Use of bacillus velezensis in preparing microbial inoculants with plant growth promoting effect

Abstract

The application provides application of Bacillus velezensis in preparation of a microbial agent with a plant growth promoting effect, the Bacillus velezensis is Bacillus velezensis IFST-221 strain, the preservation number is CGMCC No.22955, and the preservation date is July 26, 2021. The strain has the effects of bacteriostasis, nitrogen fixation, phosphorus dissolution, potassium solution, biofilm formation and auxin production, and can be used for preparing a product for inhibiting pathogenic bacteria, promoting plant growth and reducing plant soil-borne diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial preparations, and particularly relates to application of Bacillus velezensis IFST-221 strain in preparation of a microbial agent with the function of promoting plant growth, application of Bacillus velezensis IFST-221 strain in preparation of a biocontrol microbial agent for preventing and / or treating soil-borne diseases of plants, a culture of Bacillus velezensis for preventing and / or treating soil-borne diseases of plants and promoting plant growth, and a microbial agent for plants with the functions of preventing diseases and promoting growth. BACKGROUND

[0002] A soil-borne disease of crops refers to a disease caused by pathogenic bacteria invading crops from the root system or stem base under appropriate conditions with soil as a survival medium, mainly including root rot, stem base rot, wilt, and yellow wilt. For such diseases, the current prevention and control means mainly relies on chemical pesticides with carbendazim and tebuconazole as main components, but the environmental pollution and pesticide residue problems caused by chemical pesticides cannot be ignored, and the large amount of use of chemical fertilizers has caused the decline of soil fertility and the destruction of the microbial environment in the soil, further aggravating the occurrence of soil-borne diseases.

[0003] Soil-borne diseases can be prevented and controlled by pollution-free biological control means. Biological control refers to the prevention and control of harmful organisms by microorganisms or their metabolites, and plant growth-promoting rhizobacteria (PGPR) derived from the natural environment is an important biological control bacteria, mainly including Bacillus, Pseudomonas, nitrogen-fixing bacteria, and microbacterium.

[0004] CN101974462A discloses a Bacillus subtilis for preventing and controlling soil-borne diseases of Chinese herbal medicines and preparation of a microbial agent thereof, and relates to a Bacillus subtilis and preparation of a microbial agent thereof. The Bacillus subtilis X1 for preventing and controlling soil-borne diseases of Chinese herbal medicines is of the genus Bacillus (Bacillus sp.), has a preservation number of CGMCC No.4037, and was preserved on July 26, 2010. The microbial agent is prepared by inoculating the activated strain into NYD liquid medium for enrichment culture to obtain a bacterial liquid with a bacterial number concentration of 20-40 billion per milliliter. The strain X1 in the present application is a biocontrol strain with high prevention effect on soil-borne diseases; the microbial agent can prevent and control diseases such as root rot and sclerotium rot of ginseng, sclerotium rot of plain-bean, root rot and stem base rot of schisandra chinensis, and the prevention and control effect reaches 60%-80%. The biocontrol preparation of the microbial agent is safe, does not pollute the environment, does not harm non-target organisms, and does not induce drug resistance of pathogenic bacteria.

[0005] CN104509539A discloses a compound fungicide containing metalaxyl and carbendazim for preventing and treating main diseases of vegetables and grapes, which is characterized by comprising active ingredients and adjuvants, wherein the active ingredients comprise metalaxyl and carbendazim fungicides, and the weight ratio of the two is 1-20:1-20. The compound fungicide is mainly used for preventing and treating soil-borne diseases including pepper blight and pepper wilt, and aerial diseases including grape downy mildew, grape white rot, cucumber downy mildew, cucumber anthracnose, tomato late blight and tomato gray mold. The compound fungicide has a wide control spectrum, good quick-acting and persistent properties, and can effectively delay the occurrence and development of pathogen resistance. The compound preparation has simple preparation process, low cost, high safety and remarkable economic benefits.

[0006] Therefore, in order to reduce the pollution problems caused by chemical pesticides and fertilizers to the environment, it is very meaningful to develop biological agents with disease prevention and growth promotion effects. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide Bacillus velezensis and a microbial agent, in particular, the application of Bacillus velezensis IFST-221 strain in the preparation of a microbial agent with plant growth promoting effect, the application of Bacillus velezensis IFST-221 strain in the preparation of a biocontrol microbial agent for preventing and / or treating plant soil-borne diseases, a culture of Bacillus velezensis for preventing and / or treating plant soil-borne diseases and promoting plant growth, and a plant microbial agent with disease prevention and growth promotion effect.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides the application of Bacillus velezensis in the preparation of a microbial agent with plant growth promoting effect, wherein the Bacillus velezensis is Bacillus velezensis IFST-221 strain, the preservation number is CGMCC No.22955, the preservation date is July 26, 2021, and the preservation unit is China General Microbiological Culture Collection Center, located at No.3, Beichen West Road, Chaoyang District, Beijing.

[0010] The Bacillus velezensis IFST-221 strain is isolated from a healthy corn rhizosphere soil sample in a corn ear rot disease serious field plot in Qujing City, Yunnan Province, China, and is named Bacillus velezensis IFST-221 strain. The strain has the effects of nitrogen fixation, phosphorus dissolution, potassium dissolution, biofilm formation and auxin production, can significantly increase the fresh weight, dry weight and plant height of corn, cotton, tomato, cauliflower and the like, and has an obvious effect of promoting plant growth, thereby providing a new selection strategy for preparing a plant growth promoting agent.

[0011] In a second aspect, the present application provides an application of the Bacillus velezensis IFST-221 strain in preparing a biocontrol agent for preventing and / or treating plant soil-borne diseases. The Bacillus velezensis IFST-221 strain has a preservation number of CGMCC No. 22955, a preservation date of July 26, 2021, and a preservation unit of China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing.

[0012] The present application also finds that the Bacillus velezensis IFST-221 strain has the effects of significantly inhibiting pathogenic bacteria and promoting plant growth, thereby reducing plant soil-borne diseases. Specifically, the inhibition rates of the Bacillus velezensis IFST-221 strain on six pathogenic bacteria, i.e., Fusarium pseudotrichia, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Phytophthora nicotianae and Verticillium dahliae, can reach 61.84%, 68.54%, 61.51%, 71.61%, 77.88% and 74.14%, respectively. In combination with the effects of nitrogen fixation, phosphorus dissolution, potassium dissolution, biofilm formation and biotin production of the Bacillus velezensis IFST-221 strain, the Bacillus velezensis IFST-221 strain has a good application prospect in preparing a biocontrol agent for preventing and / or treating plant soil-borne diseases.

[0013] In a third aspect, the present application provides a culture of Bacillus velezensis for preventing and / or treating plant soil-borne diseases and promoting plant growth, and a preparation method of the culture comprises the following steps:

[0014] The Bacillus velezensis IFST-221 strain in the first aspect or the second aspect is inoculated into a culture medium and cultured at 30-39℃ for 12-24h to obtain the culture.

[0015] The specific value in the range of 30-39℃ can be selected from 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃ or 39℃, and other specific point values in the above-mentioned value range can be selected, which will not be described one by one here.

[0016] The specific value in the range of 12-24h can be selected from 12h, 14h, 16h, 18h, 20h, 22h or 24h, and other specific point values in the above-mentioned value range can be selected, which will not be described one by one here.

[0017] Preferably, the culture medium is LB medium, CM medium or NB medium.

[0018] The culture condition is preferably described above, and the Bacillus velezensis IFST-221 strain has a fast growth rate and strong carbon source utilization ability in the culture condition.

[0019] In a fourth aspect, the present application provides a plant microbial agent with disease prevention and growth promotion effects, and the strain in the plant microbial agent with disease prevention and growth promotion effects comprises the Bacillus velezensis IFST-221 strain according to the first aspect, the second aspect or the third aspect.

[0020] The Bacillus velezensis IFST-221 strain can be applied alone in related products, or can be combined with other strains and applied in related products. The plant microbial agent containing the Bacillus velezensis IFST-221 strain has a significant effect of promoting plant growth and preventing and / or treating soil-borne diseases of plants.

[0021] Preferably, the dosage form of the plant microbial agent with disease prevention and growth promotion effects comprises a liquid microbial agent, a powder microbial agent or a granular microbial agent.

[0022] Preferably, the viable cell count of the Bacillus velezensis IFST-221 strain in the liquid microbial agent is not less than 1×10 9 cfu / mL, for example, 1×10 9 cfu / mL, 1.5×10 9 cfu / mL, 2×10 9 cfu / mL, 2.5×10 9 cfu / mL, 5×10 9 cfu / mL, 10×10 9cfu / mL, other values within the numerical point can be selected, and here we will not repeat them one by one.

[0023] Preferably, the number of viable bacteria of the Bacillus velezensis IFST-221 strain in the powdery bacterial agent is not less than 1.5 x 10 9 cfu / g, for example, 1.5 x 10 9 cfu / g, 2 x 10 9 cfu / g, 2.5 x 10 9 cfu / g, 5 x 10 9 cfu / g, 10 x 10 9 cfu / g, other values within the numerical point can be selected, and here we will not repeat them one by one.

[0024] Preferably, the plant bacterial agent with disease prevention and growth promotion effect further comprises a protective agent.

[0025] Preferably, the protective agent comprises any one or a combination of at least two of skimmed milk powder, mannitol, sucrose or lactose; the combination of at least two includes a combination of skimmed milk powder and mannitol, a combination of mannitol and sucrose, or a combination of sucrose and lactose, etc., and other any combination mode can be selected, and here we will not repeat them one by one.

[0026] Preferably, the protective agent is skimmed milk powder.

[0027] Preferably, the mass ratio of the Bacillus velezensis IFST-221 strain to the protective agent in the powdery bacterial agent is (5-20):1.

[0028] The specific value in the (5-20) can be selected as 5, 7, 9, 11, 13, 15, 17, 19 or 20, and other specific point values within the above numerical range can be selected, and here we will not repeat them one by one.

[0029] Preferably, the strain in the plant bacterial agent with disease prevention and growth promotion effect further comprises any one or a combination of at least two of bacillus, pseudomonas, nitrogen-fixing bacteria or microbacterium; the combination of at least two includes a combination of bacillus and pseudomonas, a combination of pseudomonas and nitrogen-fixing bacteria, or a combination of nitrogen-fixing bacteria and microbacterium, etc., and other any combination mode can be selected, and here we will not repeat them one by one.

[0030] Preferably, the bacterial strain in the plant bacterial agent with disease prevention and growth promotion effects also includes a Bacillus subtilis GW-450 strain, the Bacillus subtilis GW-450 has a preservation number of CGMCC No. 26671, a preservation date of February 21, 2023, and a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Xili, Beichen West Road, Chaoyang District, Beijing.

[0031] The present application also creatively finds that the above-mentioned Bacillus velezensis IFST-221 strain can be used in combination with the Bacillus subtilis GW-450 strain for preventing and treating soil-borne diseases of plants, and has a significantly superior effect compared to single bacterial agents or other combinations, which indicates that the Bacillus velezensis IFST-221 strain and the Bacillus subtilis GW-450 strain have a synergistic effect in inhibiting the growth of soil-borne disease pathogens.

[0032] Preferably, the ratio of viable bacterial numbers of the Bacillus velezensis IFST-221 strain to the Bacillus subtilis GW-450 strain is (1-10):1.

[0033] The specific value in (1-10) can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and other specific point values within the above-mentioned value range can also be selected, which will not be described one by one here.

[0034] In the plant bacterial agent related to the present application, the Bacillus velezensis IFST-221 strain and the Bacillus subtilis GW-450 strain have a more optimal synergistic effect when meeting the above-mentioned specific viable bacterial number ratio.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] 1. The application isolates and preserves a Bacillus velezensis strain from a healthy corn rhizosphere soil sample in a corn ear rot disease severe field in Qujing City, Yunnan Province, China, which is named Bacillus velezensis IFST-221 strain. The strain has the effects of nitrogen fixation, phosphorus dissolution, potassium dissolution, biofilm formation and auxin production, and can significantly increase the fresh weight, dry weight and plant height of corn, cotton, tomato, cauliflower and other plants, showing obvious effect of promoting plant growth, which provides a new selection strategy for preparing plant growth promoting inoculant.

[0037] 2. The application also found that the Bacillus velezensis IFST-221 strain has the effects of significantly inhibiting pathogenic bacteria, promoting plant growth and reducing plant soil-borne diseases. Specifically, the inhibition rates of the Bacillus velezensis IFST-221 strain on six pathogenic bacteria, i.e. Fusarium pseudotrichia, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Phytophthora nicotianae and Verticillium dahliae, can reach 61.84%, 68.54%, 61.51%, 71.61%, 77.88% and 74.14%, respectively. Combined with the nitrogen fixation, phosphorus dissolution, potassium dissolution, biofilm formation and growth hormone release of the Bacillus velezensis IFST-221 strain, the strain has good application prospect in the preparation of biocontrol agents for preventing and / or treating plant soil-borne diseases.

[0038] 3. The Bacillus velezensis IFST-221 strain can be applied alone in related products or combined with other strains in related products. The plant bacterial inoculant containing the Bacillus velezensis IFST-221 strain has the effects of significantly promoting plant growth and preventing and / or treating plant soil-borne diseases. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The figure is the growth result diagram of the Bacillus velezensis IFST-221 in the nitrogen-free medium, NBRIP medium and potassium-dissolving bacterial medium in Test Example 1.

[0040] Figure 2 The figure is the Bacillus velezensis IFST-221 biofilm crystal violet staining result diagram in Test Example 1.

[0041] Figure 3 Figure for testing the inhibition results of Bacillus velezensis IFST-221 on six pathogenic fungi, i.e. F. pseudograminearum, F. graminearum, F. oxysporum, F. solani, P. nicotianae and P. daci in Example 3;

[0042] Figure 4 Figure for the growth of corn, cotton, tomato and cauliflower after 30 days in Example 4.

[0043] Figure 5 Figure for the growth of corn, cotton, tomato and cauliflower after 30 days in Example 4.

[0044] Figure 6 Figure for the growth of corn, cotton, tomato and cauliflower after 30 days in Example 4. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0046] The process, conditions, reagents, experimental methods, etc. for implementing the present application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the present application has no special limitations. The experimental methods not specified in the specific conditions in each embodiment are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0047] Unless otherwise specified, the meanings of all professional terms and scientific terms used in the present specification are the same as those generally understood by the skilled person in the technical field to which the present application belongs. However, if there is a conflict, the present specification including the definition shall prevail.

[0048] The F. pseudograminearum, B. subtilis, F. oxysporum and F. solani involved in the following preparation examples, examples and test examples are F. pseudograminearum ATCC24378, B. subtilis ATCC6051, F. oxysporum ATCC64530 and F. solani ATCC36031 products purchased from Shanghai Bai Feng Biological Technology Co., Ltd. The F. graminearum is F. graminearum B80303 product purchased from Ningbo Mingzhou Biological Technology Co., Ltd. The P. nicotianae and P. daci are P. nicotianae Bio-52996 and P. daci Bio-20525 products purchased from Beijing Baioubo Biological Technology Co., Ltd.

[0049] Preparation Example 1

[0050] The present preparation example provides a preparation method of the culture medium and crystal violet staining solution in the following preparation examples, examples or test examples.

[0051] LB liquid medium: 10 parts of tryptone, 5 parts of yeast extract, 10 parts of NaCl, and sterile H2O to 1000 parts, pH adjusted to 7, 121℃, 20min high temperature and high pressure sterilization and stored for later use.

[0052] LB solid medium: add 15g / L agar powder to the LB liquid medium, 121℃, 20min high temperature and high pressure sterilization and stored for later use.

[0053] CM medium: 6 parts of yeast extract, 6 parts of enzyme hydrolyzed casein, 10 parts of sucrose, add sterile H2O to 1000 parts, 121℃, 20min high temperature and high pressure sterilization and stored for later use.

[0054] PDA medium: wash the potatoes, peel and cut into small pieces, weigh 200 parts, add 800 parts of sterile H2O and boil for 25min, filter through four layers of gauze, add 20 parts of glucose and 15 parts of agar powder to the filtrate, continue to heat and stir until uniform, cool down, add sterile H2O to 1000 parts, 121℃, 20min high temperature and high pressure sterilization and stored for later use.

[0055] Nitrogen-free medium: 10 parts of sucrose, 0.12 parts of NaCl, 0.5 parts of K2HPO4·3H2O, 1 part of CaCO3, 0.2 parts of MgSO4·7H2O, 0.5 parts of yeast extract, sterile H2O to 1000 parts, pH adjusted to 7, 121℃, 20min high temperature and high pressure sterilization and stored for later use.

[0056] National Plant Institute phosphate growth medium (NBRIP): 10 parts of glucose, 5 parts of Ca3(PO4)2, 5 parts of MgCl2·6H2O, 0.25 parts of MgSO4·7H2O, 0.2 parts of KCl, 0.1 parts of (NH4)2SO4, sterile H2O to 1000 parts, pH adjusted to 7.2, 121℃, 20min high temperature and high pressure sterilization and stored for later use.

[0057] Potassium-dissolving bacteria medium: 0.5 parts of (NH4)2SO4, 0.5 parts of yeast extract, 0.3 parts of MgSO4·7H2O, 0.03 parts of FeSO4·7H2O, 0.03 parts of MnSO4·H2O, 2 parts of potassium feldspar powder, sterile H2O to 1000 parts, pH adjusted to 7, 121℃, 20min high temperature and high pressure sterilization and stored for later use.

[0058] 0.5% crystal violet staining solution: 0.5 parts of crystal violet powder was weighed and dissolved in 100 parts of absolute ethanol.

[0059] Preparation Example 2

[0060] This preparation example provides a Bacillus velezensis IFST-221 strain, and the isolation method is as shown below:

[0061] (1) Isolation of the strain in the corn rhizosphere soil: soil samples were obtained from the rhizosphere of healthy corn in a corn ear rot disease serious field in Qujing City, Yunnan Province, China, and a 10-fold volume of sterile H2O was added to prepare a suspension under the condition of 30°C and 180 rpm for 15 min. Then the suspension was diluted 10-fold, 20-fold, and 50-fold with sterile H2O, and then spread on LB solid medium and cultured at 30°C for 24 h. The strains with different morphology, size, color, and transparency on the plate were picked with a loop and streaked on a plate to obtain single colonies.

[0062] (2) Screening of biocontrol strains with antibacterial effect: the F. verticillioides strain was inoculated on a PDA plate and cultured at 25°C for 5 days. When the colonies covered the plate, the agar cake was punched with a sterile puncher at the edge and picked into a new PDA plate with a sterile toothpick. At a distance of 25 mm from the center of the new PDA plate, two points were selected, 5 μL of the bacterial solution obtained by culturing the single colony at 30°C for 12 h in step (1) was inoculated, and a plate inoculated with F. verticillioides only was used as a control group, and three parallel groups were set in each group. After 6 days of culture at 25°C, the formation of the inhibition zone was observed. According to the calculation of the inhibition zone, a strain with strong antibacterial effect was obtained, which was named as IFST-221 strain, and the inhibition rate was 61.84%.

[0063] (3) Identification of the IFST-221 strain: according to the methods described in the eighth edition of "Berge's Bacterial Identification Manual" and "Common Bacterial System Identification Manual", the colony morphology and physiological and biochemical characteristics of the strain were identified as follows:

[0064] Morphological characteristics: the single colony on the LB solid medium was milky white with a slightly yellow color, opaque, and round; the edge was raised to form wrinkles after long-term culture. The result of Gram staining was positive, and the bacilli were about 1-5 μm long.

[0065] Physiological and biochemical characteristics: starch hydrolysis reaction, positive, glucose hydrolysis reaction, positive, xylose hydrolysis reaction, positive, arabinose hydrolysis reaction, positive, mannitol hydrolysis reaction, positive, citrate utilization reaction, positive, methyl red test reaction, positive, V.P. test reaction, positive, casein hydrolysis reaction, positive, ONPG reaction, positive, can tolerate 2%, 5%, 7% NaCl aqueous solution, cannot tolerate 10% NaCl aqueous solution, can grow in liquid medium with pH = 6.8 or 5.7, can grow under temperature conditions of 15-40℃.

[0066] Genome identification: The genome of the strain was extracted using the Tian Gen bacterial DNA extraction kit, PCR amplification was performed using bacterial 16S rDNA universal primers (27F / 1492R, Table 1), and a fragment of about 1500bp was obtained. The 16S rDNA sequence is shown in SEQ ID NO: 7. The amplification product was recovered using the Megabio gel recovery kit, and the 1500bp fragment was ligated to the pMD18-T Vector plasmid purchased from TaKaRa company. After sequencing, the gene sequencing results were analyzed and compared using the BLAST tool in NCBI, and the phylogenetic tree was constructed using MEGA X. The results showed that the IFST-221 strain belongs to Bacillus.

[0067] Since the sequencing results of the IFST-221 strain on 16S rDNA can only identify the IFST-221 strain as Bacillus, the gyrA universal primer (gyrA-F / R, Table 1) and the gyrB universal primer (the primer sequence is referenced from the literature: Yu Guohui et al. 2010 May published in the journal of China Biological Control, the journal name is "Rapid identification of biocontrol Bacillus R31 using 16S rDNA combined with gyrA and gyrB genes", UP1 sequence is

[0068] GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA; UP2R sequence is AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCA) were used for PCR amplification, the amplification product was recovered using the Megabio gel recovery kit, and was ligated into the pMD18-T Vector plasmid and sent to Shengong Bioengineering Co., Ltd. for sequencing. The sequencing results were analyzed and compared using the BLAST tool in NCBI, and the phylogenetic tree of the two genes was constructed using MEGA X.

[0069] Based on the above characteristics, the IFST-221 strain is identified as Bacillus velezensis. The strain was deposited with the China General Microbiological Culture Collection Center (CGMCC) on July 26, 2021, and has the accession number CGMCC No. 22955 and the classification name Bacillus velezensis.

[0070] Table 1

[0071] Primer name Sequence name in sequence listing Oligonucleotide sequence (5'-3') 27F SEQ ID No: 1 AGAGTTTGATCCTGGCTCAG 1492R SEQ ID No: 2 TACGGCTACCTTGTTACGACTT gyrA-F SEQ ID No: 3 CAGTCAGGAAATGCGTACGTCCTT gyrA-R SEQ ID No: 4 CAAGGTAATGCTCCAGGCATTGCT

[0072] The nucleotide sequence (5'-3') shown in SEQ ID NO: 5 is as follows:

[0073] GGTTACCTTGTTACGACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCSGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACT

[0074] ACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCA

[0075] GTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC

[0076] GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTT

[0077] CCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGA

[0078] CTTWAGAAACCGCCTGCGAGCCcTTTACGCCCAATAATTCCGGACAACGCT

[0079] TGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTC

[0080] TGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCC

[0081] CTAACAACAGAGCTTTACAATCCGAAAACCTTCATCACTCACGCGGCGTTG

[0082] CTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGT

[0083] AGGAGCCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGG

[0084] TCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAAT

[0085] GCGCCGCGGGTCCATCTGTAAGCGGTAGCCGAAGCCACCTTTTATGTCTGA

[0086] ACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTA

[0087] TCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTA

[0088] ACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATTAGGCA

[0089] CGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTCTA

[0090] Example 1

[0091] The present embodiment provides a plant bacterial agent with disease prevention and growth promotion effects, and the preparation method thereof is shown as follows:

[0092] The Bacillus velezensis IFST-221 strain glycerol bacteria stored at -80°C were streaked on LB solid medium and cultured in a 37°C incubator for 20 h. Single colonies were picked from the solid medium and inoculated into 100 mL of LB liquid medium, which was cultured at 37°C and 250 rpm for 12 h to prepare the seed liquid of the Bacillus velezensis IFST-221 strain. The seed liquid was inoculated into 400 mL of LB liquid medium at a 3% (v / v) inoculation amount, and was cultured at 37°C and 250 rpm with pH adjusted to 7 for 12 h. After the fermentation was stopped, the fermentation broth was collected, sterile water was added to adjust the viable cell count to 1×10 9 cfu / mL, and the Bacillus velezensis IFST-221 liquid bacterial agent was prepared.

[0093] The Bacillus subtilis GW-450 strain glycerol bacteria stored at -80°C were streaked on LB medium and cultured in a 37°C incubator for 20 h. Single colonies were picked from the solid medium and inoculated into 100 mL of LB liquid medium, which was cultured at 37°C and 250 rpm for 12 h to prepare the seed liquid of the Bacillus subtilis GW-450 strain. The seed liquid was inoculated into 400 mL of LB liquid medium at a 1% (v / v) inoculation amount, and was cultured at 37°C and 250 rpm with pH adjusted to 7 for 12 h. After the fermentation was stopped, the fermentation broth was collected, sterile H2O was added to adjust the viable cell count to 1×10 9 cfu / mL, and the Bacillus subtilis GW-450 liquid bacterial agent was prepared.

[0094] The Bacillus velezensis IFST-221 liquid bacterial agent was mixed with the Bacillus subtilis GW-450 liquid bacterial agent at a ratio of 10:1 to prepare a plant liquid bacterial agent with disease prevention and growth promotion effects.

[0095] Example 2

[0096] The present embodiment provides a plant bacterial agent with disease prevention and growth promotion effects, and the preparation method thereof is shown as follows:

[0097] The Bacillus velezensis IFST-221 strain glycerol bacteria stored at -80°C were streaked on LB solid medium and placed in a 30°C incubator for 16 hours of culture; single colonies were picked from the solid medium and inoculated into 500 mL of LB liquid medium, which was cultured at 37°C and 250 rpm for 16 hours to prepare the seed liquid of the Bacillus velezensis IFST-221 strain; the seed liquid was inoculated into 50 L of CM liquid medium at an inoculation amount of 0.8% (v / v), and was fermented at 30°C, 250 rpm, and pH 7 for 24 hours; after the fermentation was stopped, the fermentation broth was collected; the fermentation broth was centrifuged to collect the bacterial precipitate, which was mixed with skimmed milk powder at a ratio of 9 parts of bacterial precipitate to 1 part of skimmed milk powder, and the mixture was placed in a freeze-drying instrument; after 5 hours of freeze-drying, the Bacillus velezensis IFST-221 powder bacterial agent (5 x 10 9 cfu / g) was prepared.

[0098] The Bacillus subtilis GW-450 strain glycerol bacteria stored at -80°C were streaked on LB medium and placed in a 37°C incubator for 18 hours of culture; single colonies were picked from the solid medium and inoculated into 100 mL of LB liquid medium, which was cultured at 37°C and 250 rpm for 14 hours to prepare the seed liquid of the Bacillus subtilis GW-450 strain; the seed liquid was inoculated into 400 mL of CM liquid medium at an inoculation amount of 1% (v / v), and was fermented at 37°C, 250 rpm, and pH 7 for 16 hours; after the fermentation was stopped, the fermentation broth was collected; the fermentation broth was centrifuged to collect the bacterial precipitate, which was placed in a freeze-drying instrument; after 5 hours of freeze-drying, the Bacillus subtilis GW-450 strain powder bacterial agent (2 x 10 9 cfu / g) was prepared.

[0099] The Bacillus velezensis IFST-221 powder bacterial agent 10 parts and the Bacillus subtilis GW-450 powder bacterial agent (2 x 10 9 cfu / g) 10 parts were mixed to prepare a powder bacterial agent for plants with disease prevention and growth promotion effects.

[0100] Example 3

[0101] The present embodiment provides a bacterial agent for plants with disease prevention and growth promotion effects, and the preparation method of the bacterial agent is as follows:

[0102] The Bacillus velezensis IFST-221 strain glycerol bacteria stored at -80°C were streaked on LB solid medium and cultured in a 37°C incubator for 18 h; single colonies were picked from the solid medium and inoculated in 100 mL of LB liquid medium, which was cultured at 37°C and 250 rpm for 14 h to prepare the seed liquid of the Bacillus velezensis IFST-221 strain; the seed liquid was inoculated in 400 mL of LB liquid medium at an inoculation amount of 1% (v / v), which was cultured at 37°C and 250 rpm with pH adjusted to 7 for 16 h; after the fermentation was stopped, the fermentation broth was collected, sterile H2O was added to adjust the viable count to 5×10 9 cfu / mL, and the Bacillus velezensis IFST-221 liquid inoculant was prepared.

[0103] The Bacillus subtilis GW-450 strain glycerol bacteria stored at -80°C were streaked on LB medium and cultured in a 37°C incubator for 18 h; single colonies were picked from the solid medium and inoculated in 100 mL of LB liquid medium, which was cultured at 37°C and 250 rpm for 14 h to prepare the seed liquid of the Bacillus subtilis GW-450 strain; the seed liquid was inoculated in 400 mL of LB liquid medium at an inoculation amount of 1% (v / v), which was cultured at 37°C and 250 rpm with pH adjusted to 7 for 16 h; after the fermentation was stopped, the fermentation broth was collected, sterile H2O was added to adjust the viable count to 5×10 9 cfu / mL, and the Bacillus subtilis GW-450 liquid inoculant was prepared.

[0104] The Bacillus velezensis IFST-221 liquid inoculant and the Bacillus subtilis GW-450 liquid inoculant were mixed at a ratio of 10:10 (v / v) (the viable count was 5×10 9 cfu / mL), and the plant liquid inoculant with disease prevention and growth promotion effects was prepared.

[0105] Example 4

[0106] The plant inoculant with disease prevention and growth promotion effects of the present example is different from that of example 1 only in that it does not contain the Bacillus subtilis GW-450 liquid inoculant, and the mass fraction of the Bacillus subtilis GW-450 liquid inoculant is allocated to the Bacillus velezensis IFST-221 liquid inoculant, and the rest is the same as that of example 1.

[0107] Example 5

[0108] The present example provides a plant bacterial agent with disease prevention and growth promotion effects, which is different from example 1 only in that the Bacillus subtilis GW-450 strain is replaced by the Bacillus subtilis ATCC6051 strain, and the rest is the same as example 1.

[0109] Comparative example 1

[0110] The present example provides a plant bacterial agent with disease prevention and growth promotion effects, which is different from example 1 only in that the Bacillus subtilis GW-450 strain is replaced by the Bacillus subtilis ATCC6051 strain, and the rest is the same as example 1.

[0111] Test example 1

[0112] Bacillus velezensis IFST-221 strain nitrogen fixation, phosphorus dissolution, potassium dissolution and biofilm formation

[0113] Pick up Bacillus velezensis IFST-221 single colony in the solid LB medium described in examples 1-3, inoculate 4mL nitrogen-free medium, NBRIP medium and potassium- dissolving bacterial medium respectively, 37℃, cultivate for 32h, respectively with un-inoculated medium as blank group, compare the growth of strains in examples 1-3 Figure 1 )。

[0114] The results show that the culture medium inoculated with Bacillus velezensis IFST-221 strain is more turbid than the blank group, and the strain can grow in nitrogen-free medium, NBRIP medium and potassium-dissolving bacterial medium, indicating that Bacillus velezensis IFST-221 strain has the effects of nitrogen fixation, phosphorus dissolution and potassium dissolution.

[0115] Picking a single colony of Bacillus velezensis IFST-221 in the solid LB medium described in Examples 1-3, respectively inoculating 4 mL of LB liquid medium, 37°C, incubating for 12 h, then taking the bacterial solution and adding it to a 48-well plate containing LB solid medium, 10 μL per well, 37°C, incubating for 32 h, and using LB solid medium without bacterial solution as a blank group. After incubation, the solid medium was rinsed with PBS buffer to wash off the surface cells, 100 μL of methanol was added to each well for 20 min of fixation, the formaldehyde was discarded, 100 μL of 0.1% crystal violet solution was added to each well for 20 min of staining, and the crystal violet solution was removed by washing with PBS buffer. The staining of the solid medium in the blank group and Examples 1-3 was compared. Figure 2

[0116] The solid medium in Examples 1-3 was stained purple, and the blank group showed the original color of the solid medium, as shown in Figure 2 The surface of the blank group LB medium was smooth, and the solid medium in Example 1 had wrinkled biofilm, indicating that Bacillus velezensis IFST-221 strain had the effect of promoting biofilm formation.

[0117] Test Example 2

[0118] Auxin secretion of Bacillus velezensis IFST-221 strain

[0119] Take 20 mg of auxin IAA, dissolve it with a small amount of anhydrous ethanol, then dilute it to 100 mL with distilled water, and store it in a 4°C refrigerator. Prepare IAA gradient dilutions by diluting in stages, with concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, and 50 mg / L of IAA solution. Take 1 mL of IAA at different concentrations and add it to 3 mL of Salkowski reagent (take 3 mL of 0.5 M anhydrous FeCl3 4.075 g dissolved in 50 mL of distilled water), add 37 mL of distilled water, take 60 mL of concentrated sulfuric acid and slowly add it along the wall, shake well before use, and store it in the dark. It should be yellow-green at this time;), avoid light, and let it stand for 30 min. Measure the absorbance at 530 nm. After the measurement is complete, use OD 530 value as the vertical coordinate and IAA concentration (mg / L) as the horizontal coordinate to draw a standard curve.

[0120] ​The Bacillus velezensis IFST-221 strain was inoculated into the IAA production determination medium (1 g / mL of L-tryptophan was added to LB liquid medium that had been autoclaved and left to room temperature, to a final concentration of 100 mg / L) and cultured for 24 h, then centrifuged at 1000 rpm for 10 min, 1 mL of supernatant was taken and added to 3 mL of Salkowski reagent, and the absorbance was measured at 530 nm after 30 min of dark incubation. The IAA production concentration of the Bacillus velezensis IFST-221 strain was calculated to be 5.59 mg / L through a standard curve.

[0121] Test Example 3

[0122] Antibacterial effect of Bacillus velezensis IFST-221 strain

[0123] Fusarium pseudograminearum, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Phytophthora nicotianae, and Sclerotinia homeocarpa were inoculated on PDA plates and cultured at 25°C until the colonies covered the plates. The colonies were then inoculated on new PDA plates using a sterile puncher and a sterile toothpick.

[0124] Two points were selected 25 mm from the center of the new PDA plate, and 10 μL of Bacillus velezensis IFST-221 seed liquid prepared in Examples 1-3 was inoculated at each point. Plates inoculated with only the pathogenic fungi were used as controls. Each group had three parallel groups, and the plates were cultured at 25°C until the colonies of the control group covered the plates. The antibacterial effect of the experimental group was observed, and the antibacterial rate was calculated according to the antibacterial ring. In the above experiment, the inhibition rates of Bacillus velezensis IFST-221 strain on Fusarium pseudograminearum, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Phytophthora nicotianae, and Sclerotinia homeocarpa were 61.84%, 68.54%, 61.51%, 71.61%, 77.88%, and 74.14%, respectively, indicating that Bacillus velezensis IFST-221 strain has a significant effect on inhibiting plant pathogenic fungi and has a good application prospect in biological control of plant microbial diseases. Figure 3

[0125] Test Example 4

[0126] Growth-promoting effect of plant microbial agent with disease-preventing and growth-promoting effects

[0127] ​Sow corn, cotton, tomato, cauliflower in the culture pot, after germination, use the root irrigation method to inoculate 40 mL of the plant microbial agent with disease prevention and growth promotion effect prepared in Example 4. One blank group is set for each of the four plants, and the plant microbial agent is replaced with 40 mL of sterile water in the blank group. Each group is inoculated with 14 plants, and after 30 days of inoculation, the plant height, root length, aboveground fresh weight and dry weight, underground fresh weight and dry weight of each group are recorded, the experiment is repeated three times, and the average value is calculated according to the recorded data (Table 2, Figure 4 and Figure 5 ). The average value of the aboveground fresh weight of a certain plant = the total aboveground fresh weight of a certain plant / the total number of a certain plant.

[0128] Table 2

[0129]

[0130]

[0131] From the data in Table 2, after using the plant microbial agent with disease prevention and growth promotion effect in Example 4, the average value of the aboveground fresh weight of the plant is higher than that of the blank group; in addition, Figure 4 , Figure 5 Among them, the corn plant height, cauliflower aboveground dry weight and cotton underground fresh weight of Example 4 are significantly higher than those of the blank group, and more notably, the aboveground fresh weight of corn, cotton, tomato and cauliflower in Example 4 is higher than that of the blank group, indicating that the use of the plant microbial agent with disease prevention and growth promotion effect of the application, i.e., the use of the microbial agent containing Bacillus velezensis IFST-221 strain, significantly promotes the growth of plants.

[0132] Test Example 5

[0133] Disease prevention and growth promotion effect of the plant microbial agent

[0134] The activated L. cylindracea cake was inoculated into CM medium, and after 3 days of culture at 25°C in a constant temperature shaker at 120 rpm, the spore suspension was obtained by filtering the mycelium with sterilized three-layer lens paper, and the concentration of the spore suspension was adjusted to 5×10 6 / mL with a blood cell counting plate for standby.

[0135] Model group: cotton susceptible to Verticillium wilt (Gumian No. 1) was sowed in paper pots, and when the cotton grew to one true leaf, 20 mL of L. circinata spore suspension was used for irrigation inoculation; blank group was not inoculated with L. circinata spore suspension, but 20 mL of sterile water was used instead; IFST-221 group: not inoculated with L. circinata spore suspension, but 20 mL of sterile water was used instead, and 3 days later, 40 mL of the plant fungicide with disease prevention and growth promotion effects prepared in Example 4 was used for irrigation inoculation; example or comparative example group: 3 days after inoculation with L. circinata spore suspension, 40 mL of the plant fungicide with disease prevention and growth promotion effects prepared in the example or comparative example was used for irrigation inoculation.

[0136] Each group was inoculated with 14 cotton plants, and 30 days after inoculation, photographs were taken and disease index was recorded, and the experiment was repeated 3 times (Table 3, Figure 6 ).

[0137] The disease rating standard was divided into 5 grades: 0 = healthy plant, no symptoms on leaf; 1 = one or two cotyledons showed symptoms, but no symptoms on true leaves; 2 = both cotyledons and one true leaf showed symptoms; 3 = both cotyledons and two true leaves showed symptoms; 4 = all leaves showed symptoms, symptomatic leaves fell off, apical meristem necrosis or plant death. Disease index = (0n0+1n1+2n2+3n3+4n4) / 4n x 100%, wherein n0 to n4 are the number of plants corresponding to each respective disease grade, and n is the total number of plants in each treatment group.

[0138] Table 3

[0139] Group Disease index (%) Blank group 0 Model group 73.21 IFST-221 group 0 Example 1 8.93 Example 2 10.91 Example 3 16.74 Example 4 20.49 Example 5 29.85 Comparative Example 1 40.56

[0140] From the data in Table 3, compared with the model group, the disease index of cotton caused by Verticillium dahliae infection was reduced by 64.28% after the root irrigation of the liquid inoculant containing Bacillus velezensis IFST-221 strain and Bacillus subtilis GW-450 strain at a ratio of 10:1 viable cell count in Example 1, and the control effect was as high as 87.8%, indicating that the liquid inoculant containing Bacillus velezensis IFST-221 strain and Bacillus subtilis GW-450 strain at a ratio of 10:1 had a significant disease prevention effect; the disease index of Example 1 was reduced compared with the model group, and the reduction degree was higher than that of Example 4 and Comparative Example 1, indicating that Bacillus velezensis IFST-221 strain could be combined with Bacillus subtilis GW-450 strain, synergistic effect, and the prepared inoculant had better disease prevention effect. Compared with Example 1, Example 5 used commercially available Bacillus subtilis, and the disease index was higher, indicating that the combination of commercially available Bacillus subtilis and Bacillus velezensis IFST-221 strain was worse than the combination of Bacillus subtilis GW-450 strain and Bacillus velezensis IFST-221 strain.

[0141] The applicant declares that the preparation process of the present application is illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0142] The above describes the preferred embodiments of the present application in detail, but the present application is not limited to the specific details in the above embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0143] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A plant fungicide with disease prevention and growth promotion effects, characterized in that: The bacterial strains in the plant-use microbial agent with disease prevention and growth promotion efficacy are composed of Bacillus velezensis IFST-221 strain and Bacillus subtilis GW-450 strain; the ratio of the number of viable bacteria of the Bacillus velezensis IFST-221 strain to the number of viable bacteria of the Bacillus subtilis GW-450 strain is (1-10):1; The deposit number of the Bacillus velezensis IFST-221 strain is CGMCC No. 22955, and the deposit date is July 26, 2021; the deposit number of the Bacillus subtilis GW-450 strain is CGMCC No. 26671, and the deposit date is February 21, 2023.

2. The plant-use fungicide with disease prevention and growth promotion efficacy according to claim 1, characterized in that: The dosage form of the plant microbial agent with disease prevention and growth promotion efficacy includes liquid microbial agent, powdery microbial agent or granular microbial agent.

3. The plant-use fungicide with disease prevention and growth promotion efficacy according to claim 2, characterized in that: The number of viable bacteria of Bacillus velezensis IFST-221 in the liquid inoculum is not less than 1×10 9 cfu / mL.

4. The plant-use fungicide with disease prevention and growth promotion efficacy according to claim 2, characterized in that: The number of viable bacteria of Bacillus velezensis IFST-221 in the powdered bacterial agent is not less than 1.5×10 9 cfu / g.

5. The plant fungicide with disease prevention and growth promotion efficacy according to claim 1, characterized in that: The plant fungicide with disease prevention and growth promotion effects also includes a protective agent.

6. The plant fungicide with disease prevention and growth promotion efficacy according to claim 5, characterized in that: The protective agent includes any one of skimmed milk powder, mannitol, sucrose or lactose, or a combination of at least two of them.

7. The plant-use fungicide with disease prevention and growth promotion efficacy according to claim 6, characterized in that: The protective agent is skimmed milk powder.

8. The plant fungicide with disease prevention and growth promotion efficacy according to claim 5, characterized in that: The mass ratio of the Bacillus velezensis IFST-221 strain to the protective agent in the plant microbial agent with disease prevention and growth promotion efficacy is (5-20):1.

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