Preparation method and application of a siam bacillus SL-K1 and its mycorrhizal extract

Abstract

The present invention discloses a strain of Siamese Bacillus SL-K1 and a preparation method and application of its mycorrhizal extract. The Siamese Bacillus SL-K1 was isolated from the surface silt of the Lotus Pond in Haiyang City Wetland Park, and its deposit number is CGMCC 28450. The Siamese Bacillus SL-K1 not only has excellent antibacterial properties against Ralstonia solanacearum, but also has an antibacterial effect on Fusarium, and its fermentation broth can be effectively extracted with mycorrhizal compounds. The preparation method of the mycorrhizal compound extract utilizes a microbial fermentation process, which is convenient for industrial production and control, and has high preparation efficiency. The extracted mycorrhizal compounds can be used to promote the symbiotic formation of mycorrhizae between plants and probiotic fungi, thereby promoting the absorption function of plant roots, promoting plant growth, and improving the plant's ability to resist adversity.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a siam bacillus SL-K1 and a preparation method and application of a mycorrhizin extract thereof. Background Art

[0002] Ralstonia solanacearum, as a soil-borne bacterial pathogen of plants, is widely distributed throughout the world. It infects a very wide range of hosts and can infect more than 450 plant species from four families, causing serious damage to the production of many economic crops.

[0003] Under natural conditions, Ralstonia solanacearum can invade through wounds in plant roots or stems, or directly through the root cap of uninjured secondary roots, causing disease. Ralstonia solanacearum can penetrate the sheath formed between the epidermis of the secondary root cap and the primary root, causing the cell walls of the adjacent parenchyma tissue to swell. It then invades the cortex and grows in the intercellular spaces, destroying the intercellular gel layer. This causes the host plant's cell walls to disintegrate, undergo plasmolysis, and deform, forming cavities. Ralstonia solanacearum is then distributed within the parenchyma cells and cavities. Ralstonia solanacearum can also invade through intact or uninjured ducts, stimulating the small cells within them to form invasive bodies. The bacteria then migrate into the invasive bodies, which then rupture and release the bacteria into the ducts, where they multiply in large numbers. When growing in trachea, Ralstonia solanacearum produces large amounts of exopolysaccharides, which directly affect and hinder water transport within the plant. This can clog the smaller tracheal perforations at the petiole junction and leaflets, causing plant wilt. The various cell wall-degrading enzymes secreted by Ralstonia solanacearum can also damage tracheal tissue, leading to wilting and death of the host plant. Furthermore, the motility of Ralstonia solanacearum is related to its host affinity. Luo Huanliang et al. found that lipopolysaccharides in the bacterial outer membrane help Ralstonia solanacearum recognize and adhere to the surface of Casuarina equisetifolia roots. This suggests that Ralstonia solanacearum poses a serious threat to numerous economic crops in agriculture, necessitating effective control measures.

[0004] Currently, domestic and international researchers are conducting research on the prevention and control of diseases caused by Ralstonia solanacearum from chemical, biological, and agricultural perspectives, with the primary focus on cultivating disease-resistant varieties and using chemical agents. However, the pathogen's physiology and virulence are highly differentiated, resistant varieties are limited, and resistance is easily lost and varies across different growing regions. Furthermore, chemical control is unstable against bacterial wilt, and long-term, high-volume application of chemicals can easily lead to resistance, resulting in decreased efficacy. Increased chemical dosage not only increases costs but also pollutes the environment. Adopting efficient and environmentally friendly control measures to control bacterial diseases is an urgent need in agricultural production.

[0005] Currently, biological control through bioinhibitors (i.e., endophytes and rhizosphere microorganisms) is considered an alternative measure to reduce the incidence of bacterial diseases. Due to its high efficiency, non-toxicity, zero residue, and low cost, biological control is a control method with good development potential and application prospects. Currently, biological control mainly controls plant bacterial diseases through the screening and utilization of antagonistic microorganisms. Antagonistic microorganisms mainly include antagonistic bacteria (Agrobacterium spp., Bacillus spp., and Pseudomonas spp., etc.), antagonistic endophytes, antagonistic actinomycetes, antagonistic fungi (Trichoderma, Penicillium, and antagonistic mycorrhizal fungi), etc.

[0006] Although a range of biocontrol resources have been explored, there are still relatively few microorganisms and related preparations that can be used effectively and efficiently on a large scale to control bacterial wilt in the field. Therefore, the existing technology needs to be further improved. Summary of the Invention

[0007] To address the above problems, the present invention provides a strain of Bacillus siamensis SL-K1 isolated for the first time. The strain has high inhibitory activity against Ralstonia solanacearum and can be used to ferment and produce mycorrhizin. The mycorrhizin can not only improve the plant's resistance to bacterial wilt, but also promote plant growth, and has good application prospects.

[0008] To solve the above problems, this application provides the following technical solutions:

[0009] In a first aspect, the present application provides a strain of Bacillus siamensis SL-K1, whose deposit number is CGMCC 28450.

[0010] Bacillus siamensis SL-K1 was isolated from the surface mud of the Lotus Pond in Haiyang City Wetland Park. It is a facultative aerobic, Gram-positive bacterium with an opaque white to slightly yellowish colony surface. Individual cells (in the logarithmic phase) are 1.3 to 2.7 microns long and lack a capsule. Spores are 0.8 to 1.5 microns long, oval in shape, and located in the center or slightly off to the side of the cell. Spores are located in the middle or off to the side of the cell. During spore formation, the cell does not expand and assumes a rod-shaped form.

[0011] After morphological and molecular biological identification, the strain was identified as Bacillus siamensis, named Bacillus siamensis SL-K1. It was deposited with the General Microbiology Center of the China Culture Collection Administration on September 12, 2023, with the deposit number CGMCC28450.

[0012] The inventors discovered for the first time that Bacillus siamensis SL-K1 not only has excellent antibacterial properties against Ralstonia solanacearum, but also effectively extracts mycorrhizal extracts from its fermentation broth, which can be used to produce mycorrhizal extracts, thus having a series of related application prospects.

[0013] In a second aspect, the present application provides a microbial preparation, the active ingredient of which includes the aforementioned Bacillus siamese SL-K1.

[0014] Optionally, in the microbial preparation, the siam Bacillus SL-K1 is the bacterial cell, bacterial liquid, fermentation filtrate or active extract thereof.

[0015] The bacterial cells, bacterial liquid, fermentation filtrate or active extract of Bacillus siamensis SL-K1 can be directly or mixed with other adjuvants and adsorbents to prepare liquid or solid biocontrol preparations for preventing and controlling diseases caused by Ralstonia solanacearum.

[0016] Preferably, the microbial preparation also includes other biocontrol bacteria that complement or synergize with Bacillus siamese SL-K1, thereby further expanding the antibacterial spectrum of the biocontrol preparation. Alternatively, an insecticide may be added to create a comprehensive biocontrol preparation with insecticidal and fungicidal properties.

[0017] In a third aspect, the present application further provides the use of the aforementioned Bacillus siamensis SL-K1 or the aforementioned microbial preparation in preventing and controlling plant diseases caused by Ralstonia solanacearum.

[0018] The disease prevention and control function of Siamese Bacillus SL-K1 is reflected in: it can promote the formation of mycorrhizae with plants and beneficial fungi, and mycorrhizae can defend against the invasion of pathogenic microorganisms, thereby improving the plant's immunity to pathogenic microorganisms.

[0019] Fourthly, this application also provides the use of the aforementioned Bacillus siamensis SL-K1 in the preparation of mycorrhizal extracts (i.e., mycorradicin) and plant growth promoters. During the research and development process, the inventors unexpectedly extracted mycorrhizal active ingredients from the fermentation broth of this bacterium. This application also marks the first successful extraction of mycorrhizal ingredients from Bacillus siamensis SL-K1, which is of great significance for the green and environmentally friendly extraction process of mycorrhizal ingredients.

[0020] Mycorradicin is a 14-carbon dicarboxylic acid polyene. Currently, there are no relevant Chinese literature reports on this substance. Mycorradicin, which can be literally translated as "mycorrhizin," is associated with mycorrhizal colonization. However, studies have found it in root nodules where bacteria and plants coexist. Mycorrhiza generally refers to the symbiosis between fungi and plants, so its English name, mycorradicin, has been retained. Klingner et al. discovered in 1995 that mycorradicin is a carotenoid chromophore, a "yellow pigment." Carotenoids are heterocyclic compounds synthesized by photosynthetic and some non-photosynthetic organisms. They are essential for photosynthesis and contribute to many other aspects of plant life. Oxidative decomposition of carotenoids produces a variety of essential metabolites, known as non-carotenoids. Carotenoids include the plant hormones abscisic acid and gypenospermide, signaling molecules, and growth regulators. They are crucial for regulating plant growth, development, and stress responses. They are also important components in the communication between plant rhizobia and their symbionts and parasites.

[0021] In an article published in Frontiers in Plant Science in March 2018, Stauder et al. believed that mycorradicin belongs to the carotenoid class of substances and is the product of the ancient origin of the plant gene dPSY3s and its co-evolution with AM (arbuscular mycorrhiza). Arbuscular mycorrhizae (AM), which support plant mineral nutrition, are the most common mutualistic symbiotic relationship on Earth (Parniske, 2008). Carotenoids seep from the roots into the nodule layer, where they establish a symbiotic relationship with beneficial soil fungi and soil bacteria. In 2021, Juan C. Moreno, Jianing Mi, Yagiz Alagoz and Salim Al-Babili published a research paper entitled "Plantapocarotenoids: from retrograde signaling to interspecific communication" in The Plant Journal (2021) 105, 351-375, which found that carotenoids and hormones are involved in almost all aspects of plant physiology and development. Carotenoids can act as direct signaling molecules or growth regulators, and carotenoid substances are related to plant development, stress resistance, and biotic interactions.

[0022] Mycorradicin, a novel elicitor, induces plant roots to form a symbiotic relationship with beneficial bacteria and fungi. It promotes the symbiosis between plants and fungi to form AM mycorrhizae, which can promote the colonization and growth of mycorrhizae, enhance resistance to abiotic stresses such as drought and phosphorus deficiency, and prevent soil pathogens from infecting plants through the allelopathic effects of signaling molecules. The study also found that mycorradicin-like substances can stimulate plant resistance to abiotic stresses such as drought and salinity, and can also prevent soil pathogens, including bacteria, fungi, viruses, and nematodes, from invading plants. This type of substance can improve the root colonization and performance of mycorrhizal plants, ultimately reducing agriculture's reliance on chemical pesticides and inorganic fertilizers by better supporting the symbiosis between beneficial microorganisms and plant roots.

[0023] Therefore, Mycorradicin can be used to improve plant stress tolerance, enhance plant disease resistance, and promote plant growth by supporting the symbiotic relationship between beneficial microorganisms and plant roots.

[0024] However, there has been no effective extraction method for Mycorradicin before. This application focuses on providing a method for extracting and preparing Mycorradicin.

[0025] Since the mycorrhizal extract can be used as an active ingredient of a plant growth promoter, the siam bacillus SL-K1 can be used to prepare a plant growth promoter.

[0026] In a fifth aspect, the present application also provides a method for preparing a mycorrhizal extract, which comprises the following steps:

[0027] S1. Inoculating the aforementioned Bacillus siamensis SL-K1 into a liquid culture medium to prepare a fermentation broth;

[0028] S2, centrifuging the fermentation broth, collecting the fermentation supernatant and freeze-drying it to obtain freeze-dried powder of the fermentation broth;

[0029] S3, precipitating the dry powder with ethanol, filtering and collecting the precipitate to obtain a primary extract; then removing the ethanol from the precipitate and dialyzing it to remove macromolecules larger than 500 Da to obtain a secondary extract;

[0030] S4. The secondary extract is purified by HPLC chromatography to separate and obtain a refined extract, which is a mycorrhizal extract.

[0031] Preferably, in step S1, the liquid culture medium is added with 5% of 10 4 ~10 6 / ml of culture medium containing inactivated liquid of Ralstonia solanacearum cells. 4 ~10 6 / ml of R. solanacearum cell inactivation solution plays a key role in the expression of mycorrhizin in Bacillus siamensis SL-K1.

[0032] The culture medium may be an existing conventional culture medium, such as LB culture medium: 0.1-1% yeast extract, 0.1-1% peptone, 0.1-1% NaCl, and the rest water.

[0033] Preferably, the liquid culture medium comprises the following components in the following mass percentages: 1-10% of 10 4 ~10 6 / ml of Ralstonia solanacearum cell inactivation solution, 0.1-1% beef extract, 0.1-1% peptone, 1-3% glucose, 0.1-1% NaCl, and the rest is water.

[0034] More preferably, the culture medium comprises the following components in the following mass percentages: 5% of 10 4 ~10 6 / ml of Ralstonia solanacearum cell inactivation solution, 0.3% beef extract, 0.5% peptone, 2% glucose, 0.4% NaCl, and the rest is water.

[0035] Specifically, the preparation method of the Ralstonia solanacearum cell inactivation solution is: treating Ralstonia solanacearum cells at 85-95° C. for 3-10 minutes. Preferably, the inactivation condition is: treating at 90° C. for 5 minutes.

[0036] The above-mentioned inactivation treatment can keep the cell morphology of Ralstonia solanacearum intact, but lose its activity, thereby obtaining the inactivated cell liquid of the bacterium, which is used for the induced fermentation of Bacillus siamensis SL-K1.

[0037] Preferably, in step S4, the elution system is a water-acetonitrile gradient elution system, using a Hypersil GOLD C18 column at a column temperature of 38-42°C.

[0038] Preferably, the HPLC purification conditions are as follows: a Hypersil GOLD C18 column (2.1×100 mm, 5 μm; Thermo Fisher Scientific, Waltham, USA), a column temperature of 40°C, and a flow rate of 0.4 ml / min in a two-dimensional nano-liquid phase system. During the elution process, eluent A is water (containing 0.1% formic acid, v / v), and eluent B is an acetonitrile system, with the volume ratios of A:B being 19:1, 9:1, 1:19, and 19:1, respectively. The chromatographic retention time is 21.0-21.3 min, and a refined extract of Bacillus siamensis is finally purified, namely, a non-sugar extract, Mycorradicin (i.e., mycorrhizal indica).

[0039] In a sixth aspect, the present application also provides the use of the above-mentioned mycorrhizin extract in promoting plant growth and improving plant disease resistance.

[0040] In a seventh aspect, the present application also provides a plant growth promoter, the active ingredient of which is the aforementioned mycorrhizal extract.

[0041] The present invention has the following beneficial effects:

[0042] 1. The present invention provides a strain of Bacillus siamensis SL-K1 screened by the applicant. The strain and its extracts have excellent antibacterial efficacy against the pathogen Ralstonia solanacearum. The present application also extracts mycorrhizin (i.e., mycorradicin), an important beneficial substance, from this strain for the first time. Mycorradicin can stimulate plant resistance to adversity and prevent pathogens from invading plants. Experiments have shown that the mycorrhizin extract extracted and purified from Bacillus siamensis SL-K1 can improve plant disease resistance and promote plant growth.

[0043] 2. This application provides, for the first time, a method for extracting mycorrhizal compounds from Bacillus siamensis. This method is not only simple and easy to operate, has a short cycle time, uses a limited number of extraction solvents, is non-toxic, and has a short extraction process, but also delivers excellent extraction results. Furthermore, the mycorrhizal compound extract is an environmentally friendly biopharmaceutical and can be used to prepare microbial preparations (such as growth regulators and activators) to enhance plant resistance to diseases caused by Ralstonia solanacearum and promote plant growth, thus fully leveraging the application value of Bacillus siamensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a confrontation experiment between strain SL-K1 and R. solanacearum;

[0045] Figure 2 This is the colony morphology of SL-K1;

[0046] Figure 3 This is the bacterial cell morphology of SL-K1 under a microscope;

[0047] Figure 4 This is the result of SL-K1 spore staining;

[0048] Figure 5 The antibacterial effect of SL-K1 on Fusarium oxysporum; A is SL-K1, B is Fusarium oxysporum, and the left figure is the control inoculated with Fusarium oxysporum only;

[0049] Figure 6 is the positive ion mass spectrum of Mycorradicin;

[0050] Figure 7 is the chromatogram of Mycorradicin;

[0051] Figure 8 Comparison of aboveground growth of pepper seedlings under different treatments;

[0052] Figure 9 Comparison of root weights of pepper seedlings under different treatments;

[0053] Figure 10 Comparison of chlorophyll content in pepper seedlings with different treatments;

[0054] Figure 11 Comparison of malondialdehyde content in pepper seedlings treated with different methods;

[0055] Figure 12 Comparison of chlorophyll a content in pine leaves treated with different methods;

[0056] Figure 13 Comparison of chlorophyll b content in pine leaves treated with different methods;

[0057] Figure 14 Comparison of soluble sugar content in pine leaves treated with different methods;

[0058] Figure 15 Comparison of malondialdehyde content in pine leaves treated with different methods;

[0059] Figure 16 Photos of pine trees in the experimental and control groups treated with extracts; A shows the diseased pine trees in the control group; B shows the diseased pine trees in the experimental group;

[0060] Figure 17 The control group showed withering and root rot after 60 days. DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in this field. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0062] Example 1 Isolation and Identification of Siamese Bacillus SL-K1

[0063] 1. Isolation and screening of strains

[0064] 1.1 Experimental methods:

[0065] Surface silt from the Lotus Pond in Haiyang Urban Wetland Park was collected, placed in sterile plastic bags, and refrigerated before being transported to the laboratory. A dilution plate spread method was used: aseptically, the sludge sample to be isolated was serially diluted with sterile saline. 100 μL of the dilution was evenly spread on a nutrient agar plate. The plate was allowed to stand for 5 minutes, then incubated upside down in a 30°C incubator and observed after 48 hours. Single colonies were selected and purified using the plate streak method to obtain the microbial strain to be tested.

[0066] The purified strains were subjected to antagonistic experiments with Ralstonia solanacearum. Antagonistic bacteria were screened using the plate confrontation method, and finally the presence of an inhibition zone between the two colonies was observed.

[0067] 1.2 Results and Analysis

[0068] The results showed that the isolated bacteria effectively prevented the spread of R. solanacearum when cultured in a confrontation culture, but no inhibition zone appeared. The plate confrontation experiment showed that the bacteria prevented the spread of R. solanacearum by competing for space through rapid reproduction ( Figure 1 The SL-K1 strain produces spores and a powdery lawn, while the Ralstonia solanacearum does not produce spores and has a transparent lawn, which is used to distinguish the two types of lawns. However, it has no bactericidal effect on Ralstonia solanacearum. This strain is named: SL-K1 strain.

[0069] 2. Observation of morphological characteristics of antagonistic bacteria of SL-K1 strain

[0070] 2.1 Experimental methods

[0071] (1) Observation of colony appearance: The antagonistic bacteria obtained by screening were streaked on a plate and cultured in a constant temperature incubator at 30°C for 24 h. The morphological characteristics of the colonies were observed according to the Manual of Identification of Common Bacteria.

[0072] (2) Spore staining: Take a smear of antagonistic bacteria cultured for 48 hours, stain it with a saturated malachite green aqueous solution for 10 minutes, rinse it with tap water, and then stain it with safranin staining solution for 1 minute. Wash it with water, dry it with absorbent paper, and then examine it under a microscope.

[0073] 2.2 Results and Analysis

[0074] This strain is a facultative aerobic bacterium, Gram-positive, with spores located in the middle or at the end of the bacterium. When spores are produced, the bacterium does not swell and is rod-shaped. Figure 1-2 The surface of the colony of this strain is opaque, white to slightly yellow. The length of a single bacterial cell (logarithmic phase) is 1.3 to 2.7 microns, without capsule (such as Figures 2-3 The spores are 0.8 to 1.5 microns long, oval in shape, and located in the center or slightly to the side of the bacterial body. Figure 4 ).

[0075] 3. Molecular Biological Identification of SL-K1 Strain

[0076] 3.1 Experimental methods

[0077] The antagonistic strain was identified by 16S rDNA sequencing: the SL-K1 strain was sent to Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA gene sequence was compared with BLAST on the NCBI website. The strain identification and analysis process is as follows:

[0078] (1) DNA extraction: The genome of the SL-K1 strain was extracted using the TSINGKE Plant DNA Extraction Kit (Universal) (Cat. No.: TSP101).

[0079] (2) PCR amplification: The genome obtained in the previous step is amplified using universal primers for bacterial species identification. The amplified products are analyzed by agarose gel electrophoresis to determine whether the PCR product bands are consistent with the target size, whether they are single, and whether there are any dragging bands.

[0080] (3) PCR product sequencing: After the PCR product is qualified, the target band is cut and purified and recovered, and the recovered product is used for Sanger sequencing.

[0081] (4) Comparison and analysis of sequencing results: The Sanger sequencing results were spliced ​​using ContigExpress software, and the inaccurate parts at both ends were removed. The batch of spliced ​​sequences were compared and analyzed in the nucleic acid database. The latest version of the nt database was selected as the nucleic acid database. By performing blastn comparison with the nt database, the accession number of the homologous sequence and the species identification and annotation were obtained.

[0082] 3.2 Experimental results and analysis

[0083] The 16S rDNA gene sequence of the SL-K1 strain is shown in SEQ ID NO: 1 in the sequence listing. Sequence alignment revealed that the 16S rDNA gene sequence of the SL-K1 strain shared the highest homology with that of Bacillus siamensis strain cqsM9, reaching 99%, indicating that the SL-K1 strain is a new strain of Bacillus siamensis.

[0084] Example 2 Inhibitory effect of strain SL-K1 on Fusarium oxysporum

[0085] (1) Experimental methods

[0086] The confrontation culture method was used: PDA solid culture medium was used to make a plate, and Fusarium oxysporum and strain SL-K1 were inoculated at both ends of the plate at the same time. Only Fusarium oxysporum was inoculated as a control; the plates were cultured at 25℃ for 5 days and then observed.

[0087] (2) Experimental results and analysis

[0088] from Figure 5 It can be found that compared with the control, the strain SL-K1 effectively prevented the expansion of Fusarium, proving that the strain has a good antibacterial effect on Fusarium oxysporum.

[0089] Example 3 Preparation of Mycorradicin Extract

[0090] 3.1 Experimental methods

[0091] The settings of the experimental and control groups:

[0092] An experimental group was set up and operated according to the following steps; a control group was also set up. The extraction materials and steps of the control group were the same as those of the experimental group, with the only difference being that only the Ralstonia solanacearum cell induction treatment step was omitted.

[0093] (1) Culture medium configuration

[0094] Fermentation medium formula: 5% of 10 6 / ml of Ralstonia solanacearum cell inactivation solution, 0.3% beef extract, 0.5% peptone, 2% glucose, 0.4% NaCl, and the remainder water. The pH value was adjusted to 7.2.

[0095] The method for preparing the inactivated R. solanacearum cell solution involves treating a R. solanacearum cell suspension at 90°C for 5 minutes. This treatment preserves the R. solanacearum cell morphology but inactivates the cells, thereby producing an inactivated cell solution. These inactivated cells cannot infect plants but can induce Bacillus siamensis to produce antimicrobial probiotics.

[0096] The preparation method of the fermentation medium is as follows: the components of the culture medium (except the inactivated solution of Ralstonia solanacearum cells) are mixed and the pH value is adjusted to 7.2 and then sterilized. After the culture medium is cooled to below 50°C, 5% of 10 6 / ml of Ralstonia solanacearum cell inactivation solution and mix well.

[0097] (2) Cultivation and fermentation of Bacillus siamensis

[0098] The solid slant culture of Bacillus siamese SL-K1 was inoculated into 50 ml of liquid culture medium, and cultured in a shaking incubator at 30°C and 180 rpm / min for 36 h to obtain seed liquid. The seed liquid was then inoculated into 300 ml of culture medium at an inoculum size of 5%, and cultured in a shaking incubator at 30°C and 150 rpm / min for 48 h to obtain culture liquid.

[0099] The inactivated R. solanacearum cells in the culture medium help induce Bacillus siamensis to express the target metabolite. The induction of pathogen enzyme activity is carried out throughout the fermentation process.

[0100] (3) Preparation of fermentation broth powder

[0101] The fermentation liquid after the pathogen cell induction culture is centrifuged at 10,000 revolutions per minute to separate the bacteria and the fermentation liquid, and then the bacteria are removed. The fermentation liquid is freeze-dried at -20°C using a freeze dryer to obtain dry powder of the fermentation liquid.

[0102] (4) The dry powder was precipitated with 4 volumes of ethanol, maintained at 4°C for 24 hours, and then filtered with filter paper to separate the filtrate and the precipitate, which was the primary extract.

[0103] (5) The ethanol in the precipitate is removed by rotary evaporation, and the precipitate is made into a dry powder. The dry powder is dissolved in DMSO (dimethyl sulfoxide) and then dialyzed to remove macromolecules larger than 500 Da, and finally a secondary extract is obtained;

[0104] (6) The secondary extract was purified by HPLC using a Hypersil GOLD C18 column (2.1×100 mm, 5 μm; Thermo Fisher Scientific, Waltham, USA) at a column temperature of 40°C and a flow rate of 0.4 ml / min in a two-dimensional nano-liquid phase system.

[0105] During the elution process, eluent A was water (containing 0.1% formic acid, v / v), and eluent B was an acetonitrile desorption system. The chromatographic separation gradient was shown in Table 1. The chromatographic retention time was 21.0-21.3 min. Finally, a refined extract of Bacillus siamea was purified, namely a non-sugar extract - Mycorradicin.

[0106] Table 1 Chromatographic separation gradient

[0107] time Aconc(%) Bconc(%) 0 95 5 5 90 10 15 5 95 20 5 95 25 95 5 30 95 5

[0108] (7) Mass spectrometry analysis

[0109] The extract obtained above was subjected to mass spectrometry analysis using an ESI source in POS mode: electrospray mass spectrometry performed a positive ion full scan over the nucleus-to-mass ratio range of 50-1500 m / z, with nitrogen as the nebulizer gas at a flow rate of 6 L / min, a temperature of 180°C, and a pressure of 1.0 bar. Data were acquired and processed using the LC / MS Data Analysis Software (Version 4.1) provided with the instrument. Nucleus-to-mass ratio data corresponding to specific elemental compositions were calculated using the formula prediction software provided with the instrument. The error between the measured nucleus-to-mass ratio and the standard nucleus-to-mass ratio of the substance was required to be no more than 2 mD.

[0110] 3.2 Experimental results and analysis

[0111] (1) The mass spectrometry results of the extracts of the experimental group are shown in Figure 6 , chromatogram see Figure 7 .

[0112] By analyzing the positive ion mass spectrum, we know that the largest peak is the molecular ion peak of the sodium salt of the substance, and its M / Z value is 249.1108. The LC / MS data analysis software (version 4.1) provided by the instrument gives the unique molecular formula as C 14 H 17 O4; positive ion minus hydrogen: C 14 H 17 O4 is C after deducting H 14 H 16 O4, see Figure 6 .

[0113] Because the substance is weakly alkaline after saponification with sodium ions in the culture medium, the positive ion mass spectrometry signal is clear. Combining the reported data and mass spectrometry data analysis, it can be inferred that the compound is a Mycorradicin-type substance, as shown below.

[0114]

[0115] Chemical structure of Mycorradicin

[0116] (2) The extracts of the Siamese Bacillus subtilis in the control group were tested and it was found that no Mycorradicin-like compound was found in the extracts, which indicated that Siamese Bacillus subtilis did not express this compound without the induction treatment of the pathogenic bacteria Ralstonia solanacearum cells.

[0117] Example 4 Antibacterial Experiment of Extracts of Bacillus siamese

[0118] 1. Experimental methods

[0119] (1) Culture of indicator bacteria:

[0120] Ralstonia solanacearum was inoculated into LB solid culture medium and cultured at 37°C for 2 days.

[0121] (2) Sample group settings:

[0122] Potted peppers were used to determine the indirect antibacterial effect of the SL-K1 strain extract against R. solanacearum. Experimental and control groups were set up according to the following method, with at least three replicates per group:

[0123] Water control group (CK): soil and roots were irrigated with an equal amount of sterile distilled water.

[0124] Treatment group A: The method is basically the same as the control group in Example 3, omitting the R. solanacearum cell induction treatment step, and used as the sample;

[0125] Treatment group B: Mycorradicin extract prepared according to the complete experimental steps of Example 3 was used as a sample;

[0126] Treatment group C: The fermentation broth of Bacillus siamensis induced by the pathogen Ralstonia solanacearum cells was centrifuged to remove the bacterial cells. The pre-treatment was the same as that of the refined extract, but without liquid chromatography purification. The crude extract obtained by removing substances above 500D through molecular sieves was used as the sample.

[0127] The samples of each experimental group were dissolved in sterile distilled water to prepare a 10 mg / ml solution, which was then filtered through a 0.22 μm sterile filter membrane before the following experiments.

[0128] (3) Potted plant experiment method:

[0129] The pepper seedlings were irrigated with equal amounts of sterile water (CK) or sample dilutions of treatment groups A, B, and C (all at a concentration of 10 mg / ml) at a volume of 1500 ml per pot. After growing under light for 1-2 days, the activated R. solanacearum cell suspension (at a concentration of 10 mg / ml) was inoculated into the roots. 6 / ml, 5ml per plant), and observe and record the growth conditions of the plants.

[0130] Pepper seedlings in each group were measured for growth vigor (including total weight, aboveground weight, root weight, and root length), chlorophyll content (chlorophyll a, chlorophyll b, and total chlorophyll content), and malondialdehyde (a marker of cellular senescence) using a spectrophotometer. Each experiment was repeated three times, and the data were analyzed and plotted using SAS data processing software.

[0131] Chlorophyll is the primary pigment used by plants to absorb sunlight for photosynthesis, and high chlorophyll content reflects a leaf's strong photosynthesis capacity, so it was chosen for measurement. Furthermore, membrane lipid peroxidation often occurs during plant organ aging or damage under adverse conditions. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation. Lower MDA levels indicate better plant health, so MDA levels were also measured to analyze the growth status of pepper seedlings.

[0132] (4) Experimental results and analysis:

[0133] Table 2 Potted plant experiment data

[0134]

[0135] A. The experimental results are shown in Table 2 and Figures 8-11 shown.

[0136] from Figure 8 As can be seen, the control group quickly wilted after inoculation with R. solanacearum. Compared with the control, peppers in treatment B (Mycorradicin extract) showed the best resistance to R. solanacearum, followed by treatment C (induced fermentation broth filtrate), and the worst effect was treatment A (fermentation broth filtrate without induction of pathogen spore suspension).

[0137] This indicates that the Mycorradicin produced by the induction of the R. solanacearum cell suspension has a decisive influence on significantly improving the resistance of pepper seedlings to R. solanacearum. Although the A treatment without spore suspension induction also showed weak resistance to R. solanacearum, indicating that there are other substances in the fermentation liquid that have an inhibitory effect on R. solanacearum, compared with the fermentation liquid of the SL-K1 strain induced by spores and the extract of Mycorradicin, the ability of the A treatment to resist R. solanacearum is very weak, and the seedlings are weak and chlorotic (see Figure 8 ).

[0138] B. From Figures 8-11 It can be seen that compared with the control and the other two treatments, Treatment B (Mycorradicin extract) has the best effect on the stress resistance of pepper seedlings. Its chlorophyll content is the highest, so photosynthesis is stronger, more organic matter is synthesized per unit time, the plant grows faster, and its total weight, root weight and root length are significantly improved; in addition, the malondialdehyde content of Treatment B is the lowest, which also shows that the pepper seedlings treated with B have the strongest resistance to adversity.

[0139] Example 5: Rejuvenation of diseased pine trees in a nursery using an extract of Bacillus siamese

[0140] 1. Experimental methods

[0141] Experimental subjects: Diseased pine trees in a nursery, whose roots were infected with Mucor circinelloides after being flooded.

[0142] Experimental and control groups were established. On November 8, 2023, pine trees in the experimental group received a root irrigation treatment using a Mycorradicin extract solution (i.e., an aqueous solution of SL-K1 extract) at a dosage of 30g of Mycorradicin extract mixed with 15kg of water. The same treatment was repeated 15 days later. Pine trees in the control group received an equal amount of sterile distilled water.

[0143] After 60 days of treatment, the chlorophyll, soluble sugar and malondialdehyde contents of the pine trees in the experimental group and the control group were measured using the same method as in Example 4.

[0144] 2. Experimental results and analysis

[0145] The experimental results showed that the experimental group had the best rejuvenation effect on pine trees compared with the control group. The results of physiological index analysis of the experimental and control groups showed that the pine trees in the experimental group after application of Mycorradicin extract not only grew new needles (see Figure 16 ), and the newly grown needles contain higher levels of chlorophyll a, chlorophyll b and total chlorophyll (see Figure 12 and Figure 13 ), indicating that the photosynthetic capacity of the pine trees in the experimental group was stronger. However, the pine trees in the control group withered and their roots rotted (see Figure 17 ).

[0146] Since adversity can increase the content of soluble sugars in leaves, the content of soluble sugars in leaves can reflect its growth status and adaptability to the environment. When plant organs age or suffer damage under adversity, membrane lipid peroxidation often occurs. Malondialdehyde is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of damage suffered by the plant under adversity. In this embodiment, Figures 14-15 As shown, the pine needles in the experimental group contained lower soluble sugar and malondialdehyde, which indicated that the pine trees in the experimental group treated with the extract were in better growth condition, and the lower malondialdehyde content in the leaves indicated that they had not suffered from adverse damage.

[0147] This experiment proves that Mycorradicin, as an elicitor, can enhance the disease resistance of diseased plants and promote plant rejuvenation.

[0148] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solutions and concepts of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a mycorrhizal extract, characterized in that: The following steps are involved: S1, Siamese Bacillus ( Bacillus siamensis ) SL-K1 was inoculated into a liquid culture medium to prepare a fermentation broth; the liquid culture medium included the following components in percentage by weight: 1-10% of 10 4 ~10 6 / ml of Ralstonia solanacearum cell inactivation solution, 0.1-1% beef extract, 0.1-1% peptone, 1-3% glucose, 0.1-1% NaCl, and the rest water; the deposit number of the siam Bacillus is CGMCC 28450; S2, centrifuging the fermentation broth, collecting the fermentation supernatant and freeze-drying it to obtain freeze-dried powder of the fermentation broth; S3, precipitating the dry powder with ethanol, filtering and collecting the precipitate to obtain a primary extract; then removing the ethanol from the precipitate and dialyzing it to remove macromolecules larger than 500 Da to obtain a secondary extract; S4. Purifying the secondary extract by HPLC chromatography to obtain a refined extract, which is a mycorrhizal extract; The purification conditions are as follows: the chromatographic column is a Hypersil GOLD C18 column, the flow rate is 0.4 ml / min, and the column temperature is 40°C; during the elution process, the eluent A is an aqueous solution containing 0.1% by volume of formic acid, and B is an acetonitrile elution system. When the elution time is 0-5 minutes, the volume ratio of A to B is 19:1, when the elution time is 5-15 minutes, the volume ratio of A to B is 9:1, when the elution time is 15-25 minutes, the volume ratio of A to B is 1:19, and when the elution time is 25-30 minutes, the volume ratio of A to B is 19:

1. The chromatographic retention time is 21.0-21.3 minutes.

2. Use of the mycorrhizal extract according to claim 1 in promoting the growth of pepper, improving the disease resistance of pepper to Ralstonia solanacearum, and improving the disease resistance of pine trees to Mucor circinelloides.

Images