A strain of Bacillus carnegieii BC30 and its application

Abstract

The present invention provides a strain of Bacillus cabrialesii BC30 and an application thereof, which is named as Bacillus cabrialesii BC30 and has a deposit number of CCTCC NO: M 2023167. The present invention also provides an application of the Bacillus cabrialesii BC30 in preventing and controlling tobacco black shank disease, promoting tobacco plant growth and seed germination; the Bacillus cabrialesii BC30 has a good disease prevention effect on tobacco black shank disease and has a significant growth-promoting effect on tobacco plants.

Description

Technical Field

[0001] The invention relates to the academic field of microorganisms, and in particular to a strain of Bacillus carinii BC30 and application thereof. Background Art

[0002] In my country, tobacco planting is an important industry to solve the employment problem in many rural areas of my country. According to incomplete estimates, more than 1.3 million farmers grow tobacco every year in my country. Tobacco planting plays an important role in solving employment problems in many rural areas and increasing income. Due to changes in planting methods, irrational application of pesticides and fertilizers, forced continuous cropping of tobacco fields, and resistance degradation of disease-resistant varieties, tobacco black shank disease has begun to be widely distributed in my country, and infects and harms all cultivated varieties such as flue-cured tobacco, sun-cured tobacco, air-dried tobacco, and flavored tobacco at any growth stage. According to surveys, China's average annual loss due to tobacco black shank disease is over 100 million yuan. Black shank disease has also become one of the most serious root and stem diseases in China and one of the most devastating diseases in tobacco production. The spread and occurrence of the disease seriously affect the yield and quality of tobacco, have a great impact on the tobacco industry, and have become an important factor restricting the high-quality, efficient, and safe production of tobacco.

[0003] Tobacco black shank is a fungal soil-borne disease caused by Phytophthora nicotianae, which has the characteristics of high incidence, wide distribution, great harm and strong destructiveness. It has a long survival time and can carry out initial infection and multiple reinfections. High temperature and high humidity conditions are more likely to promote the occurrence of the disease, and it can occur in both the seedbed period and the field growth stage. When the disease occurs in the seedling stage, black spots first appear at the base of the tobacco plant stem. When the humidity is high, the spots are covered with white hairs and quickly spread to nearby seedlings, causing large-scale death of other seedlings. When the disease occurs in the field, the main parts of the disease are the base and roots of the stem, and often show symptoms such as black shanks, leaf wilting, brown spots, pith shrinkage, and stem necrosis. When the disease is serious, the lesions spread along the main veins to the base of the leaves and invade the pith, causing black-brown necrosis in the middle of the stem, appearing like a waist rot. When the stems of the diseased plants are cut longitudinally, black-brown disc-like necrosis occurs inside the stems, and the plants wilt, commonly known as "wearing a coat". Large round, black-brown lesions occur on the infected parts, shaped like plasters, commonly known as "black plasters". If not promptly prevented and controlled, the disease will quickly occur and cause large-scale infection.

[0004] At present, the production mainly adopts planting disease-resistant varieties and using chemical agents to prevent and control black shank disease. However, no immune varieties to tobacco black shank disease have been found so far, and the resistance of varieties will decrease with the rise and fall of the physiological subspecies of black shank pathogens. At the same time, chemical pesticides are easy to cause environmental pollution and pesticide residues in tobacco leaves, and long-term use will also make the pathogens resistant. Therefore, finding a safe, efficient and pollution-free method for the prevention and control of tobacco black shank disease has become a top priority for tobacco production. Biological control is an environmentally friendly and promising means of prevention and control, and the use of beneficial microorganisms has become a research hotspot for plant disease prevention and control. Plant endophytic bacteria are natural resource bacteria for biological control of plant diseases and insect pests, and have a very wide range of theoretical research and development and application value. Endophytic bacteria exist in plants, are protected by plants, and are not easily affected by the external environment. Many strains have a wide range of biological effects on host plants, such as disease prevention, growth promotion and biological nitrogen fixation, and can colonize and transmit in plants and play a long-term role. Summary of the invention

[0005] The present invention aims to provide a strain of Bacillus carinii BC30 and an application thereof. The strain of Bacillus carinii BC30 has a good disease prevention effect on tobacco black shank disease and has a significant growth-promoting effect on tobacco plants.

[0006] In a first aspect of the present invention, a strain of Bacillus carinii BC30 is provided. The deposit number of the Bacillus carinii BC30 is: CCTCC NO: M 2022368.

[0007] In the second aspect of the present invention, the use of the Bacillus carinii BC30 in preventing and treating tobacco black shank disease is provided.

[0008] In the third aspect of the present invention, the use of the Bacillus carinii BC30 in promoting the growth of tobacco plants is provided.

[0009] In the fourth aspect of the present invention, the use of the Bacillus carinii BC30 in promoting tobacco seed germination is provided.

[0010] In a fifth aspect of the present invention, a fermentation agent is provided, wherein the fermentation agent comprises:

[0011] Fermenting the Bacillus carinii BC30 to obtain a fermentation liquid;

[0012] Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

[0013] In a sixth aspect of the present invention, a method for degrading o-cresol and / or 2,4-di-tert-butylphenol using the Bacillus carinii BC30 is provided, the method comprising:

[0014] Activating the Bacillus carinii BC30 or the fermentation agent to prepare a fermentation liquid;

[0015] The fermented liquid is soaked in tobacco seeds, or the fermented liquid is applied to the roots of tobacco plants.

[0016] Furthermore, the live bacteria concentration in the fermentation broth is adjusted to (1-3)×108 cfu / mL.

[0017] Furthermore, when the fermentation liquid is applied to the roots of tobacco plants, the tobacco plants are irrigated with the fermentation liquid 30 days after transplantation, and the irrigating is continued for 5 times, irrigating once every three days.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] The present invention provides a strain of Bacillus carrionii BC30 and its application, wherein the Bacillus carrionii BC30 has a good disease prevention effect on tobacco black shank and has a significant growth-promoting effect on tobacco plants. It can be seen that the strain has good theoretical research value and application prospects, and can not only be used as a good experimental material for studying the interaction between endophytic bacteria and host plants and their mechanism of action, but can also be used for the research and development of endophytic biofertilizers and biofungicides, and may also become a good carrier for multifunctional engineering bacteria such as disease prevention and growth promotion, providing a new strain resource for the biological control of tobacco black shank.

[0020] The deposit date of the Bacillus cabrialesii BC30 of the present invention is February 21, 2023, and the deposit number is CCTCCNO: M 2023167. Its classification name is Bacillus cabrialesii BC30, and the depository name is China Center for Type Culture Collection, and the address is Wuhan University, Wuhan City, Hubei Province, China, Postal Code: 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 is the antibacterial effect of endophytes; Figure 1 A is the result of the initial screening; Figure 1 B is the result of rescreening; Figure 1 C is the antibacterial effect of the model strain BS168 on tobacco phytophthora;

[0023] Figure 2The antibacterial effect of sterile fermentation broth;

[0024] Figure 3 is the disease prevention effect of BC30 on tobacco black shank; Figure 3 A is the growth of tobacco plants in the control group. Figure 3 B is the growth of tobacco plants after BC30 treatment. Figure 3 C is a comparison of the stem sections of the two groups of tobacco plants

[0025] Figure 4 This is the phylogenetic tree of BC30. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0029] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0030] The inventors of the present application obtained a tobacco endogenous bacterium by separation, purification and screening from tobacco collected in the greenhouse of the School of Life Sciences of Hubei University, and found that the strain had an antagonistic effect on tobacco blight. The bacteria were analyzed by colony morphology, biochemistry and 16SrRNA sequencing. The strain had a homology of more than 96% with multiple strains of Bacillus cabrialesii. Combined with physiological and biochemical characteristics, the strain was preliminarily determined to be of the genus (Bacillus cabrialesii) and was named Bacillus cabrialesii BC30.

[0031] The Bacillus carinii BC30 of the present invention has a good promoting effect on tobacco seed germination and tobacco plant growth, has a good disease prevention effect on tobacco black shank disease, and provides a theoretical basis for the subsequent development of biological bacterial agents.

[0032] The following will describe in detail a strain of Bacillus carinii BC30 and its application in the present application in combination with examples and experimental data.

[0033] Experimental strains: Phytophthora nicotianae was purchased from the China Agricultural Microbiological Culture Collection (ACCC 36286), and Bacillus subtilis strains 168 was purchased from the Shanghai Biotechnology Collection Center (ATCC 23857).

[0034] Test medium: NA medium: 3g beef extract, 1g yeast extract, 5g peptone, 10g glucose, 15g agar, distilled water to 1000mL, pH7.2, sterilized. LB solid medium: 10g sodium chloride, 10g peptone, 5g yeast powder, 1000mL water, 15g agar, sterilized. Potato dextrose medium (PDA): 200g potato, 20g glucose, 15-20g agar, 1000mL distilled water, pH7.0-7.4.

[0035] Example 1: Directed enrichment, separation and identification of strain BC30

[0036] 1. Isolation and purification of tobacco endogenous bacteria

[0037] After washing the tobacco, 1g of root, stem and leaf samples were cut respectively, and soaked in 75% ethanol for 1min, rinsed with sterile water 3 times, soaked in 5% NaClO for 6min, rinsed with sterile water 5 times, and then ground in a sterile mortar with 1mL of sterile water. -3 , 10 -4 , 10 -5 , 10 -6 Take 0.1 mL of CFU / mL gradient dilutions and spread them on solid LB plates, and culture them at 28°C for 48 hours. Pick single colonies with obvious culture characteristics, purify them by streaking on LB plates, and store the purified strains at 4°C for later use.

[0038] 2. Isolation and screening of strains

[0039] (1) Primary screening: Using tobacco phytophthora as an indicator, the plate confrontation method was used to determine its inhibitory activity against fungal pathogens. Use a sterile puncher with a diameter of 5 mm to punch a bacterial cake at the edge of the pathogen colony, inoculate it in the center of the PDA plate, and use a puncher to punch three holes with a diameter of 5 mm at a distance of 3 cm from the center. Add 20uL of bacterial suspension to each hole, set up a clear water control, set up 3 replicates for each treatment, and place the plate in a 37°C incubator for culture. When the control colony grows to 2 / 3 of the culture dish, observe whether there is an obvious antagonistic effect, and select strains that have an inhibitory effect on the growth of pathogens for re-screening. Take out the above primary screening plate, measure the size of the inhibition zone and calculate the inhibition rate. Inhibition rate = (diameter of the inhibition zone of the treatment group - diameter of the inhibition zone of the control group) / diameter of the inhibition zone of the treatment group × 100%.

[0040] A variety of endophytic bacteria were screened out from the rhizomes of tobacco plants and 6 strains were isolated and purified. After preliminary screening, 2 strains were screened out that had antagonistic effects against tobacco blight.

[0041] (2) Rescreening: The two strains obtained from the initial screening were rescreened using the PDA plate confrontation method. The purified test bacteria were streaked in parallel on both sides 2.5 cm away from the pathogen, with the single inoculation of the target bacteria as the control. Each treatment was repeated 3 times and cultured at a constant temperature of 28°C. When the pathogen colonies in the control group covered the bottom of the plate, the comparison was made to see if there was an obvious antagonistic effect.

[0042] Finally, a strain with good antagonistic effect on tobacco phytophthora was screened out and named BC30. Figure 1 Bacillus subtilis strains 168 were purchased for antibacterial assay comparison test. The antibacterial assay results of the Bacillus subtilis strains and the BC30 of the present invention on tobacco black shank are shown in Table 1.

[0043] Table 1 Antibacterial assay of BC30 against Phytophthora nicotianae

[0044]

[0045] As shown in Table 1, the inhibition zone of strain BC30 against tobacco phytophthora is about 2.23 cm, and the inhibition rate can reach 77.52%, which is significantly increased by 52.8% compared with the common spore strain (Bacillus subtilis 168), and the antagonistic effect can be clearly observed on the plate.

[0046] 3. Identification of strain BC30

[0047] (1) Morphological characteristics identification

[0048] The strains screened out with obvious antagonistic effects were taken out of the refrigerator, streaked on NA plates, cultured at 28°C for 48 h, single colonies were picked, streaked on new NA plates, and cultured at 28°C for 60 h. The color, morphology and other characteristics of the colonies were observed and recorded, and the cell morphology was observed by optical microscopy and Gram staining.

[0049] The bacteria were identified as Gram-positive, aerobic, and single-celled, with an optimal growth temperature of 28-30°C and a growth pH of 6.0-8.0. On LB medium, the colonies cultured in the dark at 28°C for 24 hours were milky white, round, flat, opaque, with a diameter of about 4.0 mm, irregular edges, no halo, smooth and moist surface, sticky texture, and rod-shaped vegetative cells, which were consistent with the characteristics of Bacillus carinii.

[0050] (2) 16S rDNA sequence homology analysis and phylogenetic tree construction

[0051] The strain gene DNA was extracted by the bacterial suspension boiling water bath method, and PCR amplification was performed using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system included 1 μL DNA template, 1 μL upstream and downstream primers, 12.5 μL Taq polymerase, and ddH2O to 25 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min 30 s, 24 cycles, and finally 72°C extension for 10 min. The PCR reaction products were detected by 1% agarose gel electrophoresis, and the PCR amplification products of 16S rRNA were recovered and purified and sequenced by Wuhan Qingke Innovation Biotechnology Co., Ltd. The sequencing results were submitted to the NCBI database for BLAST homology analysis, and then the sequence alignment and phylogenetic tree construction were performed using MEGA 7.0 software. Data were processed and analyzed using Excel and DPS7.05 software.

[0052] The 16S rDNA sequence of the strain to be tested obtained by PCR (as shown in SEQ ID NO.1) was subjected to BLAST comparison analysis in the database. The results are as follows: Figure 4 The results showed that the sequence similarity between strain BC30 (OP905596) and Bacillus cabrialesii was as high as 100%, and the phylogenetic tree construction results showed that BC30 and Bacillus cabrialesii (OP435777.1) belonged to the same branch ( Figure 4), combined with morphological and physiological and biochemical identifications, it was identified as Bacillus cabrialesii and named Bacillus cabrialesii BC30. It was deposited in the China Center for Type Culture Collection on February 21, 2023, with the deposit number CCTCC NO: M2023167.

[0053] Example 2 Determination of antibacterial activity of aseptic fermentation filtrate of strains

[0054] 1. Preparation of strain fermentation broth

[0055] The Bacillus carlesii BC30 strain was inoculated into 100 mL LB liquid medium and cultured at 28°C, 230 r / min for 12 h to prepare seed solution, and then inoculated into LB culture medium at a 2% inoculum amount and cultured for 24 h at 28°C, 230 r / min to obtain the strain fermentation liquid. Before the pot test, the concentration of live bacteria in the fermentation liquid was adjusted to 1×10 8 cfu / mL.

[0056] 2. Determination of antibacterial activity of sterile fermentation filtrate of strains

[0057] (1) Preparation of sterile fermentation filtrate of biocontrol strains: The cultured bacterial broth was centrifuged at 12000 r / min for 10 min, and the supernatant was sterilized and filtered twice with a bacterial filter (0.22 μm) to obtain a sterile fermentation filtrate.

[0058] (2) Effect of sterile fermentation filtrate on the growth of pathogenic mycelium: The plate punching method was used to test the antibacterial effect of sterile fermentation filtrate of antagonistic bacteria. Use a plate puncher to evenly punch three holes 2.5 cm away from the center of the plate (a culture dish with a diameter of 9 mm), inject 20 μL of fermentation filtrate into each hole, and use sterile LB liquid culture medium as the control. A pathogen block with a diameter of 5 mm was placed in the center of the culture medium, and each treatment was repeated 3 times. After culturing in the dark at 28°C for 3 days, the colony radius of the target bacteria in the control group and the treatment group was measured, and the relative inhibition rate was calculated.

[0059] 3. Results

[0060] The results of the test on the effect of the aseptic fermentation filtrate of the strain on the mycelial growth of tobacco black shank pathogen are as follows: Figure 2 And as shown in Table 2.

[0061] Table 2 Antibacterial activity of BC30 sterile fermentation filtrate

[0062]

[0063] Depend on Figure 2 As shown in Table 2, the fermentation filtrate of BC30 also has an inhibitory effect on the mycelial growth of Phytophthora nicotianae ( Figure 2), the size of the inhibition zone was 1.16 cm, the relative inhibition rate was 56.34%, and the inhibition effect was lower than that of living bacteria, which was reduced by 21.18% (Table 2).

[0064] Example 3: Determination of tobacco seed germination and growth promotion effects

[0065] 1. Select tobacco seeds with uniform particles, disinfect them with 75% ethanol for 30 seconds, wash them three times with sterile water, dry them with sterile filter paper, and immerse them in the fermentation liquid of the strain for 4 hours. Take out the seeds and place them evenly and equidistantly in a culture dish covered with 4 layers of moist filter paper, 50 seeds per dish, and set 3 replicates for each concentration treatment. Culture them in the dark in a light incubator for 14 days (humidity 80%, temperature 28±2℃), add a certain amount of sterile water every 2 days to keep the filter paper moist, count the number of seeds that germinate, and calculate the germination rate.

[0066] 2. Inoculate the strains into LB medium, shake at 28°C, 230 rpm for 24 h, and adjust the concentration of the bacterial suspension to 1×10 8 cfu / mL. The tobacco seedlings at 4-5 leaf stage were irrigated with roots and sprayed on leaves, once every 5 days, for a total of 3 times. After 15 days, the differences in seedling length, leaf length, leaf width, fresh weight and root-to-crown ratio between the treatment group and the control group were measured and compared.

[0067] Seed germination rate = (number of germinated seeds ÷ number of test seeds) × 100%

[0068] Root-shoot ratio = underground fresh weight / aboveground fresh weight x 100%

[0069] 3. The results are shown in Table 3.

[0070] Table 3 Growth-promoting effect of BC30 on tobacco plants

[0071]

[0072] From the results in Table 3, it can be seen that 10 days after the seed soaking treatment with strain BC30, the seed germination rate increased significantly, reaching 92.3%, which was 4.7% higher than the control group. There were obvious differences in agronomic traits after treatment. Compared with the control group, the seedling length, leaf length, leaf width, fresh weight and root-crown ratio were all increased after treatment with strain BC30, which shows that BC30 has a good promoting effect on tobacco seed germination and tobacco plant growth.

[0073] Example 4: Analysis of the effect of strains on the prevention of tobacco black shank

[0074] 1. Field experiment

[0075] Fifty days after the tobacco plants were transplanted, the fermentation liquid of the strain obtained in Example 2 was irrigated for 5 consecutive times, once every 4 days. Fifteen days later, the soil was inoculated with tobacco phytophthora, and 50 tobacco plants were treated. Clear water was used as a control. The incidence of the disease was counted after 90 days.

[0076] 2. The results are as follows Figure 3 As shown in the data, application of BC30 fermentation liquid to tobacco plants can significantly improve the resistance inducing ability of the plants to tobacco black shank. Dark brown to black spots appeared at the base of the stems near the soil surface in the control group, and some lower leaves withered and expanded upward and horizontally. The longitudinal section of the diseased stems showed that the pith shrank into brown discs with sparse white hyphae between the discs. The disease incidence in the treatment group was significantly alleviated and the severity of the disease was significantly reduced, indicating that strain BC30 has a good disease prevention effect on tobacco black shank, providing a theoretical basis for the subsequent development of biological agents.

[0077] In summary, the Bacillus carinii BC30 provided by the present invention has a good disease prevention effect on tobacco black shank disease and has a significant growth-promoting effect on tobacco plants.

[0078] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0079] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0080] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0081] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Bacillus cabrialesii BC30, characterized in that: The deposit number of the Bacillus carinii BC30 is: CCTCC NO: M 2023167.

2. Use of the Bacillus carinii BC30 according to claim 1 in preventing and treating tobacco black shank disease.

3. Use of the Bacillus carinii BC30 according to claim 1 in promoting the growth of tobacco plants.

4. Use of the Bacillus carinii BC30 according to claim 1 in promoting tobacco seed germination.

5. A fermentation agent, characterized in that: The fermentation agent comprises: Fermentation of the Bacillus carinii BC30 as claimed in claim 1 to obtain a fermentation broth; Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

6. A method for preventing and treating tobacco black shank disease using the Bacillus carinii BC30 according to claim 1, characterized in that: The method comprises: Activating the Bacillus carinii BC30 described in claim 1 or the fermentation agent described in claim 5 to prepare a fermentation liquid; The fermented liquid is soaked in tobacco seeds, or the fermented liquid is applied to the roots of tobacco plants.

7. The method according to claim 6, characterized in that The concentration of live bacteria in the fermentation broth is adjusted to (1-3)×10 8 cfu / mL.

8. The method according to claim 6, characterized in that When the fermentation liquid is applied to the roots of tobacco plants, the tobacco plants are irrigated with the fermentation liquid 50 days after transplantation, and the irrigating is continued for 5 times, irrigating once every 4 days.

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