2.0, K₂HPO₄ 0.7，KH₂PO₄ 0.3，(NH₄)₂SO₄ 5,MgSO₄·7H₂O 0.5，FeSO₄·7H₂00.01, agar 12.0, pH 7.0.

(2) Broth (g/L): peptone 10.0, beef extract 3.0, NaCl 5.0, pH 7.0.

3. Preparing a reagent:

preparing colloidal chitosan, namely weighing 10g of fine powder chitosan, adding a proper amount of concentrated hydrochloric acid, stirring for 4-6 h on a magnetic stirrer to fully dissolve the fine powder chitosan, repeatedly cleaning and centrifuging with deionized water (4000r/min), finally forming jelly, slowly adding NaOH while stirring to adjust the pH value to be neutral, thus obtaining the colloidal chitosan, and storing at 4 ℃ for later use.

3. Preliminary screening of bacterial strains

(1) Weighing 5g of soil sample, placing into a conical flask (250mL) containing 45mL of distilled water, stirring for dissolving, boiling for 30min, and taking supernatant for gradient dilution (10)^-1,10^-2,10^-3,10^-4,10^-5Total 5 concentrations), coating 100ul of the chitosan enzyme in a chitosanase screening culture medium (2 parallel gradient devices), and culturing in a constant temperature incubator at 37 ℃ for 2-4 days.

(2) Selecting bacterial colonies which grow on a selective culture plate and are provided with obvious transparent hydrolysis rings around, streaking and inoculating the bacterial colonies on a new chitosanase screening culture medium, culturing the bacterial colonies in a constant-temperature incubator at 37 ℃ for 2 days, then selecting the bacterial colonies, streaking and inoculating, and screening for 3 generations to obtain pure strains, namely the target strains.

(3) And (3) picking the single colony of the target strain obtained in the step (2) in a meat soup culture medium, and carrying out liquid culture. Then 5ul of culture solution is placed on a filter paper sheet with the diameter of 6mm, the filter paper sheet is placed on a chitosanase screening culture medium, the culture is carried out for 2 days at the constant temperature of 37 ℃, and the size of the transparent hydrolysis ring is measured. The clear circles formed are shown in FIG. 1, and the sizes of the clear circles produced by the target strains are shown in Table 1.

TABLE 1 size of transparent hydrolysis circles produced by the target strains

(4) Purifying and preserving the target strain, and storing at-80 ℃.

Example two: identification of strains

1. 16SrDNA sequencing identification

Genomic DNA of the target strain was extracted, and 16SrDNA was cloned from the genomic DNA using a universal primer (27F: AGA GTT TGA TCC TGG CTC AG; 1492R: TAC GGT TAC CTT GTT ACG ACT T) for bacterial 16 SrDNA. And (3) sending the PCR result to Shanghai bio-engineering company for sequencing, carrying out BLAST comparison on the sequencing result on a Gene Bank website (http:// www.nicbi.nlm.nih.gov /), finding and downloading a Gene sequence of a standard strain, calculating the genetic distance between different strains by using MEGA7.0 software, and drawing a phylogenetic tree of bacteria. The sequencing result of the 16SrDNA is shown as SEQ ID NO 1.

16SrDNA of a target strain and a Bacillus model strain are compared, and a phylogenetic tree is constructed by using MEGA7.0, wherein the genetic distances of a strain d1, which is 0.003 and 0.009, to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and Bacillus subtilis (Bacillus subtilis) are nearest, as shown in FIG. 3.

2. Sequencing identification of gyrA and gyrB

A subunit and B subunit of gyrase, as housekeeping proteins, are conserved in evolution, approximately 0.7-0.8% of gene evolution every 100 ten thousand years, and the speed is faster than 1% of gene evolution every 5000 ten thousand years of 16SrDNA, so that the gyrase is commonly used for identifying strains which have closer relationships and cannot be distinguished by 16 SrDNA. And (3) amplifying gyrase A and B genes of the bacteria by taking the genome DNA of the target strain as a template.

(1) gyrA is a universal primer, gyrA-F: 5'-CAG TCA GGA AAT GCG TAC GTC CTT-3', respectively; gyrA-R: 5'-CAA GGT AAT GCT CCA GGC ATT GCT-3' are provided.

Sequencing the amplified product, wherein the sequencing result of gyrA is shown as SEQ ID NO 2. The sequencing result was subjected to multiple sequence alignment with the gyrA gene of the model strain, and a phylogenetic tree was constructed using MEGA7.0, as shown in FIG. 4. Wherein the genetic distance between the strain to be detected and Bacillus amyloliquefaciens is closest and is 0.053 (the genetic distance between the strain to be detected and other strains is more than 0.2).

(2) According to the identification result of 16SrDNA, primers of gyrB are designed according to conserved regions of gyrB sequences of several related species, namely gyrB-F: 5'-AAC AGC AAA GGC CTT CAC CA-3', respectively; gyrB-R: 5'-GCA GAG TCA CCC TCT ACG ATA TA-3' are provided.

Sequencing the amplified product, wherein the sequencing result of gyrB is shown as SEQ ID NO 3. The sequencing result was subjected to multiple sequence alignment with the gyrB gene of the model strain, and a phylogenetic tree was constructed using MEGA7.0 as shown in FIG. 5. Wherein d1 has the nearest genetic distance to Bacillus amyloliquefaciens of 0.045 (the genetic distance to other strains is more than 0.2).

3. And (3) combining the results to determine that the target strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

The strain is preserved in 26.7.2017 in the common microorganism center of China Committee for culture Collection of microorganisms, the name of the strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MY001, and the preservation number is as follows: CGMCC NO. 14460.

Example three: indoor test for inhibiting pathogenic bacteria of stem blight of asparagus by using bacillus amyloliquefaciens alone

1. Preparing pathogenic bacteria: phomopsis asparagi, Pestalotiopsis (Pestalotiopsis) and Curvularia (Curvularia) strains are separated from the asparagus stem blight strain.

2. Preparing PDA culture medium (g/L), cutting 200g peeled potato into small pieces, boiling for 30min, filtering with gauze to remove residue, adding glucose 20.0g/L and agar 15.0g/L, and adjusting pH to natural.

3. Inoculating the three types of pathogenic strains in the center of a PDA culture medium, respectively culturing in a constant-temperature incubator at 28 ℃ until the diameter of a fungus colony is 3-4 cm, and respectively marking the fungus colony in four directions and 0.5cm away from the edge of the fungus colony as 1,2,3 and 4. Wherein the 1,3 sites are used as a reference, inoculated with Bacillus subtilis 168strain, Bacillus subtilis 168strain purchased from German strain collection center DSMZ with strain number DSM 23778; 2,4 sites are inoculated with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MY001. And (4) after treatment, placing the mixture in a constant-temperature incubator at 28 ℃ for inverted culture for 3-4 days, and observing the growth condition of pathogenic bacteria.

4. The bacteriostatic results are shown in FIGS. 6-8. On the 3 rd to 4 th day of inoculation, the control strain is covered by the fungus, and the MY001 strain forms an obvious inhibition zone for the three pathogenic bacteria. The bacillus amyloliquefaciens MY001 has a strong inhibiting effect on pathogenic bacteria of the stem blight of the asparagus and can be used for preventing and treating the stem blight diseases of the asparagus.

Example four: chitin degradation by bacillus amyloliquefaciens

In this example, the bacterial suspensions all refer to the effective bacterial content of 1X 10⁷cfu/mL of a suspension of Bacillus amyloliquefaciens MY001.

1. Preparing a low-nutrient medium (g/L): glucose 1.0, K₂HPO₄ 0.7，KH₂PO₄ 0.3，(NH₄)₂SO₄ 5.0，pH＝7.0。

The treatment group 1 was prepared by adding the bacterial suspension to a liquid medium containing a low nutrient medium and a flaky chitin (20g/L) in an amount of 1% by volume based on the volume of the liquid medium, and culturing at 37 ℃ and 200rpm for 3 days.

Taking 1ml of bacterial suspension, centrifuging to remove supernatant, and carrying out heavy suspension cleaning for several times by using sterilized double distilled water to ensure that the bacterial suspension does not contain nutrient components in a culture medium, thereby obtaining pure bacterial suspension. Two pure bacterial suspensions, 10ul each, were inoculated onto sterilized flaky chitin and placed at 37 ℃ for 3 days and 15 days, respectively, as treatment groups 2 and 3.

Sterilized chitin without any treatment was used as a control.

2. After the treatment group and the control group are treated by the steps, the treated group and the control group are respectively washed by PBS buffer solution (0.1M, pH is 7.1) to remove surface impurities and attached thalli, then 2.5% (v/v) glutaraldehyde is used for fixing for 4 hours at 4 ℃, after the fixing, the fixed group is washed by sterilized double distilled water for 3 times, then 4 samples are respectively soaked in 25%, 50%, 75%, 95% and 100% (v/v) ethanol for 5min in sequence, gradient dehydration is carried out, and finally the samples are dried by a critical point drying method. The dried sample was subjected to gold plating treatment to increase the conductivity of the sample, and then observed and photographed under an electron microscope (SU3500, HITACHI). The results are shown in FIG. 8.

3. And observing the surface of the chitin after the MY001 strain is degraded by using a scanning electron microscope. As shown in fig. 8, the chitin sample without any treatment (control) had a relatively smooth and dense surface (fig. 8A). The surface structure of the chitin (treatment groups 1 and 2) degraded by the MY001 strain for 3d is damaged to a certain degree, and cracks are generated and are similar to fibers (figure 8B, C); there was no significant difference in the degree of chitin degradation between the two experimental conditions of liquid shake culture (treatment 1, FIG. 8B) and solid culture inoculated MY001 strain (treatment 2, FIG. 8C). After the culture time of the solid culture inoculum was prolonged to 15 days (treatment group 3), the chitin was degraded to a greater extent, and not only fibrous cracks but also many small pores were formed on the surface (FIG. 8D).

The result shows that the bacillus amyloliquefaciens MY001 strain can directly degrade and utilize chitin. In addition, it should be noted that 15d is cultured for the convenience of electron microscope observation and photographing, and the MY001 bacteria is not limited to be required to be 15d for effectively degrading chitin. In combination with the test of the following fifth example, the inventor finds that the MY001 bacteria and the chitin can achieve good bacteriostatic effect when used immediately without waiting for fermentation.

Example five: indoor test for inhibiting pathogenic bacteria of stem blight of asparagus by using bacillus amyloliquefaciens and chitin in combination

1. Preparing pathogenic bacteria: phomopsis asparagi.

2. The activated phomopsis asparagi was inoculated in the center of a PDA medium (diameter 9cm) (the components and cultivation method of the PDA medium are the same as those in the examples, the following treatment groups are the same and will not be described further), the strain of Bacillus amyloliquefaciens MY001 was not inoculated, and the control group was cultured at 28 ℃ for 3 days.

Adding colloidal chitin into PDA culture medium components, inoculating activated Phomopsis asparagi in the center of PDA culture medium (diameter 9cm), wherein the final concentration of the colloidal chitin in the PDA culture medium is 1%, and culturing at 28 deg.C for 3d to obtain treatment group 1 without inoculating Bacillus amyloliquefaciens MY001 strain, and the rest treatment is identical to control group.

Inoculating the activated phomopsis asparagi in the center of a PDA culture medium (with the diameter of 9cm), and respectively inoculating the bacillus amyloliquefaciens MY001 strains on the left side and the right side of a colony and at a position 0.5cm away from the edge of the colony when the diameter of the colony reaches 2-3 cm. The cells were cultured at 28 ℃ for 3d to prepare treatment group 2.

Adding colloidal chitin into PDA culture medium components, inoculating activated Phomopsis asparagi in the center of PDA culture medium (diameter 9cm), wherein the final concentration of the colloidal chitin in the PDA culture medium is 1%, inoculating Bacillus amyloliquefaciens MY001 strain (same as treatment group 2 in the manner of point inoculation), and culturing at 28 deg.C for 3d to obtain treatment group 3.

3. The results are shown in FIG. 9.

(1) Phomopsis asparagi grew slowly in the culture medium containing colloidal chitin (treatment group 1), and the diameter of the formed colony was about 5.4cm (FIG. 9B), which is significantly smaller than that of the control group (FIG. 9A), indicating that the colloidal chitin has significant inhibitory effect on the main pathogenic bacteria of stem blight of asparagus officinalis L.

(2) As can be seen from treatment group 2 (fig. 9C): in the non-bacterium-inoculating direction, the germ hyphae are expanded to the edge of the plate, and in the bacterium-inoculating direction, the length of the germ colony is about 2.5cm, the germ colony is almost not extended, and an obvious arc-shaped inhibition zone is generated around the colony of the bacillus amyloliquefaciens MY001, so that the bacillus amyloliquefaciens MY001 strain has an antagonistic effect on main pathogenic bacteria of stem blight of asparagus.

(3) As can be seen from treatment group 3 (fig. 9D): when the colloidal chitin and the bacillus amyloliquefaciens MY001 strain coexist, the growth inhibition effect on the phomopsis asparagi is extremely obvious.

According to the fifth and sixth embodiments, the inventor finds that the bacillus amyloliquefaciens strain MY001 can be directly degraded and utilized by chitin, has unknown synergistic interaction with degraded colloidal chitin, and can exert strong inhibition effect on main pathogenic bacteria of stem blight of asparagus.

Sequence listing

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caa 1143

Claims

1. A strain of Bacillus amyloliquefaciens is characterized in that: is preserved in China general microbiological culture Collection center in 7 months and 26 days in 2017, and the name of the strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillusamyloliquefaciens) MY001 with the preservation number: CGMCC NO. 14460.

2. A strain of Bacillus amyloliquefaciens is characterized in that: the 16S rDNA sequencing sequence is shown as SEQ ID NO 1, the gyrA sequencing sequence is shown as SEQ ID NO 2, and the gyrB sequencing sequence is shown as SEQ ID NO 3.

3. Use of the bacillus amyloliquefaciens of claim 1 or 2 for degrading chitin.

4. Use of bacillus amyloliquefaciens according to claim 1 or 2 for the control of fungal plant diseases, characterized in that: the fungi are: phomopsis asparagi, Pestalotiopsis sp, Curvularia sp.

5. Use of bacillus amyloliquefaciens according to claim 4 for the control of fungal plant diseases, characterized in that: the bacillus amyloliquefaciens is applied to prevention and control of stem blight of asparagus.

6. Use of bacillus amyloliquefaciens according to claim 5 for the control of fungal plant diseases, wherein: the bacillus amyloliquefaciens and chitin are combined for preventing and treating stem blight of asparagus.

7. A bacterial preparation for preventing and treating stem blight of asparagus is characterized in that: the preparation method comprises the following steps: the Bacillus amyloliquefaciens of claim 1 or 2 formulated to a concentration of 1 x 10⁷cfu/mL of bacterial suspension, namely the bacterial preparation.

8. A compound bacterium preparation for preventing and treating stem blight of asparagus is characterized in that: the preparation method comprises the following steps: the Bacillus amyloliquefaciens of claim 1 or 2 formulated to a concentration of 1 x 10⁷Adding chitin into cfu/mL bacterial suspension, and mixing to obtain a composite bacterial preparation; in the compound bacterium preparation, the final concentration of chitin is 1%.

9. The compound bacterial preparation for preventing and treating phomopsis asparagi as claimed in claim 8, wherein: the chitin is colloidal chitin.