Bacillus amyloliquefaciens BAY-11 and use thereof

Abstract

The present application relates to Bacillus amyloliquefaciens BAY-11 and its application. It is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 25425. It can be used for preventing and treating plant diseases caused by Paraphoma chrysanthemicola.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant disease control, in particular to the field of plant fungal disease control. BACKGROUND

[0002] Paraphoma chrysanthemicola belongs to Paraphoma of Sphaeropsidaceae of Sphaeropsidales of Coclomycetes of Ileutemmyootina of eumycetes, and can cause infectious disease of Atractylodes chinensis (DC.) Koidz.

[0003] Atractylodes chinensis (DC.) Koidz, also known as Atractylodes lancea (Thunb.) DC., is a perennial herb of the family Asteraceae, and its rhizome is used as medicine. It has the effects of anti-inflammatory and analgesic, treatment of rheumatism, digestive dysfunction, acute enteritis, night blindness, influenza, and anti-tumor, and plays an important role in the treatment of COVID-19. Wild Atractylodes chinensis grows at an altitude of 300 to 900 meters on the mountain slope, under sparse broad-leaved forest or coniferous mixed forest, near the cliff or grassland on the mountain slope, and is mainly distributed in the mountainous areas of the three northeastern provinces, Hebei, Shandong, and Inner Mongolia, and also has a small amount of distribution in Shanxi and Shaanxi provinces.

[0004] In recent years, wild Atractylodes chinensis has gradually decreased, and cultivated Atractylodes lancea has continuously increased in market share. It has become an inevitable trend for wild Atractylodes lancea to become domesticated. In order to ensure the yield of Atractylodes lancea, most medicinal material growers use chemical pesticides to control infectious diseases caused by Paraphoma chrysanthemicola during the cultivation of Atractylodes lancea, but there is no effective control. SUMMARY

[0005] One of the present application provides a Bacillus amyloliquefaciens strain, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 25425.

[0006] The genetically modified Bacillus amyloliquefaciens strain of the present application can be endowed with more optimal and / or more properties, for example, the properties of the strain itself can be combined, the disease resistance performance can be increased and / or broadened according to the actual application, or the strain can be endowed with insecticidal or insect-resistant performance. That is, at least one of the above-mentioned performances is endowed to the genetically modified strain of the present application. Since the genetically modified strain is based on the Bacillus amyloliquefaciens strain of the present application, that is, the specific genes and / or sequences are introduced into and / or knocked out from the strain, the genetically modified strain is still Bacillus amyloliquefaciens.

[0007] Therefore, the second aspect of the present application provides an engineered strain obtained by genetically modifying the strain of the first aspect of the present application. For example, the genetically modified engineered strain can be obtained by introducing a plasmid carrying a functional gene, or the engineered strain can be obtained by recombining a functional gene into the genome of the wild-type strain.

[0008] Therefore, in a specific embodiment, the engineered strain is obtained by introducing a functional gene into the strain of the first aspect of the present application.

[0009] In a specific embodiment, the functional gene is at least one of a gene for controlling plant pests, a gene for controlling plant pathogenic microorganisms, and a gene for enhancing the effect of the strain on controlling plant pathogenic microorganisms.

[0010] The third aspect of the present application provides a composition comprising the strain of the first aspect of the present application or the engineered strain of any one of the second aspect of the present application.

[0011] In a specific embodiment, the dosage form of the composition is at least one of a suspension, a powder, and a granule.

[0012] In a specific embodiment, the dosage form of the composition is an oil suspension or a wettable powder.

[0013] The fourth aspect of the present application provides use of at least one of the strain of the first aspect of the present application, the engineered strain of any one of the second aspect of the present application, and the composition of any one of the third aspect of the present application in controlling plant diseases caused by Paraphoma chrysanthemicola.

[0014] In a specific embodiment, the use is for controlling diseases of Atractylodes chinensis (DC.) Koidz caused by Paraphoma chrysanthemicola.

[0015] In the present application, the terms are the general terms as indicated in the prior art, unless otherwise specified.

[0016] Advantages of the present application:

[0017] The present application first found that Bacillus amyloliquefaciens strain can be used for preventing and treating plant diseases caused by Paraphoma chrysanthemicola, in particular for preventing and treating diseases of Atractylodes chinensis (DC.) Koidz caused by Paraphoma chrysanthemicola. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The inhibition of biocontrol strain BAY-11 on Paraphoma chrysanthemicola CZ-5 strain is shown, wherein the left figure is the confrontation culture result of biocontrol strain BAY-11, and the right figure is the culture result of the control.

[0019] Figure 2 The inhibition of biocontrol strain BAY-11 on Paraphoma chrysanthemicola CZ-7 strain is shown, wherein the left figure is the confrontation culture result of biocontrol strain BAY-11, and the right figure is the culture result of the control.

[0020] Figure 3 The phylogenetic tree of biocontrol strain BAY-11 based on 16S rDNA is shown.

[0021] Figure 4 The phylogenetic tree of biocontrol strain BAY-11 based on gyrB gene is shown.

[0022] Strain preservation

[0023] The BAY-11 strain screened in the present application is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 25425, the preservation date is July 27, 2022, and the preservation address is: No. 1, Beichen West Road, Yard 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. Its systematic classification is Bacillus amyloliquefaciens. DETAILED DESCRIPTION

[0024] The above content of the present application is further described in detail in the form of preferred embodiments, but it does not constitute a limitation on the present application.

[0025] The reagents in the embodiments of the present application can be purchased through commercial channels unless otherwise specified.

[0026] Potato medium (PDA): 200 g of potato was cut into pieces, boiled in boiling water for 15 min, filtered, 20 g of glucose and 15 g of agar powder were added to the filtrate, and water was added to make up to 1000 mL, and high-pressure steam sterilization was performed for 20 min.

[0027] LB medium: 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, 15 g of agar, water was added to dissolve and make up to 1000 mL, pH 7.0, high-pressure steam sterilization was performed for 20 min.

[0028] The pathogenic fungi Paraphoma chrysanthemicola strains CZ-5 and CZ-7 were isolated from diseased plants in Chengde Niaocang Atractylodes planting sites.

[0029] Example 1

[0030] Isolation of biocontrol bacteria

[0031] A total of more than 300 soil samples were collected from orchards, Chinese herbal medicine planting sites, edible fungus planting sites, parks, and Xiaowutaishan Mountain in Hebei Province.

[0032] 1 g of each collected soil sample was weighed and added to 50 mL of sterilized water, and a soil suspension was obtained by thoroughly shaking. The soil suspension was placed in a water bath at 75°C for 30 min, and shaken every 15 min. 200 μL of the soil suspension was pipetted and spread on LB solid medium plates, and incubated at 28°C for 3 d. The colonies with a milky white color, rough surface, and wrinkled edges were selected and streaked on the surface of the LB solid medium for purification.

[0033] A total of 233 strains were obtained and numbered respectively.

[0034] Example 2

[0035] Bioassay experiment

[0036] Plate confrontation method was used to screen strains with antagonistic effect on Paraphoma chrysanthemicola.

[0037] The pathogenic fungi strains CZ-5 and CZ-7 were inoculated on PDA medium plates respectively, and incubated at 28°C for 7 days. Then, the pathogenic fungi strains CZ-5 and CZ-7 were punched into fungus cakes with sterilized punchers with a diameter of 5 mm, and inoculated on the central part of PDA medium plates with a diameter of 90 mm. The biocontrol strains isolated in Example 1 were inoculated on the four corners of the plates at a distance of 2.5 cm from the center, and sterilized toothpicks were used for inoculation. The plates inoculated with the pathogenic fungi in the center and dotted with sterilized water were used as controls. Each biocontrol strain was repeated for three times, and the plates were incubated in an incubator at 28°C. When the pathogenic fungi in the control group grew on the plates, the diameters of the inhibition zones and the colonies of each biocontrol strain in confrontation treatment and the control group were measured, and the relative inhibition rates were calculated.

[0038] Relative inhibition rate (%) = (diameter of colony in control group - diameter of colony in treatment group) / diameter of colony in control group x 100.

[0039] From the 233 strains, 41 strains with different degrees of antagonistic effect on the pathogenic fungi B. chondrillina were obtained in the preliminary screening. Seven strains with an inhibition rate of more than 65% and an inhibition zone of more than 4 mm were obtained in the rescreening. Further rescreening of the antagonistic strains obtained a stable antagonistic strain, which was numbered as BAY-11. The inhibition results of BAY-11 on B. chondrillina are shown in Table 1 and Figure 1 . The inhibition zone of BAY-11 on CZ-5 was 7.5 mm, and the inhibition rate was 77.7%. The inhibition zone of BAY-11 on CZ-7 was 7.7 mm, and the inhibition rate was 75.8%.

[0040] Table 1

[0041]

[0042] Example 3

[0043] Identification of the biocontrol strain BAY-11

[0044] Morphological identification: BAY-11 was streak-inoculated on LB medium, and incubated at 30°C for 48 h. The colony morphology of the strain was observed. The colony was milky white, opaque, irregular in edge, and relatively moist in early stage, and wrinkled in later stage.

[0045] Molecular biological identification: The total DNA of BAY-11 was extracted according to the instructions of the bacterial genomic DNA extraction kit, and PCR amplification was performed using bacterial 16S rDNA sequence primers and gyrB sequence primers. The sequence of 27F is shown in SEQ ID No. 1, the sequence of 1492R is shown in SEQ ID No. 2, the sequence of gyrB-F is shown in SEQ ID No. 3, and the sequence of gyrB-R is shown in SEQ ID No. 4.

[0046] PCR reaction system: Taq PCR Master Mix 25 μL, BAY-11 strain total DNA template 1 μL, upstream primer and downstream primer 2 μL each, supplemented with ddH2O to 50 μL, PCR reaction program: 94°C pre-denaturation for 5 min, 94°C denaturation for 40 s, 55°C annealing for 40 s, 72°C extension for 90 s, 35 cycles, 72°C extension for 10 min. The PCR product was detected by 1% agarose gel electrophoresis, and the product size was detected by DNA Marker BM5000. The target PCR product was sent to the sequencing department of Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequencing results of 16S are shown as SEQ ID No. 5, and the sequencing results of gyrB gene are shown as SEQ ID No. 6. The sequencing results were subjected to Blast comparison in the NCBI database, and sequences with high similarity were selected. The results showed that both 16S and gyrB genes were closest to Bacillus amyloliquefaciens. The nucleic acid sequence multiple sequence comparison analysis was performed by MEGA11 software, and the phylogenetic tree was constructed. Among them, Figure 3 The phylogenetic tree of biocontrol strain BAY-11 based on 16S rDNA; Figure 4 The phylogenetic tree of biocontrol strain BAY-11 based on gyrB gene. Figure 3 and Figure 4 It can be seen that the biocontrol strain BAY-11 and Bacillus amyloliquefaciens are all gathered together.

[0047] BAY-11 was identified as Bacillus amyloliquefaciens by morphology combined with molecular biology.

[0048] BAY-11 strain was preserved in China General Microbiological Culture Collection Center, and the preservation number was CGMCC No. 25425, the preservation date was July 27, 2022, and the preservation address was Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Beijing City, China. Its systematic classification is Bacillus amyloliquefaciens.

Claims

1. A type of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25425.

2. A composition comprising the strain as described in claim 1.

3. The composition according to claim 2, characterized in that, The composition is in the form of a suspension, powder, or granules.

4. The composition according to claim 2, characterized in that, The composition is in the form of an oil suspension or a wettable powder.

5. The strain as described in claim 1 or the composition as described in any one of claims 2 to 4 for the control of *Heterostilbene chrysanthum* (… Paraphoma chrysanthemicola Applications in plant diseases caused by ).

6. The application according to claim 5, characterized in that, The application is for the prevention and control of *Heterostigma chrysanthemum* (…). Paraphoma chrysanthemicola ) caused by Atractylodes macrocephala ( Atractylodes chinensis Application of (DC.) Koidz in disease.

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