Bacillus Velez BVY-148 and its application

Abstract

The present invention relates to Bacillus velez BVY-148 and its application. It is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No.25424. It can be used to prevent and treat plant diseases caused by Paraphoma chrysanthemicola.

Description

Technical Field

[0001] The present invention relates to the field of plant disease prevention and control, in particular to the field of plant fungal disease prevention and control. Background Art

[0002] Paraphoma chrysanthemicola belongs to the genus Paraphoma, belonging to the genus Paraphoma, belonging to the subdivision Ileutemmyootina, class Coclomycetes, order Sphaeropsidales, family Sphaeropsidaceae, kingdom Eumycetes, and can cause infectious diseases of Atractylodes chinensis (DC.) Koidz.

[0003] Atractylodes lancea, also known as Atractylodes lancea, mountain thorn grass, and spearhead vegetable, is a perennial herb of the Asteraceae family. Its rhizomes are often used as medicine. It has anti-inflammatory and analgesic effects, treats rheumatism, digestive disorders, acute enteritis, night blindness, influenza, and anti-tumor effects, and plays an important role in the treatment of new coronavirus pneumonia. Wild Atractylodes lancea mostly grows on hillsides at an altitude of 300 to 900 meters, under sparse broad-leaved forests or mixed coniferous forests, on cliffs near hillsides or on grasslands on hillsides. It is mainly distributed in mountainous areas of the three northeastern provinces, Hebei, Shandong, Inner Mongolia and other places, and is also distributed in small quantities in Shanxi and Shaanxi provinces.

[0004] In recent years, the number of wild Atractylodes lancea has gradually decreased, and the market share of cultivated Atractylodes lancea has continued to increase. The transformation of wild Atractylodes lancea into domesticated species has become an inevitable trend. In order to ensure the yield of Atractylodes lancea, most medicinal material growers use chemical agents to prevent and control infectious diseases caused by Chrysophora in the cultivation process of Atractylodes lancea, but they have not been able to effectively control it. Summary of the invention

[0005] One of the present invention provides a Bacillus velezensis strain, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.25424.

[0006] The second aspect of the present invention provides a composition, which comprises the strain as described in one of the present invention.

[0007] In a specific embodiment, the dosage form of the composition is at least one of a suspension, a powder and a granule.

[0008] In a specific embodiment, the composition is in the form of an oil suspension or a wettable powder.

[0009] The third aspect of the present invention provides a use of at least one of the strains described in one of the present invention and the composition described in any one of the second aspect of the present invention for preventing and controlling plant diseases caused by Paraphoma chrysanthemicola.

[0010] In a specific embodiment, the application is an application for preventing and controlling diseases of Atractylodes chinensis (DC.) Koidz caused by Paraphomachrysanthemicola.

[0011] In a specific embodiment, the application is an application for preventing and controlling Atractylodes macrocephala leaf spot disease caused by Heterostemon chrysanthemi.

[0012] Unless otherwise specified in the present invention, the terms in the present invention are all common terms in the prior art.

[0013] Beneficial effects of the present invention:

[0014] The present invention firstly finds that Bacillus velezensis strain can be used for preventing and controlling diseases of Atractylodes chinensis (DC.) Koidz, in particular diseases caused by Paraphomachrysanthemicola, such as Atractylodes chinensis leaf spot. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure shows the inhibitory effect of the biocontrol strain BVY-148 on the CZ-5 strain of Chrysanthemum heterostem mold. The left figure shows the confrontation culture results of the biocontrol strain BVY-148, and the right figure shows the culture results of the control.

[0016] Figure 2 The figure shows the inhibitory effect of the biocontrol strain BVY-148 on the CZ-7 strain of Chrysanthemum heterostem mold. The left figure shows the confrontation culture results of the biocontrol strain BVY-148, and the right figure shows the culture results of the control.

[0017] Figure 3 The phylogenetic tree of the biocontrol strain BVY-148 based on 16S rDNA is shown.

[0018] Figure 4 The phylogenetic tree of the biocontrol strain BVY-148 based on the gyrB gene is shown.

[0019] Strain preservation

[0020] The BVY-148 strain screened in this application is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 25424, a deposit date of July 27, 2022, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Bacillus velezensis. DETAILED DESCRIPTION

[0021] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0022] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0023] Potato culture medium (PDA): 200 g of potatoes were 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 the volume was made up to 1000 mL with water, and sterilized with high-pressure steam for 20 min.

[0024] LB medium: 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, and 15 g of agar. Dissolve with water and make up to 1000 mL. pH 7.0. Sterilize with high-pressure steam for 20 min.

[0025] The pathogenic fungi Paraphoma chrysanthemicola CZ-5 and CZ-7 were isolated from diseased plants grown in the Atractylodes macrocephala field in Chengde.

[0026] Example 1

[0027] Isolation of biocontrol bacteria

[0028] A total of more than 350 soil samples were collected from orchards, Chinese herbal medicine plantations, edible fungus plantations, parks, Xiao Wutai Mountain and other places in Hebei Province.

[0029] Weigh 1g of each soil sample collected and add it to 50mL of sterilized water. Shake thoroughly to obtain a soil suspension, place it in a water bath at 75℃ for 30min, and shake it every 15min. Use a pipette to draw 200μL of soil suspension and apply it on an LB solid culture medium plate. After culturing at 28℃ for 3d, select milky white, rough-surfaced, and wrinkled-edge colonies and streak them on the surface of the LB solid culture medium for separation and purification.

[0030] A total of 461 strains were obtained and numbered respectively.

[0031] Example 2

[0032] Bio-test

[0033] The plate confrontation method was used to screen the strains with antagonistic effect to Heterostyma chrysanthemi.

[0034] The pathogenic fungus CZ-5 and CZ-7 were inoculated on PDA culture medium plates, and after culturing at 28°C for 7 days, the pathogenic fungus strains CZ-5 and CZ-7 were punched into bacterial cakes with a sterilized hole puncher with a diameter of 5 mm, and were inoculated in the center of a PDA culture medium plate with a diameter of 90 mm. The biocontrol strains separated in Example 1 were inoculated at the four corners 2.5 cm away from the center with sterilized toothpicks, and the plate with the pathogen inoculated in the center and sterilized water inoculated around was used as a control. Each biocontrol strain was repeated 3 times and cultured in a constant temperature incubator at 28°C. When the pathogens in the control group covered the plate, the antibacterial bands and colony diameters of each biocontrol bacteria confrontation treatment and the control group were measured, and the relative antibacterial rate was calculated.

[0035] Relative inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100.

[0036] Ten strains with an antibacterial rate of more than 65% and an antibacterial zone of more than 5 mm against the pathogen Chrysanthemum heterostem point were screened from 461 strains. Further antagonistic strains were screened to obtain a strain with stable antagonistic function, which was numbered BVY-148. The antibacterial results of the strain against Chrysanthemum heterostem point point were shown in Tables 1 and Figure 1 Among them, the antibacterial zone for CZ-5 was 8.2 mm, and the antibacterial rate was 78.2%; the antibacterial zone for CZ-7 was 9.0 mm, and the antibacterial rate was 76.4%.

[0037] Table 1

[0038]

[0039] Example 3

[0040] Identification of the biocontrol bacterium BVY-148

[0041] Morphological identification: BVY-148 was streaked onto LB medium and cultured at 30°C for 48 hours. The colony morphology of the strain was observed. The colonies were milky white, opaque, with irregular edges. The colonies were moist in the early stage and wrinkled in the later stage.

[0042] Molecular biological identification: The total DNA of BVY-148 strain 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. Among them, the sequence of 16S rDNA primer: 27F is shown in SEQ ID No.1, the sequence of 1492R is shown in SEQ ID No.2, the sequence of gyrB primer: gyrB-F is shown in SEQ ID No.3, and the sequence of gyrB-R is shown in SEQ ID No.4.

[0043] PCR reaction system: Taq PCR Master Mix 25 μL, BVY-148 strain total DNA template 1 μL, upstream primer and downstream primer 2 μL each, supplemented with ddH 2 O to 50μL, PCR reaction procedure: 94℃ pre-denaturation for 5min, 94℃ denaturation for 40s, 55℃ annealing for 40s, 72℃ extension for 90s, 35 cycles, 72℃ extension for 10min. 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 Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results of 16S are shown in SEQ ID No.5, and the sequencing results of gyrB gene are shown in SEQ ID No.6. The sequencing results were Blast compared in the NCBI database, and sequences with high similarity were selected. The results showed that both 16S and gyrB genes were closest to Bacillus velezensis. MEGA11 software was used for multiple sequence comparison analysis of nucleic acid sequences to construct a phylogenetic tree, among which, Figure 3 Phylogenetic tree of biocontrol strain BVY-148 based on 16S rDNA; Figure 4 Phylogenetic tree of biocontrol strain BVY-148 based on gyrB gene. Figure 3 and Figure 4 It can be seen that the biocontrol strain BVY-148 and Bacillus velezensis are clustered together.

[0044] BVY-148 was identified as Bacillus velezensis by morphology and molecular biology.

[0045] The BVY-148 strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration with the deposit number CGMCC No.25424 and the deposit date July 27, 2022 at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Bacillus velezensis.

[0046] Example 4

[0047] Control effect of biocontrol bacteria BVY-148 on leaf spot disease of Atractylodes macrocephala

[0048] The seeds of Atractylodes lancea, which had been disinfected with 75% ethanol and 1% sodium hypochlorite, were planted in pots filled with sterilized soil, with 6 plants in each pot. When the Atractylodes lancea plants grew to 4 leaves, a concentration of 1.0×10 8 cfu / mL Velez Bacillus BVY-148 fermentation liquid was sprayed on the leaves of Atractylodes lancea plants once every 7 days for 3 times in succession. Spraying with clean water was used as a blank control, and spraying with 500g / L 1000-fold dilution of cyprodinil SC was used as a chemical control. Each pot was used as a treatment, and each treatment was repeated 4 times. On the second day after the last spraying, the total concentration of the spore suspension sprayed was 1×10 6 The mixture of pathogens CZ-5 and CZ-7 at a ratio of 1:1 was placed in a constant temperature culture room at 25°C for cultivation. After the control was fully diseased, the occurrence level of Atractylodes lancea leaf spot disease was investigated according to the 0-9 grade grading standard. The disease grading standard is: grade 0, no lesions; grade 1, the lesion area accounts for less than 5% of the whole leaf area; grade 3, the lesion area accounts for 6% to 15% of the whole leaf area; grade 5, the lesion area accounts for 16% to 25% of the whole leaf area; grade 7, the lesion area accounts for 26% to 50% of the whole leaf area; grade 9, the lesion area accounts for more than 50% of the whole leaf area. The incidence rate, disease index and control effect were calculated, and the results are shown in Table 2.

[0049] Table 2 Control effect of biocontrol bacteria BVY-148 on leaf spot of Atractylodes macrocephala

[0050]

[0051] The results in Table 2 show that the control effect of Bacillus Velezii BVY-148 on Atractylodes lancea leaf spot disease is 76.3%, and the control effect is obvious.

Claims

1. A Bacillus Velezii ( Bacillus velezensis ) strain, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.25424.

2. A composition comprising the strain according to claim 1.

3. The composition according to claim 2, characterized in that The dosage form of the composition is at least one of a suspension, a powder and a granule.

4. The composition according to claim 3, characterized in that The dosage form of the composition is oil suspension or wettable powder.

5. Use of the strain according to claim 1 or the composition according to any one of claims 2 to 4 for controlling plant diseases caused by Paraphoma chrysanthemicola.

6. The use according to claim 5, characterized in that: The application is application in preventing and controlling diseases of Atractylodes chinensis (DC.) Koidz caused by Paraphoma chrysanthemicola.

7. The use according to claim 5, characterized in that: The application is an application in preventing and controlling Atractylodes macrocephala leaf spot disease caused by Heterostemon chrysanthemi.

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