Tomato root rot biocontrol bacterium bacillus halotolerans M2108 and application of tomato root rot biocontrol bacterium bacillus halotolerans M2108

Abstract

The invention provides a novel strain M2108 of a salt-tolerant bacillus, the preservation number of the salt-tolerant bacillus is CGMCC (China General Microbiological Culture Collection Center) No.28283, and the novel strain M2108 of the salt-tolerant bacillus has relatively good salt tolerance and can grow and proliferate in a high-salt environment; the strain can be rapidly and stably colonized in a plant, and is rapid in propagation speed and strong in multiplication capacity in the plant. The new strain M2108 of the salt-tolerant bacillus provided by the invention has a treatment effect and a high control effect on various plant diseases: the control effect on diseases such as tomato root rot, damping-off and damping-off caused by fusarium oxysporum, pythium aphanidermatum and rhizoctonia solani is high, and the average control effect is 91.2% or above. When being used for preventing and treating plant diseases, the salt-tolerant bacillus M2108 is not easy to generate drug resistance, has good drug effect durability, is safe to human and livestock, and does not cause environmental pollution; the salt-tolerant bacillus M2108 microbial agent and the biocontrol agent provided by the invention are simple in preparation method, low in cost and simple and convenient to use. The salt-tolerant bacillus M2108 has a good application prospect in the prevention and control of plant diseases, and lays a foundation for the development of efficient and broad-spectrum biocontrol preparations.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to the technical field of a halotolerant bacillus and application thereof. Background Art

[0002] Plant diseases are one of the important factors affecting plant growth, development and yield. At present, the use of chemical pesticides is still one of the main means of plant disease prevention and control. Although the use of chemical pesticides can effectively prevent and control the harm of pathogenic bacteria to crops, they are highly toxic, have serious residues, and can cause resistance to pathogens, which is harmful to human health and damages the surrounding environment. Biological control avoids a series of environmental problems caused by chemical pesticides, and is safer, more effective and lasting. It has developed into an effective measure that is very important and increasingly valued in plant disease prevention and control. Biological control is to use the antibiotic effects, nutritional and spatial competition, hyperparasitism and microbial induction of plant systemic resistance of some microorganisms in nature to reduce the number of pathogens or weaken the pathogenic activity of pathogens, and reduce the occurrence of diseases caused by pathogens. There are many kinds of microorganisms in nature, and the development of beneficial microorganisms that can be used for biological control has practical value and important significance.

[0003] Tomato root rot is a common soil-borne disease of greenhouse tomatoes. It can occur throughout the growth period, mostly after planting, and can cause sudden wilting and stem rot in the seedling stage, root rot in the adult stage, and even death of the entire plant, causing great losses to production. Fusarium oxysporum ) is one of the pathogens that causes tomato root rot, which can cause browning and rot at the stem base and roots of tomato plants, greatly affecting tomato yields. The disease is most serious in North China and Northeast China. At present, the main prevention and control measures for root rot caused by Fusarium oxysporum are physical control, chemical control and biological control. Physical control mainly adopts the method of cultivating disease-resistant breeding and soil management. Disease-resistant breeding and soil management have a long cycle, slow effect, and cumbersome operation technology. Tomato varieties resistant to root rot have not yet been cultivated; chemical control has a fast effect and strong targeting, but chemical agents have a negative impact on human health, environment and food safety, and are prone to cause pathogenic fungi to develop drug resistance. With the advocacy of green ecological development, environmental and food safety have received keen attention from all walks of life. Therefore, researchers have turned their attention to biological control. Biological control is stable, durable and safe within its scope of use, so it has broad application prospects. It is urgent to develop green, pollution-free, efficient, sustainable and environmentally friendly biological control products. Biological control can reduce the use of chemical agents, improve tomato quality, and is conducive to the green development of agriculture and the protection of farmland environment and ecology. At present, there is a lack of biological control bacteria for tomato root rot. It is particularly important to screen biological control strains with antibacterial effects to provide high-quality strain resources for biological control of tomato root rot.

[0004] Common types of biocontrol fungi used in agricultural production include fungi, bacteria and actinomycetes, among which biocontrol fungi include Trichoderma, biocontrol bacteria include Bacillus, and biocontrol actinomycetes include Streptomyces and Micromonospora. Currently, commercialized biocontrol fungi include "Baikang", "Sudekang", "Ningdun", "Mission", MB1600, FZB24, QST713, GBO3, etc., among which GBO3 and MB1600 are mainly used for root diseases of vegetables caused by Aspergillus, Fusarium and Rhizoctonia.

[0005] In recent years, biological control has become a research hotspot because of its low environmental impact and compliance with the concept of sustainable development. Currently, the biocontrol bacteria used for the prevention and control of tomato wilt include fungi, bacteria and actinomycetes. Among the biocontrol bacteria, the most studied is Bacillus ( Bacillus spp.), such as Bacillus subtilis ( Bacillus subtilis ), Bacillus pumilus ( Bacillus pumilus ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus licheniformis ( Bacillus licheniformis ) etc. Although a variety of biocontrol agents have been developed and used, biocontrol agents are easily affected by the environment and other soil microbial communities, resulting in a strong regional effect of biocontrol agents. This urgently requires the exploration of local microbial resources that are unique and adapted to the local ecological environment for the prevention and control of tomato wilt.

[0006] In recent years, the research on halotolerant Bacillus Bacillus halotolerans ) The research on antibacterial activity has attracted more and more attention. It is reported that halotolerant Bacillus can produce a variety of antibacterial substances, including non-ribosomal peptides, polyketide compounds, lipopeptides and chitinase. The antibacterial substances produced by these halotolerant Bacillus are mainly used to prevent and treat Gram-positive bacteria and Candida that have developed drug resistance in clinical practice, but there are few reports on the inhibitory effect on plant pathogens.

[0007] In recent years, the inhibitory effect of halotolerant Bacillus on plant pathogens has attracted more and more attention. my country has successively reported the antibacterial activity of halotolerant Bacillus strains QTH8, F29, DMG-7-2, F29-3, HGR5, ASN9, DS5, T-04, KKD1, R1 and other strains against plant pathogenic fungi and bacteria. It can be seen that although the use of halotolerant Bacillus for biocontrol of plant diseases has been initially explored at home and abroad in recent years, the relevant research is still in the early stages, and there is no microbial fungicide with halotolerant Bacillus as the main ingredient, nor is there any report on the inhibitory effect of halotolerant Bacillus on tomato root rot pathogens. Summary of the invention

[0008] The purpose of the present invention is to provide a salt-tolerant Bacillus ( Bacillus halotolerans) M2108 and its application. This application uses the gradient dilution plate coating method and the plate confrontation method to screen indigenous biocontrol bacteria for tomato root rot in Xinjiang greenhouses, and determines its taxonomic status through the growth status and characteristics of the bacteria and 16Sr RNA gene sequence analysis, confirming that it has the typical characteristics of a new strain, testing the growth characteristics and growth-promoting function of the strain, and verifying the control effect of the strain using indoor pot experiments. Halophilic Bacillus ( Bacillus halotolerans ) M2108 can effectively inhibit Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani ), it has a high preventive effect on plant diseases caused by the above pathogens.

[0009] Specifically, the present invention provides the following technical solutions: The present invention provides a salt-tolerant Bacillus ( Bacillus halotolerans ) M2108, which was deposited on August 29, 2023 at the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named as Bacillus halodurans ( Bacillus halotolerans ), the deposit number is CGMCC No. 28283.

[0010] The present invention is based on the salt-tolerant Bacillus ( Bacillus halotolerant ) M2108, hereinafter referred to as “halotolerant Bacillus M2108” or “strain M2108”.

[0011] The salt-tolerant Bacillus M2108 provided by the present invention is a Gram-positive bacterium that is aerobic and has a short rod-shaped bacterial body; its colony is milky white, rough, opaque, and has irregular edges. The 16S rDNA gene sequence of the salt-tolerant Bacillus M2108 is shown in SEQ ID NO.1. The gyrB gene sequence of the strain M2108 is shown in SEQ ID NO.2.

[0012] Halophilic Bacillus M2108 can produce β-galactosidase, urease, oxidase, can use glucose, sucrose, citrate to produce acid, can decompose sulfur-containing amino acids to produce hydrogen sulfide, etc. The experimental results are similar to the biochemical results of Bacillus in the Bergey's Bacterial Identification Manual. It can use glucose, sucrose, lactose, maltose, fructose, xylose and arabinose; it can still grow at a salt concentration of 10% NaCl; it can undergo a variety of reactions and can also use starch as a carbon source. Halophilic Bacillus M2108 can grow in a culture medium containing NaCl and conventional carbon and nitrogen sources, such as LB culture medium, with a growth temperature range of 4 to 52°C, an optimal growth temperature of 28°C, and a pH of 6.1 to 8.5.

[0013] The salt-tolerant Bacillus spore-forming substance numbered M2108 provided by the present invention ( Bacillus halotolerans ) Through the molecular level identification of the strain system well known in the art, combined with the multi-phase classification system identification analysis such as morphological identification, the present invention relates to a salt-tolerant Bacillus numbered M2108 ( Bacillus halotolerans ) There are many differences between the strain with the closest homology to the standard type strain of this genus, confirming that the strain numbered M2108 belongs to Bacillus halotolerans A new species of the genus, with characteristics typical of new species.

[0014] The present invention provides a fermentation product prepared by fermenting and culturing the salt-tolerant Bacillus M2108. The fermentation product has the functions of inhibiting plant pathogens and preventing and controlling plant diseases.

[0015] The fermentation product mentioned above includes the extracellular metabolites of halotolerant Bacillus M2108, and can be prepared by conventional techniques in the art, for example, by fermenting halotolerant Bacillus M2108, centrifuging the fermentation broth and collecting the supernatant.

[0016] The present invention provides a bacterial agent, which comprises a combination of one or more of the halotolerant Bacillus M2108, its fermentation culture, and its fermentation product.

[0017] The above-mentioned microbial agent can be a liquid microbial agent or a solid microbial agent, such as a powder, and can be prepared by conventional technical means and by adding carriers or other auxiliary materials permitted in the field of microbial preparations.

[0018] The present invention provides a method for preparing the fermentation product or the bacterial agent, which comprises the step of fermenting and culturing the halotolerant Bacillus M2108 at 21-40° C., pH 6.5-8.5 and ventilation conditions.

[0019] As an embodiment of the present invention, the method for preparing the fermentation product or the bacterial agent comprises the following steps: (1) Activation of bacterial strains: Inoculate the halotolerant Bacillus sp. M2108 onto a LB solid medium containing 10 g / L NaCl, and culture by continuous streaking at 20-34° C. Pick a single colony and place it into a LB liquid medium containing 10 g / L NaCl, and culture it at 25-40° C. and 170-220 rpm on a shaker for 20-38 h to obtain an activated bacterial solution; (2) Seed culture: Inoculate the activated bacterial solution obtained in step (1) into a fermentation tank containing a seed culture medium at a volume ratio of 1:9, and culture at 25-40° C. and 170-220 rpm for 16-20 h to obtain liquid seeds; (3) Fermentation culture: Inoculate the liquid seeds obtained in step (2) into the seed culture medium at an inoculum volume ratio of 10-20%, and culture for 36-45 hours at 25-40° C. and 170-220 rpm on a shaker under ventilation conditions to obtain a live bacterial culture.

[0020] The living bacterial cells of the culture obtained in step (3) are removed by centrifugation or filtration to obtain the fermentation product of halotolerant Bacillus M2108.

[0021] The viable bacterial culture obtained in step (3) above was adjusted to a bacterial content of 10 9 ~10 10 cfu / mL to obtain a liquid bacterial agent, or add auxiliary materials to obtain a liquid bacterial agent.

[0022] The live bacterial culture obtained in the above step (3) is adsorbed with calcium carbonate to obtain a powder containing halotolerant Bacillus M2108.

[0023] The present invention also provides a biocontrol agent, which comprises a combination of one or more of the halodurable Bacillus M2108, its fermentation culture, and its fermentation product.

[0024] The active ingredients of the above-mentioned biocontrol agent may consist only of one or more combinations of halodurable Bacillus sp. M2108, its fermentation culture, and its fermentation products, or may also contain other microorganisms, compounds or plant extracts that have the effect of inhibiting plant pathogens.

[0025] The present invention proves through experiments that the salt-tolerant Bacillus M2108 can effectively inhibit Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani ) and other plant pathogens, and has a high inhibitory effect on Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphaniderm )、Rhizoctonia solani( Rhizocton solani) and other diseases caused by tomato root rot.

[0026] Based on the above functions, the present invention provides the use of halotolerant Bacillus M2108, its fermentation product, the bacterial agent or the biocontrol agent in plant disease prevention and control.

[0027] The invention provides application of halotolerant Bacillus M2108 in breeding microorganisms for preventing and controlling plant diseases.

[0028] The plant diseases described in the present invention are soil-borne diseases including but not limited to root rot. Preferably, in the above application, the plant diseases are one or more selected from root rot, brown spot and root rot.

[0029] The present invention provides the use of halotolerant Bacillus M2108, its fermentation product, the bacterial agent or the biocontrol agent in inhibiting plant pathogens. Preferably, the plant pathogen is selected from Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani )

[0030] The present invention also provides a method for preventing and controlling plant diseases, comprising: applying the halotolerant Bacillus M2108, a fermentation product thereof, the bacterial agent or the biocontrol agent to the plant.

[0031] The application methods of the salt-tolerant bacillus M2108, its fermentation product, the bacterial agent or the biological control agent include root irrigation, spraying and seed mixing.

[0032] Preferably, when applying the root, the dosage of the salt-tolerant Bacillus M2108 is 1.0×10 6 cfu~2.0×10 8 When spraying, the dosage of halotolerant Bacillus M2108 is 1.5×10 per mu. 8 cfu~3.0×10 9 When used as seed dressing, the dosage of halotolerant Bacillus M2108 is 0.5×10 per kilogram of seeds. 9 cfu~1.0×10 10 cfu.

[0033] Specifically, when used to prevent and control above-ground diseases, when sporadic diseases occur on the above-ground parts of plants, a concentration of 10 5 ~10 7 cfu / mL of salt-tolerant Bacillus M2108 liquid spray, 10-20L / mu. When used to prevent and control soil-borne diseases, when the plant seedlings grow to 3-5 leaves, spray at a concentration of 10 6 ~10 8cfu / mL of salt-tolerant Bacillus M2108 solution was used for root irrigation, 100-150mL / plant. When used to prevent seed-borne diseases, the concentration was 1.0×10 9 cfu / mL, mix seeds at a drug-seed ratio of 1:(30-50).

[0034] The salt-tolerant Bacillus M2108 provided by the present invention has at least the following beneficial effects: (1) It has a broad antibacterial spectrum against plant pathogens: Halophilic Bacillus M2108 has a broad antibacterial spectrum against Fusarium oxysporum of tomatoes ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizoctonia nightshade ) and other pathogens, with an inhibition rate of up to 91.52% to 95.61%; (2) It has a dual-effect effect on a variety of plant diseases and has a high preventive effect: Halophilic Bacillus M2108 has a high inhibitory effect on Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizoctonia nightshade ) has a high prevention effect on tomato root rot, damping-off and damping-off caused by the plant, with an average prevention effect of more than 91.2%; (3) Halophilic Bacillus M2108 can colonize plants quickly and stably, and has a fast reproduction speed and strong proliferation ability in plants; (4) Halophilic Bacillus M2108 has good salt tolerance and can grow and proliferate in high-salt environments; (5) Using halotolerant Bacillus M2108 to control plant diseases is not easy to produce drug resistance, and the drug effect is long-lasting; (6) Using halotolerant Bacillus M2108 to control plant diseases is safe for humans and animals and does not cause environmental pollution; (7) The preparation method of the bacillus halogenide M2108 bacterial agent and biocontrol agent is simple, low-cost and easy to use. Bacillus halogenide M2108 has a good application prospect in plant disease control and lays a foundation for the development of efficient and broad-spectrum biocontrol agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shown is the colony morphology of strain M1906 in Example 1 of the present invention.

[0036] Figure 2 Shown is the electrophoresis diagram of the 16S rDNA PCR amplification product of the halotolerant Bacillus M2108 in Example 1 of the present invention.

[0037] Among them, lane M is a DNA marker band, and lanes 1 and 2 are the amplified products of 16S rDNA.

[0038] Figure 3 Shown is the phylogenetic tree of the halotolerant Bacillus sp. M2108 strain obtained from the 16S rDNA sequence.

[0039] Figure 4 Shown is the electrophoresis diagram of the PCR amplification product of the gyrB gene of halotolerant Bacillus sp. M2108.

[0040] Among them, lane M is a DNA marker band, and lanes 1 and 2 are the amplified products of the gyrB gene.

[0041] Figure 5 Shown is the phylogenetic tree of the halotolerant Bacillus sp. M2108 strain obtained based on the gyrB gene sequence.

[0042] Figure 6 Shown is a graph of the growth curve of the antagonistic bacteria.

[0043] Figure 7 Shown is a graph showing the effect of temperature on the growth of antagonistic bacteria.

[0044] Figure 8 Shown is a graph showing the effect of pH on the growth of antagonistic bacteria.

[0045] Figure 9 Shown is a graph showing the effect of NaCl concentration on the growth of antagonistic bacteria. DETAILED DESCRIPTION

[0046] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0048] The percentages in the following examples are by weight unless otherwise specified.

[0049] The following examples are based on the salt-tolerant Bacillus ( Bacillus halotolerant ) M2108, hereinafter referred to as “halotolerant Bacillus M2108” or “strain M2108”.

[0050] The culture medium formulas involved in the following examples are as follows: LB solid medium (strain preservation medium): 10g tryptone, 5g yeast powder, 10g sodium chloride, 20g agar, add water to 1L, pH 6.5-7.5.

[0051] LB culture medium (bacteria activation medium): 10g tryptone, 5g yeast powder, 10g sodium chloride, add water to 1L, pH 6.5-7.5.

[0052] Seed culture medium (liquid, 1 L): K2HPO4 4.8 g, KH2PO4 3.5 g, (NH4)2SO4 2 g, MgCl2 0.16 g, CaCl2 0.02 g, Na2MoO4·2H2O 0.0024 g, FeCl3 0.0018 g, MnCl2·2H2O 0.0015 g, sodium chloride 10 g, pH = 7.0.

[0053] All the above culture media were sterilized at 121°C for 15 to 30 min.

[0054] LB plate: prepare 100 ml of the above LB solid culture medium, sterilize it by high pressure, place the melted LB solid culture medium in a 55°C water bath, and pour the plate when the temperature of the culture medium drops to 55°C. Pour 10 ml of LB solid culture medium into a sterilized culture dish, open the lid, and expose it under ultraviolet light for 10 to 15 minutes. After cooling, seal it with sealing film and place it upside down in a 4°C refrigerator for later use. PDA plate: Peel 200g of potato, cut into small pieces and put into a pot, add 1000ml of water, heat and boil for 20-30min, filter through 4 layers of gauze while hot to remove the residue, add 20g of agar powder, wait for the agar to melt, add 20g of glucose, add water to make up to 1000ml, sterilize at 121℃ for 15-30min, take out and cool to 55℃, pour 10ml of culture medium into a sterilized culture dish, open the lid, and expose under ultraviolet light for 10-15 minutes. After cooling, seal with sealing film and place upside down in a 4℃ refrigerator for later use.

[0055] The pathogenic bacteria strains used in the following examples are: Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani ) are common strains that can be purchased by the general public through public channels.

[0056] The tomato seeds used in the following examples were Heinong 53 (susceptible to root rot), Hefeng 25 (susceptible to brown spot), and Jidou 16 (moderately susceptible to root rot), all of which were purchased from Baoding Seedling Technology Co., Ltd., Hebei Province.

[0057] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0058] Example 1: Screening and identification of halotolerant Bacillus M2108 1. Isolation and screening of halotolerant Bacillus subtilis M2108 strain (1) Sample collection: Root samples of healthy plants from a tomato field in a vegetable greenhouse in Banfanggou, Urumqi, Xinjiang were collected, and the dust on the surface was washed off with sterile water. The roots of the plants were then surface disinfected by soaking them in 75% alcohol for 1 min and 8% NaClO for 4 min, and then washed with sterile water 4 times; (2) Isolation and screening: The roots were cut into 0.2 cm × 0.2 cm pieces, ground into a paste with water, allowed to stand for 10 min, and then spread on LB plates containing 10 g / L NaCl, and incubated at 28 °C for 48 h; (3) Purification: After the culture has grown, it is purified by the plate streak method. The colonies growing at the highest concentration are picked and streaked on the enrichment medium until a pure culture is isolated.

[0059] Taking tomato root rot as the target, the biocontrol bacteria were screened by plate confrontation method and field plot test method. Finally, a strain of Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphaniderm )、Rhizoctonia solani( Rhizocton solani ) caused by tomato root rot, damping-off and damping-off, and was named M2108.

[0060] 2. Classification and identification of strain M2108 (1) Morphological characteristics identification The strain M2108 is a Gram-positive aerobic bacterium cultured on LB medium containing 10g / L NaCl. The bacteria are short and rod-shaped. The colonies are milky white, rough, opaque, and have irregular edges. The streak culture on the nutrient agar slant is straight. The static culture in the liquid medium is opaque and milky white. See the attached Figure 1 These morphological characteristics are basically consistent with the morphological characteristics of Bacillus described in the Manual of Common Bacterial Systematic Identification (Dong Xiuzhu et al., Science Press, 2001), and it is preliminarily judged that the M2108 strain belongs to Bacillus.

[0061] (2) Identification and classification using 16S rDNA sequences The genomic DNA of M2108 was used as a template, and the universal primers F27 and R1492 were used as primers for PCR amplification to obtain the PCR amplification product. The sequences of primers F27 and R1492 are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; R1492: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction system (20μL) for 16S rDNA amplification was: 10×Ex Taq buffer 2.0μL; 5U Ex Taq 0.2μL; 2.5mM dNTP Mix 1.6μL; 27F 1μL; 1492 R 1μL; M2108 genomic DNA 0.5μL; ddH2O was added to 20μL. The reaction conditions of PCR were 95℃, 5min; 95℃, 30s, 56℃, 30s, 72℃, 1.5min, 30 cycles; 72℃, 10min. The obtained PCR amplification products were subjected to gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rDNA sequence of M2108 was shown in SEQID NO.1. The electrophoresis detection results of PCR amplification products are shown in the attached Figure 2 The 16S rDNA sequence of strain M2108 was used to construct a phylogenetic tree using MEGA software (Molecular Evolutionary Genetics Analysis). The phylogenetic analysis diagram is shown in the attached Figure 3 As shown in the figure, the BLAST program was used to compare the 16S rDNA gene sequences of the registered bacterial strains on the Genbank (http: / / www.ncbi.nlm.nih.gov / ) website. The results showed that the 16S rDNA of the strain was similar to that of Bacillus halodurans ( Bacillus halotolerans ) has the highest similarity, reaching 98.46%.

[0062] (3) Identification and classification based on gyrB gene sequence Using the genomic DNA of strain M2108 as a template, PCR amplification was performed using the degenerate primers gyrB-F and gyrB-R of the Bacillus gyrB gene as primers to obtain a PCR amplification product. The sequences of the gyrB-F and gyrB-R primers are as follows: gyrB-F: 5'-TTGRCGGHRGYGGHTATAAAGT-3'; gyrB-R: 5'-TCCDCCSTCAGARTCWCCCTC-3'. In the above, Y represents C / T, R represents A / G, H represents A / T / C, W represents A / T, D represents G / A / T, and S represents G / C. The PCR reaction system (50 μL) for gyrB amplification is: 10×PCR Buffer (Mg2+) 5 μL; dNTP mixture (2.5 mM) 5 μL; Taq (5 U / μL) 1 μL; gyrB-F (10 μmol / L) 1 μL; gyrB-R (10 μmol / L) 1 μL; M2108 genomic DNA 50 ng; ddH2O is added to 50 μL. The reaction conditions of PCR are 95°C, 5 min; 95°C, 30 s, 55°C, 45 s, 72°C, 1 min, 30 cycles; 72°C, 10 min. The amplified product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing, and the gyrB gene sequence of the M2108 strain was shown in SEQ ID NO.2. The electrophoresis detection results of the PCR amplification products are shown in the attached Figure 4 The gyrB gene sequence of the obtained M2108 strain was compared with that of the halodurogenic Bacillus ( Bacillus halotolerans ) has the highest homology of gyrB gene sequence, reaching 98.57%. At the same time, MEGA software (Molecular Evolutionary Genetics Analysis) was used to construct the phylogenetic tree of gyrB gene, see Appendix Figure 5 As shown, the results showed that strain M2108 was aggregated with Bacillus halotolerans, indicating that strain M2108 was a Bacillus halotolerans.

[0063] Based on the above morphological characteristics, 16S rDNA and gyrB gene sequence homology comparison analysis results, it can be seen that strain M2108 belongs to the halotolerant Bacillus ( Bacillus halotolerans ), and is different from the existing salt-tolerant Bacillus strains, and is a new salt-tolerant Bacillus strain. It can be seen that the salt-tolerant Bacillus numbered M2108 provided by the present invention ( Bacillus halotolerans) Through the molecular level identification of the strain system well known in the art, combined with the multi-phase classification system identification analysis such as morphological identification, the present invention relates to a salt-tolerant Bacillus numbered M2108 ( Bacillus halotolerant ) There are many differences between the strain with the closest homology to the standard type strain of this genus, confirming that the strain numbered M2108 belongs to Bacillus halotolerans This is a new species of the genus with typical characteristics of new species. Bacillus halotolerans )M2108 was deposited on August 29, 2023 at the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) and was classified and named as Bacillus halodurans Bacillus halotolerans , the deposit number is CGMCC No.28283.

[0064] Example 2: Preparation of bacterial solution and bacterial agent of halotolerant Bacillus sp. M2108 1. Growth curve Based on the cultivation of the above embodiment one, the participants Figure 6 It can be seen that from 0 to 4 h, the growth of the halodurable Bacillus M2108 was rapid and entered the logarithmic growth phase. After 18 h, the biomass of the strain M2108 tended to stabilize. At this time, OD 600 They are 1.7812 respectively.

[0065] 2. Effect of temperature on the growth of antagonistic bacteria See attached Figure 7 It can be seen that as the temperature gradually increases from 24℃ to 40℃, the number of live bacteria of Bacillus halophilus M2108 increases first and then decreases. The number of live bacteria of M2108 is the largest at 28℃, which is 2.10×10 9 CFU / Ml, M2108 bacteria can grow in the range of 24℃-40℃.

[0066] 3. Effect of pH on the growth of antagonistic bacteria See attached Figure 8 It can be seen that the salt-tolerant Bacillus M2108 can grow at pH 5, 6, 7, 8, and 9. Among them, the strain M2108 can grow at pH 4, but the number of viable bacteria is relatively small, which is 4.25×10 3 CFU / mL. As the pH continued to rise, the number of live bacteria of the two strains showed a trend of first increasing and then decreasing. At pH=6, the number of live bacteria of strain 3-1 was the largest, which was 3.95×10 9 CFU / mL; at pH=7, the number of viable bacteria of strain 26-8 was the largest, which was 8.89×10 8 CFU / mL.

[0067] 4. Effect of different NaCl concentrations on the growth of antagonistic bacteria See attached Figure 9 It can be seen that the halotolerant Bacillus M2108 can grow at NaCl concentrations of 0.5%, 1%, 2%, 5%, 7%, and 10%, and can tolerate a wide range of salt concentrations. The number of live bacteria is large when the salt concentration is 7% and 10%, and the number of live bacteria of halotolerant Bacillus M2108 is 3.12×10 8 CFU / mL, 6.23×10 6 CFU / mL. The salt concentrations of 0.5%, 1%, 2%, and 5% had no significant effect on the growth of halotolerant Bacillus M2108.

[0068] The preparation method of the bacterial solution and the bacterial agent of the halotolerant Bacillus sp. M2108 comprises the following steps: (1) Activation of bacterial strains: The halotolerant Bacillus M2108 strain was placed in a bacterial strain preservation medium, streaked and cultured twice at 28°C, and then a single colony was placed in a bacterial strain activation medium, and cultured at 28°C and 180 rpm for 30 h. (2) Preparation of liquid seeds: In a fermentation tank filled with a high-temperature sterilized seed culture medium, the M2108 activated bacterial solution obtained in step (1) was inoculated at an inoculum rate of 10% (V / V), and cultured at 28° C. with aeration for 12 h to obtain liquid seeds; (3) Liquid fermentation: Inoculate liquid seeds at a volume fraction of 3% into a seed culture medium that has been sterilized by high temperature, and culture at 28° C. and 180 rpm for 30 h (logarithmic growth phase) to obtain a live bacterial culture; (4) Preparation of bacterial agent: Take the live bacterial culture and centrifuge it at 4°C and 5000 r / min for 15 min. Take the bacterial precipitate and rinse it three times with 0.85% sterile saline. Add appropriate amount of seed culture medium to adjust the bacterial content to 10 9 cfu / mL, and the M2108 bacterial solution was obtained; glycerol (volume fraction 50%) was added and the solution was bottled and stored.

[0069] Example 3: Antibacterial effect of halotolerant Bacillus M2108 This example analyzes the activity of halotolerant Bacillus M2108 against Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani ), the specific method is as follows: (1) Fusarium oxysporum of tomatoes ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphaniderm )、Rhizoctonia solani( Rhizocton solani ) were inoculated on PDA plates, and Fusarium oxysporum of tomato ( Fusarium oxysporum) were cultured at 27°C, Pythium aphanidermatum ( Pythium aphanidermatum ) were cultured at 25°C, Rhizoctonia solani ( Rhizocton solani ) were cultured at 26°C. After the plates were fully grown, a 6 mm diameter punch was used to make a bacterial cake. The bacterial cake was inoculated in the center of the PDA plate. At the same time, M2108 bacterial solution (concentration 10 8 cfu / mL, 20 μL), the M2108 bacterial solution prepared in Example 2 was used as the experimental group, and the plate inoculated with only pathogenic fungi was used as the control group, and 3 replicates were set for each treatment; (2) The PDA plates inoculated with each treatment in step (1) were placed in an environment suitable for the temperature of the corresponding pathogens in step (1) and cultured. Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani ) until the pathogenic fungal colonies in the control group almost covered the plate, the diameters of the pathogenic fungal colonies in each treatment were measured, and the inhibition rate was calculated according to the following formula: Inhibition rate (%) = [(AB) / (A-6)] × 100%, where A is the diameter of the pathogenic fungal colonies in the control group, and B is the diameter of the pathogenic fungal colonies in the experimental group; (3) Antibacterial test results: The results are shown in Table 1. The strain M2108 has an antibacterial effect on Fusarium oxysporum of tomatoes ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani ) had an inhibition rate of 93.67% to 96.25%, indicating that halotolerant Bacillus M2108 had a good inhibitory effect on the above major pathogens of tomatoes.

[0070] Table 1: Antibacterial effect of strain M2108 on Fusarium spores, Pythium aphanidermatum, and Rhizoctonia solani

[0071] Example 4: Field plot test of halotolerant Bacillus sp. M2108 for controlling tomato Fusarium root rot This example provides a method for controlling Fusarium oxysporum in tomatoes using halotolerant Bacillus sp. M2108. Fusarium oxysporum ) caused by root rot in the field plot test, the specific method is as follows: (1) Tomato seeds of the Provence variety were sown at a distance of 30-50 cm in a soil inoculated with Fusarium oxysporum for many years. Fusarium oxysporum) of tomato root rot, and the field plot test was carried out in the experimental area of ​​the National Agricultural Microbiology Urumqi Observation and Experimental Station. The agent treatment agents are as follows: chemical fungicide group (carbendazim): carbendazim (50% carbendazim wettable powder diluted 800 times with water); microbial agent group (M2108): liquid agent of strain M2108 prepared in Example 2 (diluted 500 times with water to a bacterial concentration of 2×10 6 cfu / ml); control group (blank control): blank culture medium (i.e. sterilized seed culture medium). Each treatment was performed on 30 tomato seedlings, with 3 replicates. When the tomato seedlings grew 4-7 leaves, 200 mL of treatment agent was poured at the roots of the tomato seedlings. The disease situation was investigated after 30 days, and 30 plants were investigated in each plot to calculate the disease incidence and prevention effect.

[0072] (2) Results of tomato root rot control: The results are shown in Table 2. The control efficiency of halotolerant Bacillus M2108 against tomato root rot was 88.90%, which was higher than that of the chemical fungicide carbendazim, indicating that halotolerant Bacillus M2108 and its microbial agent had a good control effect on tomato root rot.

[0073] Table 2: Results of field trials of strain M2108 against tomato root rot

[0074] Note: The experimental results in Table 2 are the average values ​​of 3 repetitions; the data with different letters on their shoulders are significantly different.

[0075] Example 5: Plot field trial of halotolerant Bacillus sp. M2108 for controlling Rhizoctonia solani root rot in tomatoes This example provides a method for controlling tomato Rhizoctonia solani ( Rhizocton solani ) caused by root rot in a small plot field test, the specific method is as follows: (1) Tomatoes (Provence) were sown on May 20, 2021. Each plot was 2×3 m in size, with a row spacing of 40 cm and a plant spacing of 27 cm. When the tomato seedlings grew 2-3 leaves, each plot was inoculated with Rhizoctonia solani ( Rhizocton solani ), the inoculum volume was 1×10 6 cfu / ml bacterial solution 1000 ml, when the plant is sporadically infected, root irrigation is carried out twice, with an interval of 10 days. Different drug treatments are arranged randomly, and each treatment is repeated 3 times. Drug treatment: A: Microbial agent group (M2108): The liquid bacterial agent of halotolerant Bacillus M2108 prepared in Example 2 is diluted 500 times with water to a bacterial concentration of 2×10 6cfu / ml; B: Chemical fungicide group (Lycobacterium): 20% methyl tolclofos emulsifiable concentrate (Lycobacterium) diluted 1200 times with water; C: Blank control group: clean water. The dosage of the liquid was 120L / mu, and the disease situation was investigated 20 days after the last spraying. 30 plants were investigated in each plot, and the disease index and control effect of each treatment were calculated.

[0076] (2) Results of tomato root rot control: The results are shown in Table 3. The control effect of halotolerant Bacillus M2108 on tomato root rot was 89.48%, which was higher than that of the chemical fungicide tolclofos-methyl emulsifiable concentrate, indicating that halotolerant Bacillus M2108 and its microbial agent had a good control effect on tomato root rot.

[0077] Table 3: Results of field trials of strain M2108 against tomato root rot

[0078] Note: The experimental results in Table 3 are the average values ​​of 3 repetitions; the data with different letters on their shoulders are significantly different.

[0079] Example 6: Plot field trial of halotolerant Bacillus M2108 for controlling tomato fruit pythium root rot This example provides a method for controlling Pythium aphanidermatum in tomatoes using halotolerant Bacillus sp. M2108. Pythium aphaniderm ) caused by root rot, the specific method is as follows: (1) Seeds of tomato plants with root rot in previous years (Provence) were treated with drugs. The drugs were as follows: chemical fungicide group (Pulike): 800 times dilution of 72% Pulike water, watering the planting holes, 150-200 grams per hole; microbial agent group (M2108): M2108 liquid microbial agent prepared in Example 2, diluted 500 times with water to a bacterial concentration of 2×10 6 cfu / ml; irrigate the roots with 150-200g per hole; control group (blank control): blank culture medium (i.e. sterilized seed culture medium). Tomato seeds were sown at a distance of 30-50cm in the experimental area of ​​the National Agricultural Microbiology Urumqi Observation Experimental Station, and each treatment was repeated 3 times. The disease situation of tomatoes was investigated twice at the seedling stage and mature stage, with 30 plants investigated in each plot, and the incidence rate and prevention effect were calculated.

[0080] (2) Results of tomato root rot control: The results are shown in Table 4. The control efficacy of halotolerant Bacillus M2108 against tomato root rot at the seedling stage and mature stage was 88.89% and 85.00%, respectively. Its control effect was higher than that of the chemical fungicide thiram, indicating that halotolerant Bacillus M2108 and its microbial agent had a good control effect on tomato root rot.

[0081] Table 4: Results of field trials of strain M2108 against tomato root rot

[0082] Note: The experimental results in Table 4 are the average values ​​of 3 repetitions; the data with different letters on their shoulders are significantly different.

[0083] Example 7: Colonization and proliferation ability detection of halotolerant Bacillus sp. M2108 in tomatoes In this example, the number of live bacteria colonized on the roots of tomatoes when the salt-tolerant Bacillus M2108 was applied by root irrigation was detected. The specific method is as follows: The tomato roots and rhizosphere soil used in the root irrigation test for preventing and controlling tomato Fusarium root rot in Example 4 were sampled at different time points within 10 to 50 days after the application of the halotolerant Bacillus M2108. The roots were surface disinfected, rinsed with 75% alcohol for 1 minute, soaked in 1% sodium hypochlorite for 2 minutes, and then washed 4 times with sterile water. 100 μL of the last sterile water rinse was spread on the LB medium and cultured at 30°C for 24 hours to check the disinfection effect and observe microscopically whether miscellaneous bacteria were produced. After absorbing the surface water, grind it with 2 mL of sterile water to a paste. Let it stand for 15 minutes to fully release the halotolerant Bacillus in the tissue, then place it in a centrifuge tube and count it using the dilution coating plate method. Use sterile distilled water to make gradient dilutions, and take 10 －1 , 10 －2 and 10 －3 100 μL of the dilution gradient was spread on NA plates, with 3 replicates per gradient. After overnight culture at 34°C, the number of single colonies was counted to determine the colonization dynamics of antagonistic bacteria in different varieties of tomatoes.

[0084] Ten days after the inoculation of Bacillus halodurans M2108, soil from the roots of three tomato seedlings was collected from each replicate, with three replicates for each treatment. The soil was mixed and the water content of the soil was measured. Then, 5 g of the soil was weighed into a 250 mL conical flask, 45 mL of sterile saline was added, and the soil was shaken at 25 °C and 180 r / min for 2 h. –4 , 10 –5 The gradient dilutions were spread on NA solid culture medium and cultured in a 30 ℃ constant temperature box for 1 day. The bacterial colonies (30-300 CFU) were counted and statistically analyzed. Samples were taken once every 10 days, and the counts were performed 5 times in total.

[0085] The results of the dilution plate analysis of the salt-tolerant Bacillus M2108 under different application methods are shown in Table 5. The results showed that the colonization and proliferation abilities of the salt-tolerant Bacillus M2108 were strong, and it could colonize the rhizosphere and roots of tomatoes quickly within 10 days. The maximum number of the rhizosphere and roots during root irrigation was 6.52×10 6 CFU·g-1 and 2.68×10 5 CFU·g -1 The rapid colonization and proliferation of M2108 reduced the space occupied by pathogens in the roots, thus giving full play to the spatial competition of halotolerant Bacillus M2108.

[0086] Table 5: The number of viable bacteria of halotolerant Bacillus colonized in the rhizosphere and roots by dilution plate method

[0087] Note: The experimental results in Table 5 are the average values ​​of 3 repetitions; ± is the standard deviation of 3 repetitions; the data with different letters on their shoulders are significantly different.

[0088] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A strain of halotolerant Bacillus ( Bacillus halotolerans ) M2108, characterized in that, The salt-tolerant Bacillus M2108 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee with a deposit number of CGMCC No.28283 and a deposit date of August 29, 2023. The 16S rDNA gene sequence is shown in SEQID NO.1, and the gyrB gene sequence is shown in SEQID NO.

2.

2. The salt-tolerant Bacillus according to claim 1 ( Bacillus halotolerans ) The fermentation product prepared by fermentation culture of M2108.

3. A bacterial agent, characterized in that: The bacterial agent comprises halodurable Bacillus ( Bacillus halotolerans )M2108, its fermentation culture, its fermentation product, or a combination of one or more thereof can be a liquid bacterial agent or a solid bacterial agent, which can be prepared by conventional technical means and by adding carriers or other auxiliary materials permitted in the field of microbial preparations.

4. The method for preparing any one of the fermentation product according to claim 2 or the bacterial agent according to claim 3 comprises the following steps: (1) Bacterial activation: Halophilic Bacillus ( Bacillus halotolerans ) M2108 was inoculated onto LB solid medium containing 10 g / L NaCl, and cultured by continuous streaking at 20-34°C. A single colony was picked and placed in LB liquid medium containing 10 g / L NaCl, and cultured at 25-40°C and 170-220 rpm on a shaker for 20-38 h to obtain an activated bacterial solution; (2) Seed culture: Inoculate the activated bacterial solution obtained in step (1) into a fermentation tank containing a seed culture medium at a volume ratio of 1:9, and culture at 25-40° C. and 170-220 rpm for 16-20 h to obtain liquid seeds; (3) Fermentation culture: inoculating the liquid seeds obtained in step (2) into the seed culture medium at an inoculum amount of 10 to 20% by volume, and culturing for 36 to 45 hours at 25 to 40° C. and 170 to 220 rpm on a shaking table under ventilation conditions to obtain a live bacterial culture; The living bacterial cells obtained in step (3) are removed by centrifugation or filtration to obtain a fermentation product of the halotolerant Bacillus M2108; The viable bacterial culture obtained in step (3) above was adjusted to a bacterial content of 10 9 ~10 10 cfu / mL, and obtain a liquid bacterial agent, or add auxiliary materials to obtain a liquid bacterial agent; The live bacterial culture obtained in the above step (3) is adsorbed with calcium carbonate to obtain a powder containing halotolerant Bacillus M2108.

5. A biocontrol agent comprising the halodurable Bacillus ( Bacillus halotolerans ) M2108, its fermentation culture, its fermentation product, or a combination of one or more thereof may also contain other microorganisms, compounds or plant extracts that have the effect of inhibiting plant pathogens.

6. A salt-tolerant Bacillus according to claim 1 ( Bacillus halotolerans ) Use of any one of M2108, or the fermentation product according to claim 2, or the bacterial agent according to claim 3, or the biological control agent according to claim 5 in plant disease prevention and control.

7. The salt-tolerant Bacillus according to claim 1 ( Bacillus halotolerans ) Use of M2108 in breeding microorganisms for preventing and controlling plant diseases, characterized in that: The plant diseases are one or more selected from root rot, brown spot and root rot.

8. A salt-tolerant Bacillus according to claim 1 ( Bacillus halotolerans ) M2108, or the fermentation product according to claim 2, or the bacterial agent according to claim 3, or the biocontrol agent according to claim 5, in the use of any one of them in inhibiting plant pathogens, characterized in that: The plant pathogenic bacteria is selected from Fusarium oxysporum ( Fusarium oxysporum )、Pythium aphanidermatum( Pythium aphanidermatum )、Rhizoctonia solani( Rhizocton solani ) 9. A method for preparing a product for preventing and controlling plant diseases, characterized in that: The method comprises: applying the halotolerant Bacillus M2108, a fermentation product thereof, the bacterial agent or the biological control agent to the plant.

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