A paenibacillus-like bacterial strain and use thereof

Abstract

The application provides a Paenibacillus-like strain and application thereof, the Paenibacillus-like strain is preserved in Guangdong Microbial Culture Collection Center on August 4, 2025, and the preservation number is GDMCC No: 66801; the application is to prepare a preparation comprising the Paenibacillus-like strain, a preparation for preventing or inhibiting plant pathogenic fungi from invading plants; the Paenibacillus-like strain can effectively and durably inhibit the growth of plant pathogenic fungi, can be used as a broad-spectrum antibacterial agent, enhances the resistance of plants, and promotes the growth of plants.

Description

Technical Field

[0001] This invention relates to a Bacillus strain and its application, belonging to the field of microbial technology. Background Technology

[0002] Plant fungal diseases are caused by plant pathogenic fungi. They account for approximately 70-80% of all plant diseases. Several or even dozens of fungal diseases can be found on a single crop.

[0003] Fungal diseases are the most numerous known diseases, exhibiting a wide variety of symptoms and appearing on various parts of the plant. The characteristics of the fungal infection on a plant are called symptoms, which are the primary basis for diagnosing whether a disease is fungal. The pathogens of fungal diseases can be spread through spores, which can be dispersed in the air or by rainwater and insects. Fungi germinate under suitable temperature and humidity conditions, invade the plant, and cause disease. Generally, high temperature and high humidity environments are conducive to the occurrence and spread of fungal diseases. Examples of fungal diseases include: rot, anthracnose, ring spot, powdery mildew, black spot, and dry rot.

[0004] Current control methods include agricultural control, biological control, and chemical control, each with its own advantages and disadvantages. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the first objective of this invention is to provide a Bacillus-like strain that can effectively and persistently inhibit the growth of plant pathogenic fungi, and can be used as a broad-spectrum antibacterial agent to enhance plant resistance and promote plant growth.

[0006] The second objective of this invention is to provide an application of the above-mentioned Bacillus strain, which is to use the Bacillus strain to prepare a formulation to realize the function of the strain.

[0007] The first objective of this invention can be achieved by adopting the following technical solution: a strain of the genus Bacillus, classified and named Paenibacillus This strain of Bacillus sp. was deposited on August 4, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66801.

[0008] Furthermore, the 16S rDNA of the Bacillus spp. strain is shown in SEQ ID NO.1.

[0009] The second objective of this invention can be achieved by adopting the following technical solution:

[0010] The application of a strain of the genus Bacillus, for the preparation of preparations containing strains of the genus Bacillus.

[0011] Furthermore, the preparation is a preparation for preventing or inhibiting plant pathogenic fungi from infecting plants.

[0012] Furthermore, the fungus is at least one of *Colletotrichum gloeosporioides* and *Colletotrichum candida*.

[0013] Furthermore, the plant is at least one of the following: green beans, melons, bananas, sweet oranges, apples, pears, grapes, and peppers.

[0014] Furthermore, the formulation is applied to the seeds or planting substrate of plants.

[0015] Furthermore, the formulation is prepared by inoculating a Bacillus spp. strain into a fermentation substrate for fermentation, and then drying the obtained fermentation broth.

[0016] Furthermore, the concentration of the fermentation broth is 7 × 10⁻⁶. 6 -9×10 6 cfu / mL.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. The Bacillus strain of the present invention can be used to inhibit plant pathogenic fungi, and can be used as a broad-spectrum antibacterial agent to enhance plant resistance and promote plant growth;

[0019] 2. The Bacillus strains of the present invention can be used to prepare reagents that can be applied to plants to prevent or inhibit plant pathogenic fungi. Attached Figure Description

[0020] The Bacillus strains involved in this invention are classified and named as follows: Paenibacillus This strain of Bacillus sp. was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2025, with accession number GDMCC No: 66801.

[0021] Figure 1 The colony morphology of strain HDS3-Ba4;

[0022] Figure 2 Microscopic morphology of HDS3-Ba4 strain;

[0023] Figure 3 The growth curve of HDS3-Ba4;

[0024] Figure 4-5 Photos of the antibacterial test of Fusarium oxysporum tomato at CK and 4 days, respectively;

[0025] Figure 6-7 These are photos of the antibacterial test of *Colletotrichum gloeosporioides* CK and 4 days after its initial release.

[0026] Figure 8-9 Photographs of the antibacterial test of *Coccidioidomyces chinensis* CK and 4d, respectively;

[0027] Figure 10-11 These are photos of the antibacterial test of Fusarium moniliforme CK and 8 days, respectively;

[0028] Figure 12-13 Photos of the antibacterial test of Helicobacter pylori at CK and 8 days, respectively;

[0029] Figure 14-15 Photos of the antibacterial test of Curvularia esculenta (CK) and 10 days after its initial growth, respectively;

[0030] Figure 16-17 These are photos of the antibacterial test of *Russula decipiens* CK and 10 days after exposure to dew. Detailed Implementation

[0031] The present invention will now be further described with reference to the accompanying drawings and specific embodiments:

[0032] Example 1:

[0033] A strain of the genus Bacillus was obtained by the following method:

[0034] 1) Isolation and purification of the strain: After sampling, fermentation broth was prepared. 100 μL of the sample solution was added to 900 μL of sterile water, and then the sample was diluted to 10 μL. -4 10 -5 The concentration was determined by incubating the medium in a 70°C water bath for 20 minutes. 100 μL of the resulting liquid was then spread onto NA plates and incubated at 28°C for 72 hours. Bacterial growth was observed on the plates. Once colonies appeared, typical single colonies were streaked onto NA solid medium for isolation and purification. Single colonies were inoculated into 5 mL of sterilized NB broth and incubated at 28°C for 72 hours. The culture was then stored at -80°C with 50% v / v glycerol for later use.

[0035] The isolated and purified single colonies were identified using the following primers:

[0036] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2)

[0037] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3);

[0038] The 16S rDNA sequence of the Bacillus spp. strain HDS3-Ba4 was obtained, as shown in SEQ ID NO.1, and named as Bacillus spp. strain HDS3-Ba4.

[0039] 2) The sequence was compared and analyzed using the EZBioCloud database. The results showed that the HDS3-Ba4 strain was closely related to known type strains in the database. Paenibacillus ginsengarvi KCTC 13059 T The homology is 97.48%, which is suspected to be... Paenibacillus A new species of the genus Bacillus.

[0040] 3) Upload the genome of the target strain to the EzBioCloud website in South Korea. Download the genome sequence of a type strain with high similarity and use the EzBioCloud online tool ANICalculator to calculate the ANI value, using 95-96% as the cutoff value to determine whether the strains are different species. Upload the genomes of the target strain and related type strains to the online tool Genome-to-Genome Distance Calculator 3.0 on the German Culture Collection website, using 70% as the cutoff value, and calculate the dDDH value to determine whether the strains are different species.

[0041] Table 1 Calculation of ANI and dDDH in the HDS3-Ba4 genome

[0042]

[0043] The genome of HDS3-BA4 and closely related type strains Paenibacillus ginsengarvi KCTC 13059 T In comparison, the ANI value is 72.82%, which is less than the threshold of 95%, and the dDDH value is 22.30%, which is less than the threshold of 70%. Therefore, HDS3-Ba4 is a new species of Bacillus spp., named Bacillus spp. Paenibacillus sp.)HDS3-Ba4. Figure 1 This refers to the colony morphology of the bacterial strain. Figure 2 It is in microscopic form.

[0044] 4) Determination of the growth curve of HDS3-Ba4: The bacterial culture in the cryopreservation tube was inoculated into NB medium at an inoculation rate of 2% v / v and cultured in an incubator at 30℃ for 72 h. After two subcultures, the bacteria were washed twice by centrifugation at 5000 r / min for 5 min with PBS buffer. Then, the OD 600 nm of the strain was adjusted to 1.0 with PBS buffer. The strain was then inoculated into NB medium at an inoculation rate of 2% v / v and cultured in a growth curve analyzer at 30℃. The growth curve of HDS3-Ba4 was measured every 10 minutes to obtain the growth curve (3 replicates).

[0045] like Figure 3As shown, HDS3-Ba4 took 14.5 hours to grow to an OD of 1.0 at 600 nm and 30 hours to reach a stable state.

[0046] Detection:

[0047] 1) Antibacterial effect detection of HDS3-Ba4 strain:

[0048] The bacterial culture in the cryopreservation tube was inoculated into NB medium at an inoculation rate of 2% v / v and cultured at 30°C for 72 h. After two subcultures, the bacteria were washed twice by centrifugation at 5000 r / min for 5 min with PBS buffer. Then, the OD600nm of the strain was adjusted to 1.0 with PBS buffer. The strain was then inoculated into NB medium at an inoculation rate of 2% v / v and cultured at 30°C for 30 h. The resulting fermentation broth was then used for further processing.

[0049] Add 100 μL of fermentation broth of strain HDS3-Ba4 to each of the four wells. Finally, place a uniformly sized bacterial block in the center of the solidified plate and incubate at 30°C for 1-10 days, observing whether the strain grows.

[0050] Prepare mycelial blocks of pathogenic fungi such as Fusarium oxysporum tomatoense, Colletotrichum gloeosporioides, Coccidioides praecox, Fusarium solani, Helicobacter spp., Curvularia zedoaria, and Lactotrichum dewys and repeat the experiment according to the above method.

[0051] Table 2. Antibacterial rate (%) of HDS3-Ba4 in antibacterial experiment.

[0052]

[0053] Figure 4-17 The images show the control (CK) and inhibitory tests of various pathogens, including *Fusarium oxysporum* (4 days), *Colletotrichum gloeosporioides* (4 days), *Coccidioidomyces coccidioidomyces* (4 days), *Fusarium oxysporum* (8 days), *Helicobacter pylori* (8 days), *Curvularia esculenta* (10 days), and *Eucalyptus globulus* (10 days).

[0054] 2) The control effect of HDS3-Ba4 strain on pathogenic bacteria infecting detached green bean leaves:

[0055] Prepare the HDS3-Ba4 strain suspension for later use. Inoculate the strain into NB medium at an inoculation rate of 2% v / v and incubate at 30°C for 17 h. Prepare the fermentation broth for later use.

[0056] The pathogenic fungus *Colletotrichum gloeosporioides* was inoculated onto PDA medium and cultured at 30°C for 3 days. Mycelial blocks from the colony edges were collected using an inoculation spatula as inoculum. Isolated beans were immersed in fermentation broth for half an hour, then removed, the leaf roots wrapped with moistened cotton balls, and placed in disposable petri dishes lined with moistened cotton balls. After standing for 24 hours, the pathogen was inoculated using a three-needle puncture treatment. *Colletotrichum gloeosporioides* mycelial blocks were inoculated onto the puncture sites of the isolated leaves, and cultured at 30°C for 11 days to observe disease progression. HDS3-Ba4 showed a significant control effect of 93.84% against *Colletotrichum gloeosporioides* infection in isolated beans. The same detection procedure was used against the pathogen *Colletotrichum candida*, with a control effect of 95.36%.

[0057] Table 2 shows that the HDS3-Ba4 strain exhibits good antifungal effects, particularly against *Colletotrichum gloeosporioides* and *Colletotrichum candida*, compared to other fungi. Significant effects were observed within 4 days, and sustained inhibitory activity was observed after 10 days. Based on the in vitro infection experiment on detached bean leaves, it is believed that the HDS3-Ba4 strain can be used to prepare agricultural products, especially those susceptible to both anthracnose and anthracnose rot, such as bean, providing targeted inhibition.

[0058] Example 2:

[0059] Application of HDS3-Ba4 strain preparations:

[0060] The HDS3-Ba4 strain was inoculated into a fermentation substrate and cultured at 28-32℃ for 60-84 h, yielding a fermentation broth concentration of 7×10⁻⁶. 6 -9×10 6 The concentration of cfu / mL is obtained by drying the sample.

[0061] Testing the antibacterial efficacy of plant antibacterial agents:

[0062] Experimental Group 1: The antibacterial agent powder was used to coat the seeds. The antibacterial agent and the bean seeds could be mixed at a mass ratio of 1:60-100 before sowing.

[0063] Experimental Group 2: Antibacterial powder was sprinkled into the substrate soil for sowing, with a mass ratio of antibacterial agent to substrate of 1:40-80.

[0064] Control group: No treatment was performed.

[0065] Experimental group 1, experimental group 2, and the control group were placed in the same experimental field. Field monitoring required at least 3 quadrats, with 100 plants sampled each time. Field cultivation and other planting management were uniform and consistent. No disease control was carried out throughout the entire growth period. The use of pesticides for pest control should comply with general regulations.

[0066] During the pod-setting stage of common beans, the control group showed symptoms of anthracnose and / or anthracnose. The incidence rates of the control group, experimental group 1, and experimental group 2 were statistically analyzed, and the disease index and control effect were calculated.

[0067] Grading method: Grade 0, no lesions; Grade 1, scattered lesions on pods; Grade 3, small lesions on pods, 1-2 mm long; Grade 5, large lesions on pods, 2.1-5.0 mm long; Grade 7, large lesions on pods, more than 5.1 mm long; Grade 9, most lesions on pods are connected, and the pods are rotten.

[0068] Incidence rate = (Number of cases / Total number of surveyed samples) × 100%;

[0069] Disease index = [∑(number of diseased plants at each level × corresponding level value)] / (total number of plants surveyed × highest disease level) × 100%;

[0070] Relative efficacy = (disease index in control area - disease index in experimental area) / disease index in control area × 100%.

[0071] Table 3. Field incidence rate, disease index, and relative control efficacy (%) of plant antibacterial agents against anthracnose / charcoal rot in common beans.

[0072]

[0073] This antibacterial agent can play a preventive role in crop cultivation. It can be diluted and applied by spraying, dipping, and / or direct powdering to the planting substrate, seeds, seedlings, leaves, and fruits.

[0074] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A type of Bacillus genus ( Paenibacillus strain (sp.), characterized in that, The Bacillus strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2025, with accession number GDMCC No: 66801.

2. The Bacillus strain as described in claim 1, characterized in that, The 16S rDNA of the Bacillus strain is shown in SEQ ID NO.

1.

3. The application of a Bacillus strain as described in claim 1, characterized in that, The application is for preparing formulations containing strains of the genus Bacillus.

4. The application as described in claim 3, characterized in that, The preparation is an agent for preventing or inhibiting plant pathogenic fungal infection of plants; the fungus is at least one of *Colletotrichum gloeosporioides* and *Colletotrichum candida*; the plant is *Colletotrichum candida*.

5. The application as described in claim 4, characterized in that, The preparation is applied to the seeds or planting substrate of plants.

6. The application as described in claim 3, characterized in that, The preparation is obtained by the following method: inoculating a Bacillus spp. strain into a fermentation substrate for fermentation, and drying the obtained fermentation broth.

7. The application as described in claim 6, characterized in that, The concentration of the fermentation broth is 7×10⁻⁶. 6 -9×10 6 cfu / mL.

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