CN114437994A

CN114437994A - Biological control bacterium Bacillus siamensis HT1 and application thereof in preparation of biological control bacterium agent

Info

Publication number: CN114437994A
Application number: CN202210261346.1A
Authority: CN
Inventors: 梁爽; 何海涛; 张�浩; 唐亚楠; 郑丽宁; 吴宪; 潘洪玉
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2022-05-06

Abstract

A biological control bacterium Bacillus siamensis HT1 and application thereof in preparing biological control bacterium agent, belonging to the technical field of agricultural microbiology. The strain is preserved in China center for type culture Collection with a preservation date of 2021, 12 months and 3 days, and the preservation number is CCTCC NO: m20211539, classified and named as Bacillus siamensis HT 1. Experiments show that the strain can effectively inhibit the soybean root rot caused by fusarium oxysporum and phytophthora sojae, thereby effectively preventing and treating the soybean root rot caused by fusarium oxysporum and phytophthora sojae. Compared with the existing chemical synthetic pesticide, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like due to the natural environment, and has wide application prospect and good practical application value.

Description

Biological control bacterium Bacillus siamensis HT1 and application thereof in preparation of biological control bacterium agent

Technical Field

The invention belongs to the technical field of agricultural microbiology, and particularly relates to a biological control bacterium Bacillus siamensis HT1 and application thereof in preparation of a biological control bacterium agent.

Background

Soybean root rot is mainly caused by Fusarium sp (Fusarium spp.), Phytophthora sojae (Phytophthora sojae), Pythium sp (Pythium spp.), and Rhizoctonia solani (Rhizoctonia solani). Among them, Fusarium oxysporum (Fusarium oxysporum) and Phytophthora sojae (Phytophthora sojae) are the main pathogenic bacteria of soybean root rot. The disease can occur in the whole soybean growth period, and the whole plant can be directly killed when the disease is serious, so that huge economic loss is caused.

For a long time, the method for preventing and treating soybean root rot mainly comprises the steps of applying a large amount of chemical pesticides and breeding disease-resistant varieties, but the characteristics of fast propagation, large variation degree and the like of root rot germs enable chemical agents to easily generate drug resistance, and in addition, the disease-resistant degree of the varieties is not high, people improve the understanding of the drug resistance and the food safety problem, and the like, the method for preventing and treating the soybean root rot by using organisms is more and more concerned. Compared with chemical pesticides, microbial pesticides have the characteristics of strong selectivity, no pollution, difficulty in generating drug resistance, no damage to ecological environment and the like, and are the research hotspots.

Among many biocontrol bacteria, Bacillus spp is still the most studied type so far, and because of its strong stress resistance and antibacterial activity, it is an important member of rhizosphere bacteria (PGPR) and plays an important role in promoting plant growth and inhibiting plant diseases. And the bacillus has high propagation speed and easy artificial culture, and is widely applied to production practice as a biocontrol microbial inoculum.

Currently, there are reports of Siamese bacillus for preventing and treating soybean root rot. Therefore, the invention provides a larger theoretical basis for searching a new strain for preventing and treating the soybean root rot.

Disclosure of Invention

The invention aims to provide a biological control bacterium Bacillus siamensis HT1, the preservation number of which is CCTCC NO: m20211539, classified and named as Bacillus siamensis HT 1.

The invention also aims to provide application of the biological control bacterium Bacillus siamensis HT1 in preparation of a biological control bacterium agent, in particular application in preparation of a Soybean root rot (Soybean root rot) control agent.

The Bacillus siamensis HT1 is separated from soybean rhizosphere soil in a soybean field of a teaching and scientific research base of Jilin agriculture university in Changchun city, Jilin province, the strain is delivered to a China Center for Type Culture Collection (CCTCC) for preservation, the preservation date is 2021 years, 12 months and 3 days, and the preservation number is CCTCC NO: m20211539, classified and named Bacillus siamensis HT1, address: wuhan university (430072) in Wuhan city, Hubei province, China. Colony characteristics: colonies were white, round, surface wrinkled, wet, protruding, opaque (FIG. 1).

The application of Bacillus siamensis HT1 comprises the preparation of the biocontrol microbial inoculum for controlling phytopathogen by using the strain, wherein the phytopathogen comprises but is not limited to: soybean rhizoctonia rot (Fusarium oxysporum), soybean Phytophthora (Phytophthora sojae), soybean Phomopsis longifola Hobbs) soybean Sclerotinia (sclerotiorum) corn microsporum (Bipolaris maydis), corn stalk rot (Fusarium graminearum), ginseng root rot (Fusarium solani) and tomato Botrytis cinerea (Botrytis cinerea).

Compared with the prior art, the invention has the following advantages: the Bacillus siamensis HT1 provided by the invention can effectively inhibit the growth of important pathogenic bacteria on soybeans, and has good inhibition effect. Avoiding the potential adverse effects of chemical control on the environment and crop safety.

Drawings

FIG. 1 is a schematic diagram showing the colony characteristics of HT1 strain (LB solid, 30 ℃, 18 h);

FIG. 2 is a scanning electron micrograph of HT1 strain (LB solid, 30 ℃, 18 h);

FIG. 3 is a phylogenetic tree of strain HT1 (based on the 16S rDNA sequence);

FIG. 4 is a growth curve of HT1 strain (LB liquid, 30 ℃, 48 h);

FIG. 5 is a schematic diagram showing the cultivation of HT1 strain against soybean root rot (PDA, 25 ℃,7 d);

FIG. 6 is a schematic diagram showing the cultivation of HT1 strain on a variety of pathogenic bacteria (PDA, 25 ℃,7 d);

FIG. 7 is a graph showing the inhibition effect of various concentrations of HT1 strain on soybean root rot (PDA, 25 ℃,7 d);

FIG. 8 is a curve of the inhibitory effect of different concentrations of HT1 strain on the growth of soybean root rot hypha (PDA, 25 deg.C, 7 d);

FIG. 9 is a photograph showing the effect of HT1 strain on the hyphal morphology of soybean root rot (PDA, 25 ℃,2 d);

FIG. 10 is a graph showing the inhibition effect of HT1 strain on soybean root rot at different temperatures (PDA, 25 ℃,7 d);

FIG. 11 is a graph showing the inhibition effect of HT1 strain on soybean root rot at different pH (PDA, 25 ℃,7 d);

Detailed Description

In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples. Technical schemes related to the embodiments of the present invention are all conventional schemes in the art if not specifically stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1: isolation and identification of Bacillus siamensis HT1(HT1 strain) and biological characteristic analysis

First, separation of strain and analysis of biological characteristics thereof

A soybean rhizosphere soil sample is collected from a Jilin agriculture university teaching and scientific research base (2888 of New City, Changchun, Jilin province). Placing 10g of the soil sample into a triangular flask containing 90mL of sterile water, and placing at 30 ℃ for 150r min^-1Shaking on shaking bed for 30min to mix thoroughly. Standing the sample for 10min, sucking 1mL of supernatant, and gradually diluting with sterile water to 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7Suspensions of gradient concentrations, 0.2mL of suspension per gradient, three replicates per concentration, were plated onto NA solid medium plates and incubated in an inverted position in a 28 ℃ incubator for 24 h. Selecting bacterial colonies with different forms, purifying the bacterial colonies on an LB solid culture medium by a plate marking method, numbering and storing the bacterial colonies after purification.

Respectively inoculating soybean root rot fungus cakes (fusarium oxysporum and phytophthora sojae) with the diameter of 5mm at the central position of a PDA solid culture medium, and inoculating purified bacterial strains as experimental groups at four positions which are about 20mm away from the fungus cakes on the PDA solid culture medium respectively from the upper part, the lower part, the left part and the right part of the fungus cakes; taking PDA solid culture medium inoculated with soybean root rot fungus cake as control. Repeating the treatment for 3 times, repeating the experiment for two times, performing inverted culture in a constant temperature box at 28 ℃, and observing whether the flat plate of the experimental group has a bacteriostatic zone or not when the flat plate of the control group is full. A strain with an obvious bacteriostatic zone is selected as a soybean root rot biological control strain and named as an HT1 strain, namely Bacillus siamensis HT 1.

The NA solid culture medium comprises: 30.0g of beef extract, 50.0g of peptone, 30.0g of sodium chloride, 20.0g of agar and 1000.0mL of distilled water.

The LB solid culture medium is: 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 20.0g of agar powder and 1000.0mL of distilled water.

The PDA solid culture medium is as follows: 200.0g of potato, 20.0g of glucose, 20.0g of agar powder and 1000.0mL of distilled water.

Secondly, morphological characteristics of the strain

The HT1 strain is cultured (LB solid medium, 30 ℃) for 18h, and the colony is white, round, wrinkled on the surface, wet, convex and opaque. (FIG. 1)

Thirdly, observing results by a scanning electron microscope

The HT1 strain is a rod-shaped bacterium with a non-smooth surface observed by an electron microscope. (FIG. 2)

Fourth, analysis of physiological and biochemical characteristics

The physiological and biochemical characteristics of the HT1 strain were determined with reference to "microbiological experiments" (Shen, Fanxuo, Li Guangwu. microbiological experiments (third edition); Beijing: advanced education Press, 1999.) and "Manual of identification of common bacterial systems" (Dongxu bead, Chuaimiao. Manual of identification of common bacterial systems; Beijing: scientific Press, 2011.).

The test result shows that the bacterial strain is negative in methyl red reaction, arginine double hydrolysis reaction, starch hydrolysis reaction, urease reaction and V-P reaction; gram, casein hydrolysis reaction, methionine hydrolysis reaction, maltose utilization, sucrose utilization and gelatin liquefaction are all positive; citrate is unavailable, nitrate is available; indole can be produced.

Fourth, classification attribute identification of HT1 Strain

HT1 strain DNA was extracted by bacterial genome DNA extraction kit (Tiangen Biochemical technology (Beijing) Ltd., Beijing, China).

The 16S rDNA gene was amplified using the Polymerase Chain Reaction (PCR) with the universal primers 27F (5'-AGAGTTGATCCTGCTCAG-3'), 1492R (5'-GTTACCTTTACGACTT-3'). 50 μ L amplification system: premix taq 25. mu.L, template (HT1 strain DNA) 5. mu.L, primers 2.5. mu.L each, and primer extension using ddH₂O make up to 50. mu.L. The PCR cycle parameters were: preheating at 95 deg.C for 5min, denaturing at 95 deg.C for 30s, annealing at 56 deg.C for 30s, extending at 72 deg.C for 1.5min, extending at 72 deg.C for 10min, and purifying by 1% agarose gel electrophoresis and gel recovery. Extracted by a kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Beijing, China) and then sequenced by Biotechnology Co., Ltd. (Shanghai, China). The 16S rDNA sequence of HT1 strain is shown in SEQ ID NO.1 by homology comparison of 16S rDNA sequence in Blast software Genbank. A phylogenetic tree of biological control bacteria is constructed, HT1 is determined to be Siamese Bacillus, and is named as Bacillus simensis HT1 (figure 3), and the Siamese Bacillus is stored in a refrigerator at the temperature of 80 ℃.

The strain is delivered to China center for type culture collection for preservation, and the preservation number is CCTCC NO: m20211539, classified and named Bacillus siamensis HT1, address: wuhan university, Wuhan district, Wuhan city, Hubei province, China.

Example 2: HT1 strain purification, fermentation culture and growth curve drawing

(1) Strain activation: taking out HT1 strain stored at-80 deg.C, thawing on ice, inoculating into LB liquid culture medium, and culturing at 28 deg.C for 150r min^-1The culture was carried out overnight for 12 h.

(2) Strain culture: the HT1 strain cultured in step (1) for 12h was transferred to a fresh LB solid medium, cultured in an incubator at 28 ℃ for 18h, and then stored at 4 ℃.

(3) And (3) fermenting the strains: inoculating the HT1 strain in the step (2) into LB liquid medium by using an inoculating loop, and inoculating at 150 r.min^-1Shaking culture at 28 ℃ until logarithmic phase (OD)₆₀₀0.5) to obtain a seed solution. Sucking 0.5mL of the seed solution, transferring into a 250mL triangular flask containing 100mL of LB liquid culture medium, and culturing at 150 r.min^-1Shaking-culturing at 28 deg.C for 3 days, centrifuging at 8000rpm for 5min, and adjusting the concentration of the centrifuged thallus to 1 × 10⁸cfu/mL to prepare the fermentation liquor. Used in the following examples.

The LB liquid culture medium is as follows: 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 1000.0mL of distilled water.

(4) Drawing of growth curves

The HT1 strain preserved at 4 ℃ in step (2) was inoculated into a 250mL Erlenmeyer flask containing 100mL LB liquid medium with an inoculating loop at 28 ℃ for 150 r.min^-1Lower culture to logarithmic phase (OD)₆₀₀0.5), a seed liquid was prepared. 0.5mL of the seed solution was transferred to 100mL of LB liquid medium, and the OD of the bacterial solution was measured every two hours₆₀₀Values, measured for 48h consecutively, were plotted for growth (FIG. 4).

Example 3: determination of bacterial inhibition spectrum of HT1 strain

The concentration prepared by the step (3) of example 2 was 1X 10⁸cfu/mL fermentation liquid, adding 0.5mL fermentation liquid into 100mL PDA solid culture medium to prepare PDA 1 bacterial liquid-containing PDA solid culture medium, after the PDA solid culture medium is solidified, respectively inoculating Fusarium oxysporum (Fusarium oxysporum) and Phytophthora sojae (P) with diameter of 5mm at central position of the culture mediumHypophthora sojae), Phomopsis longifolia Hobbs, Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) Zea mays (Bipolaris maydis), Sclerotinia mays (Fusarium graminearum), Panax ginseng (Fusarium solani), Botrytis cinerea (Botrytis cinerea) pathogenic bacterium block as treatment group. The control group was prepared by inoculating the above pathogen block with a diameter of 5mm at the center of PDA solid medium without HT1 bacterial liquid. Culturing at 28 deg.C for 7 days, observing antibacterial condition, and calculating inhibition rate. Each treatment was repeated 3 times and the experiment was repeated twice.

The PDA solid culture medium is as follows: 20.0g of glucose, 20.0g of agar powder, 200.0g of potato and 1000.0mL of distilled water.

Inhibition (%) - (colony diameter of control-colony diameter of treated group)/colony diameter of control × 100%.

After 7 days, the colony diameters of the control group and the treated group were measured. It can be shown that the HT1 strain has obvious inhibiting effect on various pathogenic bacteria. FIG. 5 shows the inhibition effect of HT1 strain on two pathogenic bacteria of soybean root rot, and Table 1 shows the inhibition rate data of HT1 strain on various pathogenic bacteria. Wherein A and B are Phytophthora sojae (Phytophthora sojae), and the inhibition rate of the HT1 strain to the Phytophthora sojae is 82.44%; c and D are Fusarium oxysporum (Fusarium oxysporum), and the inhibition rate of the HT1 strain is 80.91%; (wherein A, C is the control group and B, D is the treatment group). FIG. 6 shows the inhibition effect of HT1 strain on various pathogenic bacteria, wherein A and B are Phomopsis longicolla Hobbs, and the inhibition rate of HT1 strain is 57.89%; c and D are soybean Sclerotinia sclerotiorum (sclerotiorum), and the inhibition rate of the HT1 strain is 82.22%; e and F are corn small leaf spot (Bipolaris maydis), and the inhibition rate of the HT1 strain on the corn small leaf spot is 76.67%; g and H are corn stalk rot (Fusarium graminearum), and the inhibition rate of the HT1 strain is 72.22%; i and J are ginseng root rot (Fusarium solani), and the HT1 strain has 74.44% of inhibition rate. K and L are Botrytis cinerea (Botrytis cinerea), and the inhibition rate of the HT1 strain is 80.1% (wherein A, C, E, G, I, K is a control group, and B, D, F, H, J, L is a treatment group).

TABLE 1 inhibition of various pathogenic bacteria by HT1 strain

Serial number	Pathogenic bacteria	Inhibition ratio (%)
			1	Phytophthora sojae	82.44
2	Fusarium oxysporum	80.91
			3	Phomopsis longissima (L.) F.sp.	57.89
4	Sclerotinia rot of soybean	82.22
			5	Southern leaf blight of corn	76.67
6	Corn stalk rot	72.22
			7	Root rot of ginseng	74.44
8	Botrytis cinerea	80.1

Example 4: inhibition of soybean root rot by HT1 strain

The concentration prepared by the step (3) of example 2 was 1X 10⁸cfu/mL fermentation broth, adjusted to 1X 10 with sterile water⁴cfu/mL、1×10⁵cfu/mL、1×10⁶cfu/mL、1×10⁷cfu/mL、1×10⁸cfu/mL. Preparing a PDA solid culture medium containing the biological Control bacterium fermentation liquid according to the proportion of adding 0.5mL of the biological Control bacterium fermentation liquid into 100mL of the PDA solid culture medium, inoculating a fusarium oxysporum cake and a phytophthora sojae cake which grow well into the central position of the PDA solid culture medium to serve as a treatment group, inoculating the fusarium oxysporum cake and the phytophthora sojae which grow well onto the PDA solid culture medium which is not added with the biological Control bacterium fermentation liquid to serve as a blank Control group (Control), culturing for 7 days at 28 ℃, observing the bacteriostasis condition, and calculating the inhibition rate. Treatment was repeated 3 times and the experiment was performed twice.

The inhibition ratio (%) - (control colony diameter-treatment colony diameter)/control colony diameter X100%

After 7 days, the colony diameters of the control group and the treated group were measured. As shown in fig. 7 and table 2, the colony diameters of fusarium oxysporum and phytophthora sojae were decreased as the concentration of the bacterial liquid was increased. When the concentration of the bacterial liquid is 1 × 10⁴The inhibition rates of cfu/mL are 46.67% and 40.82%, respectively; when the concentration of the bacterial liquid is 1 × 10⁵The inhibition rates are 57.41 percent and 57.15 percent respectively when cfu/mL; when the concentration of the bacterial liquid is 1 × 10⁶The inhibition rates of cfu/mL are 69.26% and 67.90% respectively; when the concentration of the bacterial liquid is 1 × 10⁷The inhibition rates are 75.19 percent and 75.38 percent respectively when cfu/mL; the concentration of the bacterial liquid is 1 × 10⁸The inhibition rates at cfu/mL were 80.91% and 82.44%, respectively.

TABLE 2 inhibition of soybean root rot by HT1 strains at different concentrations

Example 5: inhibition effect of HT1 strain on growth amount of soybean root rot hypha

The concentration prepared by the step (3) of example 2 was 1X 10⁸cfu/mL fermentation broth, adjusted to 1X 10 with sterile water⁴cfu/mL、1×10⁵cfu/mL、1×10⁶cfu/mL、1×10⁷cfu/mL、1×10⁸cfu/mL. Preparing a PDA solid culture medium containing the biological Control bacterium fermentation liquid according to the proportion of adding 0.5mL of the biological Control bacterium fermentation liquid into 100mL of the PDA solid culture medium, inoculating a fusarium oxysporum cake and a phytophthora sojae cake which grow well into the central position of the PDA solid culture medium to serve as a treatment group, inoculating the fusarium oxysporum cake and the phytophthora sojae which grow well onto the PDA solid culture medium which is not added with the biological Control bacterium fermentation liquid to serve as a blank Control group (Control), culturing for 7 days at 28 ℃, scraping hyphae off by using a sterile blade, weighing and calculating the inhibition rate. Treatment was repeated 3 times and the experiment was performed twice.

The inhibition (%) was (%) (weight of control hyphae-weight of treated hyphae)/weight of control hyphae × 100%

After 7 days, the weight of the hyphae was measured for the control group and the treated group. As shown in fig. 8 and table 3, the weight of the mycelia of fusarium oxysporum and phytophthora sojae was decreased (Control) as the concentration of the bacterial liquid was increased. When the concentration of the bacterial liquid is 1 × 10⁴The inhibition rates are 40.75 percent and 34.75 percent respectively when cfu/mL; when the concentration of the bacterial liquid is 1 × 10⁵The inhibition rates of cfu/mL are 57.08% and 47.87%, respectively; when the concentration of the bacterial liquid is 1 × 10⁶The inhibition rates of cfu/mL are respectively 68.42 percent and 59.75 percent; when the concentration of the bacterial liquid is 1 × 10⁷cfu/mLThe inhibition rates are respectively 79.68% and 77.66%; the concentration of the bacterial liquid is 1 × 10⁸The inhibition rates at cfu/mL were 84.77% and 86.70%, respectively.

TABLE 3 inhibition rate of HT1 strains with different concentrations on growth of soybean root rot hypha

Example 6: effect of HT1 Strain on the hyphal morphology of Soybean root rot

The concentration prepared by the step (3) of example 2 was 1X 10⁸cfu/mL fermentation broth. Preparing a PDA solid culture medium containing the biocontrol bacteria fermentation liquid according to the proportion of adding 0.5mL of the biocontrol bacteria fermentation liquid into 100mL of the PDA solid culture medium, inoculating a fusarium oxysporum (figure 9A, C) and phytophthora sojae (figure 9B, D) cake with good growth into the center of the PDA solid culture medium to serve as a treatment group, simultaneously inoculating the fusarium oxysporum and the phytophthora sojae which have good growth onto the PDA solid culture medium without the biocontrol bacteria fermentation liquid to serve as a blank control, culturing for 2 days at 28 ℃, taking down the cake, placing under an optical microscope for observation, and observing the change of hypha morphology. Treatment was repeated three times and the experiment was performed twice.

Under the low power lens, the hyphae of the treated group and the control group are obviously different. Hyphae in the treated group had a tendency to grow upward, less tendency to grow all around, shorter hyphae, and dense (FIG. 9A, C). The control group had a more pronounced tendency to grow all around with longer hyphae and sparser hyphae (FIG. 9B, D).

Example 7: inhibition of soybean root rot by HT1 strain at different temperatures

The concentration prepared by the step (3) of example 2 was 1X 10⁸cfu/mL fermentation liquid, preparing a PDA solid culture medium containing the fermentation liquid of the biological control bacteria according to the proportion of adding 0.5mL fermentation liquid of the biological control bacteria into 100mL PDA solid culture medium, inoculating fusarium oxysporum and phytophthora sojae cakes which grow well in the center of the PDA solid culture medium as a processing group, and simultaneously adding the fermentation liquid of cfu/mL into the PDA solid culture mediumInoculating Fusarium oxysporum and Phytophthora sojae with good growth on PDA solid culture medium without addition of biocontrol bacteria fermentation broth as blank control, culturing at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, and 40 deg.C for 7 days, observing bacteriostasis condition, and calculating the inhibition rate of HT1 strain on soybean root rot. Each treatment was repeated three times.

Inhibition (%) (control group colony diameter-treatment group colony diameter)/control group colony diameter × 100%

After 7 days, the colony diameters of the control group and the treated group were measured. As shown in fig. 10 and table 4, the inhibition effect of the HT1 strain on soybean root rot shows a tendency of increasing first and then decreasing with increasing temperature, and when the temperature is 25 ℃, the inhibition effect of the HT1 strain on fusarium oxysporum and phytophthora sojae is maximized, and the inhibition rates are 81.35% and 82.90%, respectively.

TABLE 4 inhibition rate of HT1 strain on soybean root rot

Example 8: inhibition of soybean root rot by HT1 strain at different pH

The concentration prepared by the step (3) of example 2 was 1X 10⁸According to the proportion that 0.5mL of biocontrol bacteria fermentation liquor is added into 100mL of PDA solid culture medium, the PDA solid culture medium with the pH of 2, 3, 4, 5, 6, 7, 8, 9 and 10 and containing the biocontrol bacteria fermentation liquor is prepared, Fusarium oxysporum and Phytophthora sojae cakes with good growth are inoculated into the center of the PDA solid culture medium to serve as a treatment group, meanwhile Fusarium oxysporum and Phytophthora sojae which are not added into the PDA solid culture medium are inoculated into the PDA solid culture medium to serve as blank controls, the PDA solid culture medium is cultured for 7 days at 28 ℃, the bacteriostasis condition is observed, and the inhibition rate of the HT1 strain on the root rot of the soybean is calculated. Each treatment was repeated three times.

After 7 days, the colony diameters of the control group and the treated group were measured. As shown in fig. 11 and table 5, the inhibition effect of the HT1 strain on soybean root rot shows a tendency of increasing first and then decreasing with increasing pH, and when the pH is 7, the inhibition effect of the HT1 strain on fusarium oxysporum and phytophthora sojae is maximized, and the inhibition rates are 81.04% and 82.83%, respectively.

TABLE 5 inhibition rate of HT1 strain on soybean root rot pathogen at different pH

Example 10: pot culture prevention effect of HT1 strain fermentation liquor on soybean root rot

A sorghum grain inoculation method is adopted, Fusarium oxysporum (Fusarium oxysporum) and Phytophthora sojae (Phytophthora sojae) are used as tested pathogenic bacteria, and the prevention effect and the treatment effect of the biocontrol HT1 strain on soybean root rot are measured. Each 25 strains were treated in triplicate.

The pathogenic bacteria inoculum is mixed with sterile soil according to 4% of the weight of the potting soil (3kg) and potted, water is added to maintain the water content of 40% -60%, and the temperature is 25 +/-2 ℃. The soybean seeds (Ji soybean No. 1) are sown after surface disinfection, 9 seeds are sown in each pot, and after emergence, 5 seeds in each pot are reserved for thinning.

Preparation of HT1 Strain fermentation broth: the HT1 strain preserved in a refrigerator at the temperature of-80 ℃ is thawed on ice, and then is inoculated into an LB liquid culture medium, after overnight culture for 12h, the strain is inoculated onto an LB solid culture medium, and activation culture is carried out for 24h at the temperature of 30 ℃. Bacterial colonies of the strain were picked with an inoculating loop, inoculated into 100mL (250mL triangular flask) of LB medium at 30 ℃ for 150 r.min^-1Shake culturing for 18h, preparing HT1 strain fermentation broth, adjusting the final concentration of HT1 strain fermentation broth to 1 × 10 with sterile water⁸cfu/mL。

Preparation of pathogen inoculum: cooking sorghum grains, filling the sorghum grains into a 250mL conical flask, sterilizing the sorghum grains for 30min at 121 ℃, repeating the sterilization for 1 time, respectively inoculating 10 PDA to two pathogenic bacteria cakes cultured for 7 days after the sorghum grains are cooled, culturing the two pathogenic bacteria cakes for 20 days at 25 ℃ in the dark, and inoculating the pathogenic bacteria inoculum after the surfaces of the sorghum grains are full of hyphae for inoculation.

Method for measuring preventive effect: the fermentation broth of the HT1 strain was applied to the treated soil at an application rate of 30mL per pot. After being kept moist for 24 hours at 25 ℃, soybean seeds were sown in the treated soil, and each treatment was repeated three times. Meanwhile, a Control (CK) test is set, the morbidity situation is investigated on the 28 th day after treatment, the root and stem morbidity situation is investigated according to the soybean root rot grading standard, the total plant number and the diseased plant number are recorded, and the disease index and the prevention and treatment effect are calculated.

TABLE 6 Soybean root rot Classification Standard

Grading	Grading standards
			0	No disease spots on the base of the seedling stem and the main root
1	The base and the main root of the stem have a small number of scabs, and the area of the scabs is below 1/4
		2	The area of the lesion spots on the base part and the main root of the stem accounts for 1/4-1/2 of the total area of the base part and the main root of the stem
3	The area of the lesion spots on the base part and the main root of the stem accounts for 1/2-3/4 of the total area of the base part and the main root of the stem
		4	The lesion spots on the stem base and the main root are connected,the phenomenon of stem winding is formed, but the root system is not necrotic
5	Necrosis of the root system, wilting or death of the overground part

Determination of therapeutic effect: the soybean seeds are sown in the treated soil, after the soybean seeds are kept moist for 24 hours at 25 ℃, the HT1 strain fermentation liquor is irrigated in the treated soil at the application rate of 30mL per pot, and each treatment is repeated three times to serve as a treatment group. Meanwhile, a control group (CK) test is set, and the disease condition is investigated and the disease index and the prevention and treatment effect are calculated on the 28 th day after treatment.

After 28 days, the disease was investigated, and as can be seen from table 7, the preventive and therapeutic effects of HT1 strain on fusarium oxysporum were 79.31% and 62.63%, respectively. As can be seen from Table 8, the preventive and therapeutic effects of the HT1 strain on Phytophthora sojae were 79.17% and 62.21%, respectively. The HT1 strain can effectively prevent and treat soybean root rot caused by two pathogenic bacteria.

Table 7: data of experimental results on prevention and treatment effects of HT1 strain on fusarium oxysporum

Note: the data in the table are mean ± standard deviation; the lower case letters a, b indicate the significance of the difference at a P <0.05 level.

Table 8: data of experimental data on prevention and treatment effects of HT1 strain on phytophthora sojae

Claims

1. A biological control bacterium Bacillus siamensis HT1, which is preserved in China center for type culture collection with the preservation date of 2021 year, 12 months and 3 days, and the preservation number is CCTCC NO: m20211539, classified and named as Bacillus siamensis HT 1.

2. The application of the biocontrol bacterium Bacillus siamensis HT1 in the biocontrol microbial inoculum, which is characterized in that: the biocontrol bacterium Bacillus siamensis HT1 is used for preparing a biocontrol bacterium agent for controlling phytopathogen.

3. The application of the biocontrol bacterium Bacillus siamensis HT1 in the biocontrol bacterium agent as claimed in claim 2, which is characterized in that: the plant pathogenic bacteria are Fusarium oxysporum (Fusarium oxysporum), Phytophthora sojae (Phytophthora sojae), Phomopsis longicola (Phytopsis nigra Hobbs), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Microsporum maydis (Bipolaris maydis), Phoma maydis (Fusarium graminearum), Rhizopus ginseng (Fusarium solani) or Botrytis cinerea (Botrytis cinerea).