A strain of Bacillus HL-2 and its application in the prevention and control of anthrax in Oregon.

Abstract

This invention discloses a strain of Bacillus HL-2 and its application in the prevention and control of anthracnose in Okinawa. The Bacillus HL-2 provided by this invention has the preservation number GDMCC No. 66222; it possesses the ability to produce heparin, solubilize inorganic phosphorus and potassium, and fix nitrogen. It can secrete proteases, amylases, cellulases, and lipases, and can also inhibit the growth of various anthracnose pathogens. Therefore, it can be used to prevent and control anthracnose in Okinawa and has significant application value in the field of mangrove conservation.

Description

Technical Field

[0001] This invention belongs to the field of microbiology technology, specifically relating to a strain of Bacillus HL-2 and its application in the prevention and control of anthrax in Oligocene. Background Technology

[0002] In recent years, affected by global climate change and human activities, mangrove diseases have become increasingly frequent, posing a serious threat to the overall health of mangrove ecosystems and profoundly impacting the ecological security of coastal areas. Anthracnose, in particular, has attracted significant attention due to its wide host range and highly destructive nature. It commonly causes leaf spots, withering and necrosis of young branches in various mangrove species, including *Avicennia marina*, *Avicennia apetalis*, and *Excoecaria agallocha*, severely affecting the normal growth and function of mangrove plants. Currently, research on the control of anthracnose pathogens in mangroves is scarce.

[0003] Traditional control methods face numerous problems, including high costs and difficulty in implementation. Biological control, as a highly safe natural control method, is the primary approach for mangrove disease control. Bacillus, with its advantages of rapid reproduction, broad antibacterial spectrum, and strong resistance, is an important group of biocontrol microorganisms. Currently, no technologies utilizing Bacillus to control mangrove diseases have been reported. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide a strain of Bacillus (… Bacillus sp.) HL-2 (the preservation number of this bacterium is: GDMCC No.66222, the preservation date is: April 27, 2025, the preservation unit is: GDMCC-Guangdong Provincial Microbial Culture Collection Center) and its application in the prevention and control of anthrax in Oligocene.

[0005] The first objective of this invention is to provide a strain of Bacillus ( Bacillus sp.)HL-2, with accession number GDMCC No.66222.

[0006] A second objective of this invention is to provide a biological agent containing a culture of the aforementioned Bacillus HL-2 as an active ingredient.

[0007] Preferably, the concentration of Bacillus HL-2 culture in the formulation is OD0.05. 600 =0.3 or above.

[0008] Preferably, the concentration of Bacillus HL-2 culture in the formulation is OD0.05. 600 =0.6 or above.

[0009] Preferably, the culture of Bacillus HL-2 is obtained by inoculating Bacillus HL-2 on PDA medium, LB medium or KMB medium and culturing it.

[0010] A third object of the present invention is to provide the use of the said Bacillus HL-2 or the said preparation in at least one of the following (1)-(2):

[0011] (1) Inhibits the growth of plant anthracnose pathogens;

[0012] (2) Promote plant growth.

[0013] Preferably, the anthrax pathogen is *Discocephalus endophyticus* (…). Colletotrichum endophyticum HL(ML)-YB-2A, Karst anthrax bacteria ( Colletotrichum karstii ML-HB-6 or Tropical Anthrax ( Colletotrichum tropicale HQ-YB-1.

[0014] Preferably, the plant growth promotion is achieved by decomposing inorganic phosphorus, potassium, fixing nitrogen, producing ironophiles, and / or secreting extracellular enzymes.

[0015] Preferably, the extracellular enzyme is a cellulase, protease, lipase, and / or amylase.

[0016] A fourth object of the present invention is to provide a method for preventing and treating anthrax in Rhizoctonia rubra, comprising the step of applying the Bacillus HL-2 or the preparation thereof to Rhizoctonia rubra.

[0017] The beneficial effects of this invention are:

[0018] The culture of Bacillus HL-2 of the present invention has an inhibitory effect on the pathogenic fungus of anthrax in mangroves and can prevent and control anthrax in olive trees; it has the ability to produce iron, solubilize inorganic phosphorus, potassium and fix nitrogen, and can secrete protease, amylase and cellulase.

[0019] The Bacillus HL-2 of this invention is of great significance for the prevention of diseases in the mangrove plant *Rhizophora stylosa*, and has important application value in the construction and protection of coastal protection systems.

[0020] Preservation Instructions

[0021] The present invention Bacillus sp. HL-2 (Bacillus HL-2) was deposited on April 27, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 66222. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0022] Figure 1The images show the colony morphology and Gram staining of strain HL-2 on PDA, LB, and KMB media; where A is the colony morphology of strain HL-2 on PDA medium, B is the colony morphology of strain HL-2 on LB medium, C is the colony morphology of strain HL-2 on KMB medium, and D is the Gram staining of strain HL-2.

[0023] Figure 2 The activity of strain HL-2 was determined.

[0024] Figure 3 Phylogenetic tree analysis of 16S rRNA from Bacillus HL-2.

[0025] Figure 4 The spectrum of Bacillus HL-2 is shown; where A represents the antagonistic effect of Bacillus HL-2 against different anthrax pathogens, and B represents the radial growth inhibition rate of Bacillus HL-2 against different anthrax pathogens.

[0026] Figure 5 The study compared the efficacy of different concentrations of Bacillus HL-2 in controlling anthrax in Oligocene; where A represents the efficacy of Bacillus HL-2 in controlling anthrax. C. endophyticum The control effect of HL(ML)-YB-2A, B is the effect of Bacillus HL-2 on C. karsti The control effect of ML-HB-6, C represents Bacillus HL-2 against C. tropicale The preventive and control effects of HQ-YB-1. Detailed Implementation

[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0028] Example 1: Bacillus ( Bacillus Isolation and Identification of HL-2 (sp.)

[0029] 1. Isolation of strain HL-2

[0030] The strain HL-2 was isolated from the rhizosphere soil of *Rhizophora stylosa* in a mangrove nature reserve.

[0031] 2. Identification of strains

[0032] 2.1 Morphological identification

[0033] Colony morphology and size were observed according to the methods described in the "Manual of Systematic Identification of Common Bacteria" and the "Microbiology Laboratory Manual", and physiological and biochemical characteristics were tested at the same time.

[0034] The culture medium used for the characterization of the above-mentioned strain HL-2 was prepared as follows:

[0035] Potato Dextrose Agar (PDA) medium: Take 200 g peeled potatoes, 20 g glucose, and 15 g agar powder, add them to deionized water to adjust the total volume to 1000 mL, pH 7.0-7.2. After sterilization, cool and pour into plates for later use.

[0036] Selection medium (LB medium): Take 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar powder, add them to deionized water to adjust the total volume to 1000 mL, pH 7.0-7.2. After sterilization, cool and pour into plates for later use.

[0037] King's Medium (KMB): Take 20 g peptone, 20 g glucose, 1.5 g anhydrous magnesium chloride, 1.5 g dipotassium hydrogen phosphate, and 15 g agar powder, add them to deionized water to adjust the total volume to 1000 mL, pH 7.0-7.2. After sterilization, cool and pour into plates for later use.

[0038] On PDA medium, strain HL-2 colonies are pale yellow, flat, and have intact edges. Figure 1 (A) On LB medium, the colonies are pale yellow, flat, and have radially diffused edges. Figure 1 (B in the text); on KMB medium, the colonies are yellowish-white, wrinkled, and have radial diffusion at the edges ( Figure 1 (C in the middle).

[0039] The results of its physiological and biochemical characterization showed that strain HL-2 belongs to Gram-positive bacteria. Figure 1 D in the sample can oxidize glucose, utilize xylose, arabinose, mannitol, and citrate, and has the ability to hydrolyze starch, reduce arginine, and reduce nitrate, as well as produce gelatinase and amylase; VP is negative, cannot utilize propionate, and cannot grow under anaerobic conditions, 7% sodium chloride, and pH 5.7 (Table 1).

[0040] The results of its active substance detection showed that strain HL-2 has the functions of solubilizing inorganic phosphorus, potassium, and fixing nitrogen, but not organic phosphorus. It can secrete proteases, amylases, cellulases, and lipases, and has the ability to produce heparin. Figure 2 ).

[0041] Table 1. Physiological and biochemical results of strain HL-2

[0042]

[0043] Note: "+" indicates a positive result; "-" indicates a negative result.

[0044] 2.2 Molecular biological identification

[0045] The 16S rRNA gene sequence was used to identify strain HL-2 molecularly. Colony PCR amplification of the target gene fragment was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). The amplified products were sequenced by Sangon Biotech (Shanghai) Co., Ltd.

[0046] The sequencing results were corrected and assembled using Bioedit and DNAman software to obtain the 16S rRNA nucleotide sequence of strain HL-2 (as shown in SEQ ID NO.3). The sequence was then aligned using BLAST in the GenBank database, downloaded, and aligned again using ClustalW in MEGA-X software. A phylogenetic tree was constructed using the neigbor-joining method for analysis. A bootstrap test was performed during phylogenetic tree construction, with 1000 repeated samplings. The phylogenetic tree is shown below. Figure 3 .

[0047] from Figure 3 It can be seen that strain HL-2 and Bacillus velezensis Clustered in the same branch, and based on the results of morphological identification and 16S rRNA sequence analysis, strain HL-2 was named Bacillus (Bacillus). Bacillus sp.)HL-2.

[0048] Example 2: Determination of the antibacterial effect of Bacillus HL-2 against anthrax in mangroves

[0049] The tested anthrax pathogen strain: *Discocephalus endophyticus* ( Colletotrichum endophyticum HL(ML)-YB-2A, Karst anthrax bacteria ( Colletotrichum karsti ML-HB-6 was isolated from diseased plants of *Rhizophora stylosa*, and *Anthracnose* (*Bryophyllum typhimurium*) was also isolated. Colletotrichum tropicale HQ-YB-1 was isolated from diseased plants of Echeveria elegans.

[0050] The antagonistic effect of Bacillus HL-2 on the tested anthrax pathogen strain was determined using the plate confrontation method. Treatment group: A small amount of Bacillus HL-2 was streaked (2.0 cm long) onto a PDA plate and incubated at 30℃ for 1 day. Then, an anthrax pathogen mycelium was placed 2.5 cm away from the HL-2 inoculation site and incubated upside down at 30℃. A blank control group was prepared by inoculating the tested anthrax pathogen mycelium onto a blank PDA plate, with three replicates. After incubation at 30℃, the distances R1 and R2 were measured, and the percentage inhibition of radial growth (PIRG) was calculated.

[0051] The formula for calculating the radial growth inhibition rate of colonies is: PIRG (%) = [(R1-R2) / R1] × 100; where: R1 represents the distance between the inoculation point of the pathogen in the blank control group and the radial growth along the edge of the plate; R2 represents the distance between the inoculation point of the pathogen in the treatment group and the radial growth along the direction of the antagonistic bacteria.

[0052] Table 2. Inhibition rate of Bacillus HL-2 against mangrove anthracnose pathogens

[0053]

[0054] Note: SD stands for standard deviation. Lowercase letters in each treatment group indicate statistical significance (p<0.05).

[0055] The test results showed that Bacillus HL-2 had a good inhibitory effect on different anthrax pathogens, all reaching more than 60%, and could effectively inhibit the growth of anthrax pathogens (Table 2). Figure 4 ).

[0056] Example 3: Pathogenicity determination of different pathogens to Rhododendron simsii

[0057] According to Koch's postulates, pathogenicity was determined using an in vitro inoculation method.

[0058] Healthy leaves of similar size from current-year shoots of *Rhizophora stylosa* were selected. The surface was repeatedly rinsed with tap water, disinfected with a 2% sodium hypochlorite solution for 30 seconds, rinsed three times with sterile water, and then air-dried for later use. Mycelial cakes (5 mm diameter) were taken from the edges of colonies of a strain cultured on PDA agar plates for 7 days using a punch. A puncture inoculation method was used, creating a wound on the leaf at the inoculation site using a sterile needle. The mycelial side of the pathogenic fungal cake was applied to the puncture site. A blank PDA agar plate was applied to the puncture site as a control. The leaves were incubated in a light-controlled incubator with humidity (light conditions: 80% light intensity, 80% relative humidity, 28℃, 12 h; darkness conditions: 0 light intensity, 80% relative humidity, 28℃, 12 h). Leaf disease development was observed daily. Once lesions appeared on the leaves, the diseased tissue was isolated and cultured, and morphological and molecular identification was performed to determine if it was consistent with the original inoculated strain. Each treatment has 3 replicates, and each replicate has 1 leaf.

[0059] The pathogenicity test results (Table 3) show that all tested anthrax pathogens exhibited certain pathogenicity against *Rhizoctonia solani*. Among them, strains... C. endophyticum HL(ML)-YB-2A, C. Karsti ML-HB-6 and C. tropicaleHQ-YB-1 caused lesions exceeding 10 cm² on the leaves of *Rhizophora stylosa* within 10 days after inoculation. 2 (Table 3) shows that it has strong pathogenicity. Therefore, anthrax pathogens were subsequently selected. C. endophyticum HL(ML)-YB-2A, C. karsti ML-HB-6 and C. tropicale HQ-YB-1 is undergoing efficacy testing.

[0060] Table 3. Pathogenicity of different pathogens to red sea olive

[0061]

[0062] Note: Data are expressed as mean ± standard deviation, and lowercase letters in each treatment group indicate statistical significance (p<0.05).

[0063] Example 4: Determination of the control effect of Bacillus HL-2 on anthrax in Oligocene

[0064] The control effect of Bacillus HL-2 on anthrax in Rhodotorula rubra was tested using an in vitro indoor experiment.

[0065] (1) Select activated Bacillus HL-2 and inoculate it into a 250 mL Erlenmeyer flask containing 200 mL of LB liquid medium. Place the flask in a shaker at 30°C and 200 rpm for 15 h. After the culture is complete, adjust the bacterial solution to an OD concentration. 600 =0.3 is the treatment group I, and the concentration OD is adjusted to 0.3. 600 =0.6 represents treatment group II, and the diluted Bacillus HL-2 bacterial solution was obtained for later use.

[0066] (2) Culture highly pathogenic anthrax pathogens on PDA agar plates. C. endophyticum HL(ML)-YB-2A, C. karsti ML-HB-6 C. tropicale HQ-YB-1, incubate at 25℃ for 5 days, then use a 5 mm punch to create mycelial cakes for later use.

[0067] (3) Cut healthy, disease-free leaves of red olive, rinse them repeatedly with running water, place them in a clean bench and soak them in a sodium hypochlorite solution with a solute mass fraction of 2% for 30 seconds, rinse them three times with sterile pure water, and dry them. Seal the base of the petiole with wax to prevent moisture loss and set aside for later use.

[0068] (4) Wound inoculation was performed on the leaves of the red olive after step (3). The leaves of the red olive were soaked in the diluted bacterial solution of Bacillus HL-2 prepared in step (1) for 45 s. After being incubated in an incubator at 28℃ for 24 h, the pathogenic bacterial cake from step (2) was inoculated into the wound of the red olive leaf.

[0069] Red olive leaves were soaked in sterile LB liquid medium for 45 s and incubated at 28°C for 24 h. The anthracnose pathogen mycelium from step (2) was inoculated at the wound site as a positive control group; blank PDA medium was inoculated at the wound site as a negative control group. Three biological replicates were set up for each group (each replicate contained 10 leaves).

[0070] All treated Rhizophora stylosa leaves were placed in petri dishes containing sterile, water-moistened filter paper and incubated in a light incubator (light conditions: 80% light intensity, 80% relative humidity, 28℃, 12 h; darkness conditions: 0% light intensity, 80% relative humidity, 28℃, 12 h). Observations were made daily, and the disease incidence and lesion size were measured and recorded. The incidence rate was calculated, and the disease index and control effect were calculated according to the grading standards.

[0071] The grading standard is based on whole leaves. The severity grading standard for anthracnose in *Opuntia ficus-indica* is as follows: Grade 0: No symptoms on leaves; Grade 1: Lesions on affected leaves with a lesion area of ​​less than 2 cm². 2 Level 2 refers to lesions on diseased leaves with an area of ​​2 cm². 2 -4 cm 2 Level 3 indicates diseased leaves with lesions ranging in size to 4 cm². 2 -6 cm 2 Level 4 indicates that the lesion area on the affected leaves is between 6 cm². 2 -8 cm 2 Level 5 indicates diseased leaves with lesions ranging in size to 8 cm². 2 -12 cm 2 Level 6 is 12 cm. 2 The leaves above show signs of disease.

[0072] The calculation formula is as follows:

[0073] Incidence rate (%) = (Number of diseased leaves in each treatment group / Total number) × 100;

[0074] Disease severity index (%) = [∑(Disease level × Number of cases at that disease level) / (Highest disease level × Total number)] × 100;

[0075] Prevention and control effect (%) = [(positive control disease index - treatment group disease index) / positive control group disease index] × 100.

[0076] Table 4. Control efficacy of Bacillus HL-2 against anthrax in Oregon.

[0077]

[0078] The results showed that the incidence and disease index of treatment groups I and II were significantly lower than those of the positive control. The spread of disease was not significant in either treatment groups I or II. The control effect of treatment group II was over 60%, with treatment group II showing better control than treatment group I. These results indicate that Bacillus (… Bacillus HL-2 (sp.) can effectively reduce the incidence and severity of anthrax in Oligocene, demonstrating good biocontrol effects (Table 4). Figure 5 ).

Claims

1. A strain of Bacillus ( Bacillus sp.) HL-2, characterized in that, and has a preservation number of GDMCC No. 66222.

2. A biological preparation containing Bacillus sp. HL-2, characterized in that, A culture containing the Bacillus sp. HL-2 of claim 1 as an active ingredient.

3. The preparation according to claim 2, characterized in that, The concentration of the culture of Bacillus HL-2 in the preparation is OD 600 = 0.3 or more.

4. The preparation according to claim 3, characterized in that, The concentration of the culture of Bacillus HL-2 in the preparation is OD 600 = 0.6 or more.

5. The formulation of claim 2, wherein, The culture of the Bacillus sp. HL-2 is obtained by inoculating the Bacillus sp. HL-2 into a PDA medium, a LB medium or a KMB medium and culturing.

6. A method of controlling glomerella cingulata of red sea grapes, characterized by, The method comprises the step of applying the Bacillus sp. HL-2 of claim 1 or the preparation of claim 2 to Rhizophora. The method comprises the step of applying the Bacillus sp. HL-2 of claim 1 or the preparation of claim 2 to Rhizophora.

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