A new broad-spectrum disease-resistant Bacillus species, bacterial-drug compound and its application

Abstract

The present invention relates to the field of microbial technology, and specifically discloses a new broad-spectrum disease-resistant Bacillus species, a fungicide compound, and applications thereof. The new broad-spectrum disease-resistant Bacillus species is Bacillus species 129-52, which was deposited on April 1, 2025, at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 34059, and is classified as Bacillus sp.; the strain 129-52 of the present invention has a living inhibition rate of 66.82% on branches with apple rot disease. After using fungicide-drug synergistic treatment, it was found that the inhibition rate of two tested drugs, carbendazim and pyraclostrobin, on apple rot disease reached more than 80% after the dosage of the two tested drugs, carbendazim and pyraclostrobin, was reduced by half. The fungicide-drug synergistic treatment can effectively achieve the purpose of reducing the dosage, maintaining the effect, and efficiently preventing and controlling apple rot disease.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a new broad-spectrum disease-resistant Bacillus species, a bacterial-drug compound and applications thereof. Background Art

[0002] With the development of agriculture and intensive planting, plant diseases have become one of the main reasons limiting its production. For example, apple rot caused by Valsa mali, gray mold and ring rot of fruits after harvest, and root rot caused by continuous cropping of plants have caused serious economic losses. Chemical control is still the most direct and effective method for the prevention and control of forest and plant diseases. Chemical fungicides represented by tebuconazole, difenoconazole, carbendazim, myclobutanil and pyraclostrobin have the advantages of high efficiency, rapid effect, convenient application and significant economy. However, long-term single use can easily lead to multiple problems: (1) Resistance risk: Target pathogens (such as Valsa mali, Botrytis cinerea, etc.) have developed varying degrees of resistance to fungicides such as carbendazim, resulting in reduced control efficacy; (2) Phytotoxicity risk: Excessive or improper application can easily lead to plant phytotoxicity and human and animal poisoning incidents; (3) Ecological and health risks: Chemical fungicide residues can easily accumulate in the environment and agricultural products, destroying soil microbial communities and threatening non-target organisms and human health.

[0003] Biological control technology refers to the use of antagonistic microbial resources for biological control of plant diseases. my country's abundant microbial resources provide a basis for the screening of biocontrol bacteria. However, existing biological control technologies still have unstable field effects. The efficacy of a single strain is restricted by environmental factors such as temperature and humidity, and the physical and chemical properties of the soil. In addition, the effect cycle is long, making it difficult to meet the needs of disease outbreaks. Most biocontrol bacteria are only effective against specific pathogens and lack broad-spectrum disease resistance.

[0004] Existing research indicates that biocontrol bacteria combined with low-dose chemical agents can achieve both rapid and sustained effects, thereby achieving synergistic efficacy. However, most chemical agents inhibit the growth of biocontrol bacteria, and highly effective and compatible combinations are lacking. Furthermore, reported synergistic systems are mostly based on conventional Bacillus spp. (such as Bacillus subtilis), with limited inhibitory spectra and without the identification of new species. The synergistic mechanisms underlying these combined systems have yet to be systematically elucidated, hindering their targeted development. Based on these findings, the present invention provides a new broad-spectrum disease-resistant Bacillus spp., a bacterial-drug combination, and its applications. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a new broad-spectrum disease-resistant Bacillus species, a bacterial-drug compound and applications thereof.

[0006] In a first aspect, the present invention provides a new broad-spectrum disease-resistant Bacillus species, which adopts the following technical solution:

[0007] A new broad-spectrum disease-resistant Bacillus species is Bacillus species 129-52, which was deposited on April 1, 2025, at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 34059 and a taxonomic name of Bacillus sp.

[0008] Preferably, the 16S rRNA sequence of the Bacillus sp. 129-52 is shown as SEQ ID NO: 1.

[0009] In a second aspect, the present invention provides a use of a new broad-spectrum disease-resistant Bacillus species in the preparation of an apple rot-resistant product.

[0010] In a third aspect, the present invention provides a use of a new broad-spectrum disease-resistant Bacillus species in the preparation of a product for inhibiting plant pathogens.

[0011] Preferably, the plant pathogens include Botryosphaeria cinerea, Botryosphaeria berengerianade, Fusarium solani WQ1, Phytophthora parasitica var. nicotianae, Rhizoctonia solani, and Fusarium pseudograminearum.

[0012] In a fourth aspect, the present invention provides a fungus-drug compound, which adopts the following technical solution:

[0013] A bacterial-drug compound comprises the new broad-spectrum disease-resistant Bacillus species.

[0014] Preferably, the broad-spectrum disease-resistant new Bacillus species active ingredient includes live bacteria, fermentation liquid or bacterial suspension of the new Bacillus species 129-52.

[0015] Preferably, the fungicide-drug combination further comprises carbendazim or pyraclostrobin.

[0016] Preferably, the active ingredient of the fungicide compound further includes a dilution of carbendazim or a dilution of pyraclostrobin.

[0017] Preferably, the bacterial drug compound comprises OD 600 =0.8 of Bacillus sp. 129-52 bacterial suspension and 4000-fold dilution of carbendazim.

[0018] Preferably, the bacterial drug compound comprises OD 600 =0.8 of Bacillus sp. 129-52 bacterial suspension and 4000-fold dilution of pyraclostrobin.

[0019] Preferably, the formulation of the fungus-drug compound is a water dispersant, a water suspension or a dispersible oil suspension.

[0020] In summary, the present invention has the following beneficial effects:

[0021] The present invention collects rhizosphere soil samples of red willow from Ruoqiang County in the Tarim Basin of Xinjiang, and screens out a new Bacillus species 129-52 from the rhizosphere soil samples. The strain 129-52 has a living inhibition rate of 66.82% on apple rot diseased branches, has certain inhibitory activity, and has high compatibility with both carbendazim and pyraclostrobin. After using bacterial and drug synergistic treatment, it is found that the inhibition rate of apple rot disease reaches more than 80% after the dosage of the two test agents is halved, and there is no significant difference in the treatment effect compared with the original dosage of the test agents. In fact, the prevention and control effect of the apple rot disease is slightly improved when the dosage of the two test agents is halved and combined with the 129-52 bacterial solution. The pollution of chemical agents is reduced and the prevention effect is improved, thereby achieving the purpose of reducing the dosage, maintaining the effect and efficiently preventing and controlling the apple rot disease.

[0022] The strain 122-9 of the present invention has certain inhibitory activity against apple rot pathogen (Cytospora mali QH2), pear ring rot pathogen (Botryosphaeria berengerianade), pear gray mold (Botryosphaeria cinerea), carrot root rot pathogen (Fusarium solani WQ1), tobacco phytophthora parasitica var. nicotianae, wheat stem rot pathogen (Fusarium pseudograminearum), and potato black mole pathogen (Rhizoctonia solani), among which the inhibitory activity against apple rot pathogen, pear ring rot pathogen, pear gray mold, and tobacco phytophthora all reaches more than 50%, indicating broad-spectrum antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the inhibition spectrum of strain 129-52 in Example 1 of the present invention;

[0024] Figure 2 The plate culture characteristics and scanning electron microscopy morphology of strain 129-52 in Example 2 of the present invention are shown;

[0025] Figure 3 This is the phylogenetic tree of strain 129-52 in Example 2 of the present invention based on the 16SS rDNA sequence and the whole genome;

[0026] Figure 4 This is a graph showing the inhibitory activity of five test agents against strain 129-52 as determined by the Oxford cup method in Example 3 of the present invention;

[0027] Figure 5 This is a graph showing the results of the in vivo protective effect determination of the fungus and drug combination against apple rot in Example 4 of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0029] The test materials involved in the embodiments of the present invention are as follows:

[0030] 1.1 Test soil samples

[0031] In July 2022, rhizosphere soil samples of red willow were collected in Ruoqiang County, Tarim Basin, Xinjiang (E40°11′06, N88°14′37″). When collecting soil samples, 5 cm of the ground surface was removed, and soil samples within 25 cm near the roots were taken. The collected soil was mixed evenly, sealed and stored in kraft paper bags, numbered, and placed in a refrigerator at 4°C in the Horticultural Crop Pathogen and Disease Control Laboratory of Inner Mongolia Agricultural University for future use.

[0032] 1.2 Test strains

[0033] Pear gray mold (Botryosphaeria cinerea) and pear ring rot (Botryosphaeria berengerianade) were isolated and identified by the Institute of Fruit Research, Chinese Academy of Agricultural Sciences (Sun et al. 2017; Sun Pingping et al., 2018). Apple rot pathogen (Cytospora mali QH2) was isolated and identified by our laboratory (Ma Qiang et al., 2020). Carrot root rot pathogen (Fusarium solani WQ1) was provided by the Institute of Vegetables and Flowers, Inner Mongolia Academy of Agricultural Sciences (Han Fengying et al., 2020). Tobacco blight (Phytophthora parasitica var. nicotianae), potato black mole pathogen (Rhizoctonia solani), and wheat stem rot pathogen (Fusarium pseudograminearum) were provided by Henan Agricultural University.

[0034] 1.3 Test culture medium:

[0035] (1) Potato dextrose medium (PDA): 200 g potato extract, 20 g glucose, 20 g agar powder, 1000 mL distilled water, pH 7.2-7.4.

[0036] (2) LBA medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 20 g agar powder, and dilute to 1 L with deionized water.

[0037] (3) LB liquid medium: Same as LBA medium, without adding agar.

[0038] 1.4 Test agents

[0039] The test drugs were purchased from the local market. The information including drug name, active ingredient content, dosage form and manufacturer is shown in Table 1 below.

[0040] Table 1 Five tested drugs

[0041]

[0042] Example 1

[0043] Isolation of antagonistic strains and determination of inhibition spectrum

[0044] Strains were isolated from the test soil samples using a serial dilution method, and single colonies were transferred to LBA medium for antagonistic activity screening. The inhibitory activity of the isolated strains against Cytospora mali QH2, the apple rot pathogen, Botryosphaeria cinerea, the pear ring rot pathogen, Botryosphaeria berengerianade, the carrot root rot pathogen, Fusarium solani WQ1, the tobacco phytophthora parasitica var. nicotianae, the potato black mole pathogen, Rhizoctonia solani, and the wheat stem rot pathogen, Fusarium umpseudograminearum, was determined using a plate standoff assay.

[0045] Pick the 7 test pathogen cakes mentioned above and place them in the center of the PDA plate. Inoculate the isolated strains in two directions 3 cm away from the test pathogen cake and incubate at 25°C. Use the plate inoculated with only pathogens as a control. When the control colonies fill the plate, observe and record the colony radius. The results are shown in Table 2 and Figure 1 .

[0046] Inhibition rate (%) = (1-average lesion radius of the treatment group / average lesion radius of the control group) × 100%

[0047] Table 2 Results of the in vitro inhibitory activity assay of strain 129-52 against the tested pathogens

[0048]

[0049] Combined with Table 2 and Figure 1 The results showed that the strain 129-52 isolated from the rhizosphere soil sample of red willow had a broad-spectrum antibacterial activity against the tested pathogens, and had certain inhibitory activity against the seven tested pathogens, among which the inhibitory activity against apple rot pathogen, pear ring rot pathogen, pear gray mold and tobacco phytophthora reached more than 50%.

[0050] Example 2

[0051] Strain identification

[0052] (1) Morphological identification

[0053] The purified strain 129-52 was inoculated into LBA medium, and the morphological characteristics of the colonies in the dish were observed. The colonies were picked and observed under a scanning electron microscope. The strain 129-52 had typical bacterial colony characteristics on LBA medium. The colonies were milky white to light yellow opaque colonies with protrusions on the colony surface, rough edges, and fast growth. Microscopic observation showed that the bacteria were short rods with a size of approximately 16-17μm × 8-9μm. Figure 2 In the middle, the left side is the positive colony characteristic diagram of strain 129-52 on LBA medium, the middle side is the negative colony characteristic diagram of strain 129-52 on LBA medium, and the right side is the result of scanning electron microscopy observation of bacterial morphology of strain 129-52.

[0054] (2) Molecular identification

[0055] Genomic DNA from strain 129-52 was extracted and whole-genome sequenced using the PacbioSequel II. Sequencing reads were assembled using SMARTLink 10.1.0 software. The evolutionary distances between the genomes of different strains were calculated using the genome alignment distance evolution method in the Type Strain Genome Server online program (https: / / tygs.dsmz.de). The minimum evolutionary distances between genomes were used to construct a phylogenetic tree of the 16S rRNA and whole-genome sequences of the strains screened in this experiment and the 10 strains (Meier-Kolthoff & Göker, 2019). The number for each branch represents the confidence level of 1000 bootstrap tests. The average nucleotide identity (ANI) of the whole-genome sequences of 129-52 and related strains was calculated using Jspecies (Goris et al., 2007). The results are shown in Tables 3 and 4. Figure 3 .

[0056] Table 3 ANI values ​​of similar strains and 129-52

[0057]

[0058] Combined with Table 3 and Figure 3 The results show that strain 129-52 has the highest ANI value of 92.98% with Bacillus vallismorti DV1-F-3 and Bacillus spizizenii TU-B-10, and 92.92% with Bacillus inaquosorum KCTC 13429. The ANI values ​​with other strains range from 91% to 92.8%. Due to the 95-96% or higher identity of the whole genome sequence, strain 129-52 is considered to be the same species (Meier-Kolthoff et al., 2013). Although strain 129-52 clusters with Bacillus inaquosorum and Bacillus inaquosorum in the phylogenetic tree, the phylogenetic distance remains relatively distant, indicating that it belongs to a different species. Therefore, based on the phylogenetic tree, strain 129-52 is designated as a new species of Bacillus and named Bacillus sp. 129-52. Figure 3 A represents the phylogenetic tree based on 16S rRNA, and B represents the phylogenetic tree based on the whole genome sequence. Different colors in species cluster and subspecies cluster represent different species or subspecies.

[0059] The 16S rRNA sequence of Bacillus sp. 129-52 is shown in SEQ ID NO: 1:

[0060]

[0061] The new Bacillus species 129-52 obtained in the present invention was deposited on April 1, 2025, at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 34059 and the classification name Bacillus sp.

[0062] Example 3

[0063] Bacteria-drug compatibility determination

[0064] Five test agents with excellent efficacy against apple rot were selected: tebuconazole, prochloraz, carbendazim, difenoconazole, and pyraclostrobin. Their inhibitory activity against strain 129-52 was determined using the Oxford cup assay. The method was as follows: First, each test agent was diluted 4000-fold according to the recommended concentration. Strain 129-52 was added to LB liquid medium and incubated at 28°C and 180 rpm for 48 hours to obtain a 129-52 bacterial suspension. Five mL of the 129-52 suspension was then added to 200 mL of thawed, but not yet solidified, LB medium, shaken thoroughly, and then transferred to a Petri dish to prepare a plate containing the bacteria. After the Petri dish solidified, an Oxford cup was placed in the plate, and 100 μL of each diluted test agent was added to the cup. Each treatment was repeated three times. After incubation at 28°C for two days, the diameter of the clear zone of each colony was counted.

[0065] Combine Figure 4 The results showed that carbendazim and pyraclostrobin had good compatibility with strain B129-52. The clear zone radii of carbendazim and pyraclostrobin against strain 129-52 were 0.10 mm and 0.1 mm, respectively, while the inhibition zone diameters of tebuconazole, prochloraz, and difenoconazole against 129-52 were 1.06 mm, 2.46 mm, and 1.5 mm, respectively. Therefore, two highly compatible carbendazim and pyraclostrobin were selected for compounding with strain 129-52.

[0066] Example 4

[0067] Effect of synergistic fungus and drug on the prevention and control of apple rot

[0068] The control agent, tebuconazole, a commonly used apple rot control agent on the market, was used as a control agent. The in vitro branch assay was used to evaluate the control effect of the test agent, carbendazim, and pyraclostrobin in combination with strain 129-52 on apple rot. The specific method is as follows: the test agents tebuconazole, carbendazim, and pyraclostrobin were diluted to 4000 times according to their recommended concentrations and then used. Strain 129-52 was selected and inoculated into 200mL LB liquid medium, cultured at 28℃ and 180r / min for 48h, and the OD was adjusted with sterile water. 600 = 0.8 to obtain a 129-52 bacterial suspension for use. Eight treatment groups were set up: CK- (LB treatment without pathogen inoculation), CK+ (LB + pathogen), 129-52 bacterial suspension, a 4000 dilution of carbendazim, a 4000 dilution of pyraclostrobin, a 4000 dilution of tebuconazole (control), a 1:1 volume ratio of carbendazim 4000 dilution + 129-52 bacterial suspension, and a 1:1 volume ratio of 4000 dilution of pyraclostrobin + 129-52 bacterial suspension; totaling eight treatment groups.

[0069] Two-year-old healthy golden red apple branches were selected and cut into twigs of about 10 cm. They were disinfected with sodium hypochlorite and alcohol respectively, rinsed with sterile water and dried, and the ends of the branches were sealed with paraffin. After heat sterilization with a sterile puncher, a 6 mm wound was burned behind the middle branch of the apple branch. After spraying the apple branches with 8 groups of treatment liquids for 30 minutes, except for the CK-group which was not inoculated with pathogens, the other 7 groups applied the apple rot pathogen QH2 bacterial cake to the wound area, wrapped it with sterile absorbent cotton soaked in sterile water, and then wrapped it with plastic wrap. All branches were placed at 25℃, 16h light / 8h dark and moisturized for incubation. 7 days after inoculation, the absorbent cotton was removed and the apple rot pathogen QH2 bacterial cake was re-inoculated and then directly wrapped with plastic wrap. After 14 days, the lesion size (longitudinal length) was measured and the inhibition rate was calculated. The results are shown in Table 4 and Figure 5 .

[0070] Table 4 The control effect of fungus and drug combination on apple rot

[0071]

[0072] Combined with Table 4 and Figure 5 The results show that the bacterial liquid of the screened strain 129-52 has a living inhibition rate of 66.82% on apple rot branches, which has a certain inhibitory activity. After the synergistic treatment of bacteria and drugs, it was found that the inhibition rate of apple rot disease reached more than 80% after the dosage of the two test agents was halved, and there was no significant difference in the treatment effect with the original dosage of the test agents. Even after the dosage was halved and combined with the 129-52 bacterial liquid, the prevention and control effect of apple rot disease was slightly improved. The bacterial and drug combination agent proposed in the present invention not only reduces the pollution of chemical agents, but also improves the prevention effect, thereby achieving the purpose of reducing the dosage, maintaining the effect and efficiently preventing and controlling apple rot disease.

[0073] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it is protected by patent law.

Claims

1. A new broad-spectrum disease-resistant Bacillus species, characterized by: The new broad-spectrum disease-resistant Bacillus species is Bacillus species 129-52, which was deposited on April 1, 2025 at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 34059, and the classification name is Bacillus sp.

2. The new broad-spectrum disease-resistant Bacillus species according to claim 1, characterized in that The 16S rRNA sequence of the new Bacillus sp. 129-52 is shown in SEQ ID NO:

1.

3. Use of the new broad-spectrum disease-resistant Bacillus species according to claim 1 or 2 in the preparation of products resistant to apple rot.

4. Use of the new broad-spectrum disease-resistant Bacillus species according to claim 1 or 2 in the preparation of a product for inhibiting plant pathogens; The plant pathogens are Botrytis cinerea, Botryosphaeria berengerianade, Fusarium solani, Phytophthora parasiticavar. nicotianae, Rhizoctonia solani, and Fusariumpseudograminearum.

5. A fungus-drug compound, characterized in that: The invention comprises the new broad-spectrum disease-resistant Bacillus species according to claim 1 or 2.

6. The fungus-drug compound according to claim 5, characterized in that: The active ingredients of the bacterial-drug compound include live bacteria of the new species Bacillus sp. 129-52, fermentation liquid or bacterial suspension.

7. The fungus-drug compound according to claim 5, characterized in that: The fungicide compound further comprises carbendazim or pyraclostrobin.

8. The fungus-drug compound according to claim 7, characterized in that: The active ingredients of the fungicide compound further include a carbendazim dilution or a pyraclostrobin dilution.

9. The fungus-drug compound according to claim 5, characterized in that: The dosage form of the fungus-drug compound is a water dispersant, a water suspension or a dispersible oil suspension.

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