A new species of biocontrol bacillus, a bacterium-drug compound and its application

Abstract

The present invention relates to the field of microbial technology, and specifically discloses a new biocontrol bacillus species, a bacterium-drug compound, and applications thereof. The new biocontrol bacillus species is Bacillus species 122-9, 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. 34057. It is classified as Bacillus sp.; the strain 122-9 of the present invention has a living inhibition rate of 79% on branches with apple rot, and has high compatibility with tebuconazole, carbendazim, and pyraclostrobin. After using bacterium-drug synergistic treatment, it was found that the inhibition rate of the three tested drugs on apple rot reached more than 80% even when the dosage was halved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a new species of biocontrol bacillus, a bacterium-drug compound and applications thereof. Background Art

[0002] With the development of agriculture and the intensification of crop cultivation, plant diseases have become one of the main limitations on production. For example, apple rot caused by Valsa mali, postharvest gray mold and ring rot, and root rot caused by continuous cropping, among other diseases, cause severe economic losses. Apple rot, in particular, occurs in major apple-producing areas in my country and around the world. In severe cases, it can even attack the fruit, causing secondary infection and resulting in a 50%-70% yield reduction in orchards, causing significant losses to the fruit industry. Chemical control remains the most direct and effective method for preventing and controlling forest and plant diseases. Currently, fungicides such as tebuconazole, difenoconazole, carbendazim, prochloraz, and pyraclostrobin are registered for the prevention and control of apple rot, postharvest gray mold, and ring rot in pears. Chemical control has the advantages of high efficiency, rapid effect, ease of use and high economic benefits. However, long-term improper use of chemical agents not only causes phytotoxicity to plants, causes poisoning in humans and animals, kills beneficial microorganisms, but also leads to drug resistance in pathogens. At the same time, high pesticide residues will also bring serious harm to human health and pollute the ecological environment.

[0003] Biological control technology refers to the use of antagonistic microbial resources for the biological control of plant diseases. It has the advantages of abundant strain resources, wide sources, safety to non-target organisms, minimal toxicity and side effects, good environmental compatibility, and sustained action against pests. However, single biological control methods have the disadvantages of slow field efficacy, unsatisfactory control results, and susceptibility to environmental influences.

[0004] The fungus-drug synergistic technology proposed in recent years combines biocontrol bacteria with low-dose chemical agents, which can not only give play to the eco-friendly characteristics of biocontrol bacteria, but also utilize the rapid antibacterial effect of chemical agents, thereby achieving synergistic efficiency. This technology can not only effectively control diseases, but also reduce the use of chemical agents, reduce potential threats to the environment and human health, and provide new ideas for the green and sustainable development of the apple industry. However, there are currently few studies and applications of fungus-drug synergy for various diseases such as apple rot. Practical applications face technical obstacles such as the lack of a scientific compatibility screening system and the existence of activity inhibition phenomena in most potential fungus-drug combinations. Based on the above statements, the present invention provides a new species of biocontrol bacteria Bacillus, a fungus-drug combination and its application. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a new species of biocontrol bacteria Bacillus, a fungus-drug compound and applications thereof.

[0006] In a first aspect, the present invention provides a new species of biocontrol bacteria Bacillus, which adopts the following technical solution:

[0007] A new species of biocontrol bacillus is provided. The new species of biocontrol bacillus is Bacillus sp. 122-9, 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. 34057, and the classification name is Bacillus sp.

[0008] Preferably, the 16S rRNA sequence of the new Bacillus sp. 122-9 is shown as SEQ ID NO: 1.

[0009] In a second aspect, the present invention provides a use of a new species of biocontrol bacteria Bacillus in the preparation of a product resistant to apple rot.

[0010] In a third aspect, the present invention provides a use of a new species of biocontrol bacteria Bacillus in the preparation of a product for inhibiting plant pathogens.

[0011] Preferably, the plant pathogens include Botryosphaeria cinerea, Botryosphaeria berengerianade, Monilinia fructigena, 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 fungus-drug compound comprises the new species of biocontrol bacterium Bacillus.

[0014] Preferably, the active ingredient of the biocontrol bacteria Bacillus sp. nov. includes live bacteria, fermentation liquid or bacterial suspension of Bacillus sp. nov. 122-9.

[0015] Preferably, the fungicide compound further comprises tebuconazole, carbendazim or pyraclostrobin.

[0016] Preferably, the active ingredients of the fungicide compound further include a diluent of tebuconazole, a diluent of carbendazim or a diluent of pyraclostrobin.

[0017] Preferably, the bacterial drug compound comprises OD 600 =0.8 of Bacillus sp. 122-9 suspension and 4000-fold dilution of tebuconazole.

[0018] Preferably, the bacterial drug compound comprises OD 600 =0.8 of Bacillus sp. 122-9 suspension and 4000-fold dilution of carbendazim.

[0019] Preferably, the bacterial drug compound comprises OD 600 =0.8 of Bacillus sp. 122-9 bacterial suspension and 4000-fold dilution of pyraclostrobin.

[0020] Preferably, the formulation of the fungus-drug compound is a water dispersant, a water suspension or a dispersible oil suspension.

[0021] In summary, the present invention has the following beneficial effects:

[0022] The present invention collects soil samples from the rhizosphere of camel thorn and red willow in the Tarim Basin of Xinjiang, and screens out a new Bacillus species 122-9 from the soil samples. The strain 122-9 has a living inhibition rate of 79% on apple rot branches and has high compatibility with tebuconazole, carbendazim and pyraclostrobin. After synergistic treatment with bacteria and drugs, it is found that the inhibition rate of apple rot reaches more than 80% after the dosage of the three test drugs is halved, and there is no significant difference in the treatment effect compared with the original dosage of the test drugs. In fact, the prevention and control effect of apple rot is slightly improved when the dosage of the drugs is halved and combined with the 122-9 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 effectively preventing and controlling apple rot.

[0023] The strain 122-9 of the present invention has a certain inhibitory effect on pear gray mold (Botryosphaeria cinerea), pear ring rot fungus (Botryosphaeria berengerianade), pear stem rot fungus (Monilinia fructigena), carrot root rot fungus (Fusarium solani WQ1), tobacco phytophthora (Phytophthora parasitica var. nicotianae), potato black mole fungus (Rhizoctonia solani), and wheat stem rot fungus (Fusarium pseudograminearum), with an inhibition rate of more than 30%. The inhibition rate of the strain 122-9 on pear gray mold is 76.99%, and the inhibition rates on pear ring rot fungus and tobacco phytophthora also reach more than 60%, indicating a broad antibacterial spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the inhibitory activity of strain 122-9 against apple rot pathogen QH2 in Example 1 of the present invention;

[0025] Figure 2 This is a graph showing the inhibition spectrum of strain 122-9 in Example 2 of the present invention;

[0026] Figure 3 The plate culture characteristics and scanning electron microscopy morphology of strain 122-9 in Example 3 of the present invention are shown;

[0027] Figure 4 This is the phylogenetic tree of strain 122-9 in Example 3 of the present invention based on the 16SS rDNA sequence and the whole genome;

[0028] Figure 5 This is a graph showing the inhibitory activity of five test agents against strain 122-9 as determined by the Oxford cup method in Example 4 of the present invention;

[0029] Figure 6 This is a graph showing the results of the in vivo protective effect test of the fungus and drug combination against apple rot in Example 5 of the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] The test materials involved in the embodiments of the present invention are as follows:

[0032] 1.1 Test soil samples

[0033] In July 2022, soil samples were collected from the rhizosphere of camel thorn and red willow in the Tarim Basin, Xinjiang (E41°09′20″, N86°25′15″). The collected soils were mixed evenly, sealed and stored in kraft paper bags, numbered, and placed in a refrigerator at 4°C in the Laboratory of Pathogens and Disease Control of Horticultural Crops of Inner Mongolia Agricultural University for future use.

[0034] 1.2 Test strains

[0035] Pear gray mold (Botryosphaeria cinerea), pear ring rot (Botryosphaeria berengerianade), and pear stem rot (Monilinia fructigena) 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 (Cytospora mali QH2) was isolated and identified by our laboratory (Ma Qiang et al., 2020). Carrot root rot (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 (Rhizoctonia solani), and wheat stem rot (Fusariumpseudograminearum) were provided by Henan Agricultural University.

[0036] 1.3 Test culture medium:

[0037] (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.

[0038] (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.

[0039] (3) LB liquid medium: Same as LBA medium, without adding agar powder.

[0040] 1.4 Test agents

[0041] 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.

[0042] Table 1 Five tested drugs

[0043]

[0044] Example 1

[0045] Isolation and screening of antagonistic strains

[0046] The strains were isolated from the test soil samples using the serial dilution method, and single colonies were transferred to LBA medium for antagonistic activity screening. The isolated strains were screened using the plate standoff method and the in vitro shoot inoculation method.

[0047] Antagonistic strains were screened using the plate standoff method. A culture of QH2, the apple rot pathogen, was placed in the center of a PDA plate. The isolated strain was inoculated 3 cm to the left and right of the QH2 cake and incubated at 25°C. A plate inoculated only with QH2 served as a control. When the control colonies filled the plate, the colony radius was observed and recorded. From these, strains with excellent antibacterial efficacy and stable antagonistic activity were selected as candidate strains for in vitro branch inoculation experiments.

[0048] The antagonistic activity of the selected candidate strains against apple rot on golden red branches was determined by in vitro branch inoculation. The candidate strains were inoculated into 200 mL LB liquid medium and cultured at 28°C and 180 rpm for 48 h. The OD was adjusted with sterile water. 600 =0.8 to obtain a bacterial solution for future use. Two-year-old, healthy golden-red apple branches were selected and cut into approximately 10 cm twigs. These branches were disinfected with sodium hypochlorite and alcohol, rinsed with sterile water, and air-dried. The ends of the branches were sealed with paraffin wax. A sterile punch was used to sterilize the branches, followed by a 6 mm wound created behind the mid-branch. After spraying the branches with the bacterial solution, a QH2 apple rot pathogen cake was applied to the wounds. The wounds were then wrapped with sterile absorbent cotton soaked in sterile water and then covered with plastic wrap. The wounds were sprayed with LB liquid medium and then inoculated with QH2 as the positive control (CK). Branches sprayed with LB liquid medium alone without inoculation with the QH2 apple rot pathogen cake served as the negative control. All branches were incubated at 25°C under a 16-hour light / 8-hour dark cycle to maintain humidity. 7 days after inoculation, the absorbent cotton was removed and the cake of apple rot pathogen QH2 was re-inoculated and then directly wrapped with plastic wrap. 14 days later, the size of the lesion (vertical length) was measured and the inhibition rate was calculated. The results are shown in Table 2 and Figure 1 .

[0049] Table 2 Antagonistic activity of strain 122-9 against apple rot pathogen QH2 in vitro and on isolated branches

[0050]

[0051] Combined with Table 2 and Figure 1 The results show that 25 bacterial strains were isolated and purified from rhizosphere soil samples of camel thorn and red willow. Using plate standoff and in vitro branch assays, an active antagonist strain, 122-9, was identified. Its inhibition zone against the apple rot pathogen QH2 was 2.41 mm in a dish, with an inhibition rate of 56.35%. In in vitro branch assays, the inhibition rate reached 77.74%. In Table 2, different letters in the same column indicate significant differences between two treatments at the P < 0.05 level (lesion size).

[0052] Example 2

[0053] Determination of the inhibitory spectrum of strains

[0054] The antibacterial spectrum of the isolated antagonistic bacteria 122-9 was determined using the plate confrontation method. A 6mm diameter cake of different pathogens that had been cultured for 5 days was selected and placed in the center of a PDA plate. Antagonistic bacteria 122-9 cakes that had been grown on the culture medium for 3 days were inoculated 3 cm to the left and right of the pathogen cakes and incubated at a constant temperature of 25°C. Each treatment was repeated 3 times, and a culture dish inoculated only with pathogens was used as the control (CK). When the control group colonies filled the plate, the diameter of the inhibition zone was observed and recorded. The results are shown in Tables 3 and 4 below. Figure 2 .

[0055] Average inhibition rate (%) = [1-(average radius of pathogens in the treatment group) / (average radius of pathogens in the control group)] × 100%.

[0056] Table 3 Results of the antibacterial spectrum determination of strain 122-9

[0057]

[0058] Combined with Table 3 and Figure 2 The results show that strain 122-9 has a certain inhibitory effect on different test pathogens, with an inhibition rate of more than 30%. Its inhibition rate against pear gray mold is 76.99%, and its inhibition rate against pear ring rot and tobacco phytophthora is also more than 60%, with a broad antibacterial spectrum.

[0059] Example 3

[0060] Strain identification

[0061] (1) Morphological identification

[0062] The purified strain 122-9 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 122-9 had typical bacterial colony characteristics on LBA medium. The colonies appeared milky white to light yellow opaque colonies with protrusions on the colony surface, rough colony edges, and fast growth. Microscopic observation showed that the bacteria were short rods with a size of approximately 14-15μm × 5-6μm. Figure 3 In the middle, the left side is the positive colony characteristic diagram of strain 122-9 on LBA medium, the middle side is the negative colony characteristic diagram of strain 122-9 on LBA medium, and the right side is the result of scanning electron microscopy observation of bacterial morphology of strain 122-9.

[0063] (2) Molecular identification

[0064] Genomic DNA from strain 122-9 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). Using the minimum evolutionary distances between genomes, a phylogenetic tree of 16S rRNA and whole-genome sequences of the strains screened in this experiment and the 12 strains was constructed using FASTME 2.1.6.1 software (Meier-Kolthoff & Göker, 2019). The number for each branch represents the confidence level of 1000 bootstrap tests. The average nucleotide identity (ANI) between the whole-genome sequences of 122-9 and similar strains was calculated using Jspecies (Goris et al., 2007). The results are shown in Tables 4 and 4 below. Figure 4 .

[0065] Table 4 ANI values ​​of similar strains and 122-9

[0066]

[0067] Combined with Table 4 and Figure 4 The results show that strain 122-9 shares an ANI value of 93.42% with Bacillus vallismorti DV1-F-3 and 93.08% with Bacillus inaquosorum KCTC13429. The ANI values ​​for strain 122-9 with other strains range from 76.07% to 92.88%. Due to a complete genome sequence identity of at least 95-96%, strain 122-9 is considered to be the same species (Meier-Kolthoff et al., 2013). A phylogenetic tree analysis revealed that strain 122-9 and related species belong to different species or subspecies. Therefore, the phylogenetic tree confirmed that strain 122-9 is a new species of Bacillus and named Bacillus sp. 122-9. Figure 4 A represents the phylogenetic tree based on 16S rRNA, and B represents the phylogenetic tree based on the whole genome. Different colors in species cluster and subspecies cluster represent different species or subspecies.

[0068] The 16S rRNA sequence of Bacillus sp. 122-9 is shown in SEQ ID NO: 1:

[0069]

[0070] The new Bacillus species 122-9 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. 34057 and the classification name Bacillus sp.

[0071] Example 4

[0072] Bacteria-drug compatibility determination

[0073] Five test agents with excellent efficacy against apple rot were selected: tebuconazole, prochloraz, carbendazim, difenoconazole, and pyraclostrobin. Their inhibitory activity against strain 122-9 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 122-9 was added to LBA medium and incubated at 28°C and 180 rpm for 48 hours to obtain a 122-9 bacterial suspension. Five mL of the 122-9 suspension was then added to 200 mL of thawed, but not yet solidified, LBA medium, shaken thoroughly, and then transferred to a Petri dish to prepare a culture plate. After the Petri dish solidified, an Oxford cup was placed, 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.

[0074] Combine Figure 5 The results showed that tebuconazole, carbendazim, and pyraclostrobin had good compatibility with strain B122-9. The clear zone radii for tebuconazole, carbendazim, and pyraclostrobin were 0.465, 0.40, and 0.4 mm, respectively. However, the inhibition zone diameters for prochloraz and difenoconazole were greater than 1 mm, at 1.645 mm and 1.545 mm, respectively. Therefore, three highly compatible agents, tebuconazole, carbendazim, and pyraclostrobin, were selected for compounding with strain 122-9.

[0075] Example 5

[0076] Effect of synergistic fungus and drug on the prevention and control of apple rot

[0077] The in vitro branch assay was used to evaluate the control effect of the test agents tebuconazole, carbendazim, and pyraclostrobin in combination with strain 122-9 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 122-9 was selected and inoculated into 200 mL LB liquid medium, cultured at 28°C and 180 r / min for 48 hours, and the OD was adjusted with sterile water. 600= 0.8 to obtain a 122-9 bacterial suspension for use. Nine treatment solutions were prepared: CK- (LB treatment without pathogen inoculation), CK+ (LB + pathogen), 122-9 bacterial suspension, a 4000 dilution of carbendazim, a 4000 dilution of pyraclostrobin, a 4000 dilution of tebuconazole, a 1:1 volume ratio of carbendazim 4000 dilution + 122-9 bacterial suspension, a 1:1 volume ratio of pyraclostrobin 4000 dilution + 122-9 bacterial suspension, and a 1:1 volume ratio of tebuconazole 4000 dilution + 122-9 bacterial suspension; a total of nine treatment solutions.

[0078] 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 9 groups of treatment liquids for 30 minutes, except for the CK-group which was not inoculated with pathogens, the other 8 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 ° C, 16 hours of light / 8 hours of darkness for moisturizing culture. After 7 days of 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 5 and below. Figure 6 .

[0079] Table 5 The control effect of fungus and drug combination on apple rot

[0080]

[0081] Combined with Table 5 and Figure 6 The results show that the bacterial liquid of the screened strain 122-9 has a living inhibition rate of 79% on apple rot branches, which has a certain inhibitory activity. After the synergistic treatment with bacteria and drugs, it was found that the inhibition rate of apple rot disease with the three test agents was more than 80% after the dosage 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 122-9 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.

[0082] 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 species of biocontrol bacterium Bacillus, characterized in that The new biocontrol bacterium Bacillus species is Bacillus species 122-9, which was deposited on April 1, 2025, at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 34057 and a taxonomic name of Bacillus sp. The 16S rRNA sequence of the new Bacillus sp. 122-9 is shown in SEQ ID NO:

1.

2. Use of the new species of biocontrol bacterium Bacillus according to claim 1 in the preparation of a product resistant to apple rot.

3. Use of the new species of biocontrol bacterium Bacillus according to claim 1 in the preparation of a product for inhibiting plant pathogens; The plant pathogens are Botrytis cinerea, Botryosphaeria berengeriana, Diplodia natalensis, Fusarium solani, Phytophthora parasitica var. nicotianae, Rhizoctonia solani, and Fusarium pseudograminearum.

4. A fungus-drug compound, characterized in that: The invention comprises the new species of biocontrol bacteria Bacillus and the drug according to claim 1; The drug is tebuconazole, carbendazim or pyraclostrobin.

5. The fungus-drug compound according to claim 4, characterized in that: The active ingredients of the bacterial-drug compound include live bacteria of the new species of Bacillus 122-9, fermentation liquid or bacterial suspension.

6. The fungus-drug compound according to claim 5, characterized in that: The active ingredients of the fungicide compound further include a tebuconazole dilution, a carbendazim dilution or a pyraclostrobin dilution.

7. The fungus-drug compound according to claim 4, 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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