A bacillus flexus strain BJ699 and its application in strawberry production

Abstract

The application belongs to the field of agricultural microorganism technology, and discloses a bacillus curvatus strain BJ699 and application thereof in strawberry production. Bacillus flexus The application provides a new bacillus curvatus (BJ699) strain, and the preservation number of the strain BJ699 in China Typical Culture Collection Center is CCTCC NO:M20252013. The application further provides a microbial inoculant containing the strain BJ699 as an active ingredient and application thereof in preparation of a microbial inoculant for preventing and controlling a strawberry root rot pathogen and / or degrading pyraclostrobin. The new strain or the microbial inoculant containing the strain in the application is from a natural environment, has no cross-resistance with existing bactericides and insecticides, has the advantages of high efficiency, low toxicity, no environmental pollution and no easy generation of drug resistance, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology, specifically relating to a type of Bacillus curvatureus (… Bacillus flexus Strawberry strain BJ699 and its application in strawberry production, especially in the control of strawberry pathogens and the degradation of pyraclostrobin residues in soil. Background Technology

[0002] strawberry( Strawberry pineapple Duch. is a perennial herbaceous plant belonging to the genus Strawberry in the family Rosaceae. Its fruit is soft, juicy, and rich in nutrients, including various vitamins, making it a favorite among consumers and earning it the reputation of "Queen of Fruits".

[0003] Due to the special nature of greenhouse cultivation environments, soil-borne diseases have become increasingly serious with the increase in continuous cropping years, causing severe economic losses to strawberry cultivation.

[0004] Root rot is a devastating soil-borne disease commonly encountered in strawberry cultivation. It is caused by a variety of pathogens, making control difficult. Among the reported pathogens causing strawberry root rot are Fusarium (…). Fusarium spp.), Phytophthora ( Phytophthora spp.), Rhizoctonia spp. Rhizoctonia spp.) and anthrax bacteria ( Colletotrichum (e.g., spp.). Among them, Fusarium is the main pathogenic bacterium.

[0005] Due to differences in soil properties, climate conditions, and management levels, the pathogens isolated from different major strawberry producing areas are also different, and in most cases, it is a mixed infection of several pathogens.

[0006] Currently, the use of chemical fungicides remains a common control measure against pathogens in strawberry cultivation. However, the problems of pesticide residues and pathogen resistance caused by chemical fungicides remain unresolved. Pyraclostrobin, a commonly used chemical fungicide for controlling diseases such as strawberry root rot, is prone to causing pesticide residues in strawberries. Therefore, biological control has become a research hotspot due to its advantages such as low cost, greenness, and environmental friendliness.

[0007] To address the aforementioned problems, the inventors isolated a new *Bacillus curvaturei* strain BJ699 from the rhizosphere soil of strawberries. Experiments confirmed that this strain is effective against *Fusarium* species (…). Fusarium spp.), Phytophthora ( Phytophthora spp.), Rhizoctonia spp. Rhizoctonia spp.) and anthrax bacteria ( Colletotrichum This invention has a good control effect on strawberry root rot caused by fungicides such as spp., and also has the ability to degrade the chemical fungicide pyraclostrobin, thus completing the present invention. Summary of the Invention

[0008] To address one or more of the aforementioned technical problems in the prior art, the present invention provides, in a first aspect, a Bacillus curvatureis (… Bacillus flexus The strain BJ699 has the accession number CCTCC NO: M20252013 at the China Center for Type Culture Collection.

[0009] The Curvular Bacillus isolated in this invention ( Bacillus flexus The detailed preservation information for strain BJ699 is as follows:

[0010] Depository: China Center for Type Culture Collection;

[0011] Location of deposit: Wuhan University, Wuhan, China;

[0012] Deposit date: September 12, 2025;

[0013] Classification and naming: Bacillus flexus BJ699;

[0014] Accession number: CCTCC NO: M20252013.

[0015] Research has revealed that the *Bacillus curvatureii* isolated in this invention (…) Bacillus flexus Strawberry strain BJ699 can significantly inhibit the growth of strawberry root rot pathogens and also has a very good pyraclostrobin degradation effect. It is particularly suitable for application in areas with pyraclostrobin soil pesticide residues. In this way, it can prevent strawberry root rot pathogens while degrading pyraclostrobin residues in the soil, playing a dual role in the control of root rot pathogens and soil improvement.

[0016] In a second aspect, the present invention provides a microbial inoculant comprising the *Bacillus curvatureii* described in the first aspect of the present invention. Bacillus flexus BJ699 strain is used as the active ingredient.

[0017] Preferably, the microbial agent is in the form of a fermentation broth. The fermentation broth can be the fermented material or the supernatant obtained after filtering the fermented material.

[0018] Preferably, the fermentation broth is grown on NB medium. Experiments have shown that the new strain isolated in this invention grows well on NA medium, with single colonies appearing pale yellow (e.g., ...). Figure 1 (As shown).

[0019] Alternatively, the culture medium used for the fermentation broth may also be a culture medium with the following formula: sweet potato powder 14-16 g / L, yeast extract 16-19 g / L, corn steep liquor 3-4 g / L, magnesium sulfate 0.8-1.2 g / L, pH 6.5-7.

[0020] The present invention provides, in a third aspect, the *Bacillus curvatureis* described in the first aspect of the present invention (…). Bacillus bent Application of strain BJ699 in the preparation of microbial agents for the control of strawberry root rot and / or degradation of pyraclostrobin.

[0021] Preferably, the microbial agent is the microbial agent described in the second aspect.

[0022] Preferably, the microbial agent is in the form of a fermentation liquid and is applied by root irrigation or spraying.

[0023] Preferably, the strawberry root rot disease is caused by fungi selected from Fusarium (…). Fusarium spp.), Phytophthora ( Phytophthora spp.), Rhizoctonia spp. Rhizoctonia spp.) and anthrax bacteria ( Colletotrichum The strawberry root rot is caused by one or more pathogens selected from the genera *Phytophthoracactorum*, *Fusarium oxysporum*, *Rhizoctonia solani*, and *Colletotrichum gloeosporioides*. The *Bacillus curvularia* of the present invention... Bacillus flexus Strawberry strain BJ699 exhibits excellent inhibitory effects against these pathogens causing strawberry root rot.

[0024] Preferably, the pyraclostrobin is in the form of pyraclostrobin residues found in the soil of strawberry growing areas.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) This invention provides a novel Bacillus curvatureus ( Bacillus flexus strain BJ699 was first reported to be effective against Fusarium. Fusarium spp., Phytophthora Phytophthora spp., Rhizoctonia spp. Rhizoctonia SPP and anthrax Colletotrichum Strawberry root rot caused by spp. and degradation of pyraclostrobin do not exhibit cross-resistance with existing fungicides and insecticides.

[0027] (2) Compared with the current use of chemically synthesized pesticides for pest control, the microbial agent of the present invention or its active ingredient BJ699 comes from the natural environment, and therefore has the advantages of high efficiency, low toxicity, no environmental pollution and low resistance. Attached Figure Description

[0028] Figure 1 Show Bacillus flexus Photograph of the colony morphology of strain BJ699.

[0029] Figure 2 Show Bacillus flexus Phylogenetic tree of BJ699. As shown in the figure, the phylogenetic tree includes 6 species, listed from top to bottom as follows: Bacillus flexus Strain HDB-2, BJ699 、Bacillus licheniformis Strain MJW-38, Priestiamegaterium strain WSH10, Bacillus megaterium Strain DZBJ02, Bacillus sp.KYJ963.

[0030] Figure 3 Show Bacillus flexus BJ699's in vitro antibacterial effect. Detailed Implementation

[0031] To better explain the present invention, specific embodiments will be used to further illustrate the invention below. However, the scope of protection of the present invention is not limited to these embodiments. Unless otherwise specified, the technical solutions described in this invention are conventional techniques in the art, and the reagents or materials described are all derived from commercial sources.

[0032] Example 1: Bacillus flexus Acquisition and identification

[0033] The inventors isolated a Bacillus strain from the rhizosphere soil of strawberries in Changping, Beijing. Through whole-genome sequencing and comparison with NCBI data, combined with physiological and biochemical identification, the Bacillus strain was determined to belong to [the Bacillus family / organism]. Bacillus flexus (like Figure 1 , Figure 2 (As shown in Table 1). The inventors deposited this strain at the China Center for Type Culture Collection, and the deposit information is as follows:

[0034] Deposit date: September 12, 2025;

[0035] Category Naming: Bacillus flexus BJ699;

[0036] Accession number: CCTCC NO: M20252013;

[0037] Location of deposit: Wuhan University, Wuhan, China.

[0038] In this application, Bacillus flexus BJ699 is sometimes simply referred to as BJ699.

[0039] Table 1. Physiological and biochemical characteristics of BJ699

[0040]

[0041] Note: +: positive reaction; -: negative reaction.

[0042] Example 2: Bacillus flexus In vitro antibacterial activity test of BJ699 against strawberry pathogens

[0043] The strawberry pathogens listed in Table 2 were activated from slant culture onto PDA plates and cultured at 30℃ for 6 days for later use. Single colonies of BJ699 were transferred to NB medium and cultured at 35℃ with shaking at 180 rpm for 12 hours to obtain the fermentation broth. 10 μL of the fermentation broth was dropped onto both ends of a PDA plate. After 24 hours, each pathogen was inoculated into a microbial dish using a punch with a 4 mm inner diameter. The microbial dish inoculated with the medium served as a blank control. The distance between each pathogen and the BJ699 microbial dish was 25 mm. After the control colonies fully colonized the plate, the diameters of the colonies in each treatment and the control colonies were counted, and the inhibition rate was calculated using the following formula:

[0044] Inhibition rate = [(Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter)] × 100%

[0045] The diameter of the mycelium cake refers to the diameter of the circular mycelium block (i.e., mycelium cake) removed by the punch, which is 4 mm in this embodiment.

[0046] In vitro antibacterial tests showed that BJ699 exhibited good antibacterial activity against strawberry pathogens such as *Phytophthora indicum*, *Fusarium oxysporum*, *Rhizoctonia solani*, and *Colletotrichum candida* (see Table 2 and...). Figure 3 ).

[0047] Table 2. In vitro antibacterial activity of BJ699

[0048]

[0049] Example 3: Bacillus flexus BJ699 for the control of strawberry root rot

[0050] After activation at 30℃, one loopful of BJ699 slant culture was inoculated into LB liquid medium and cultured for 16 hours at 30℃ and 180 rpm on a shaker to obtain seed culture for shake flask fermentation. The seed culture was then inoculated into the shake flask fermentation medium at a 1% (v / v) inoculation rate and cultured at 30℃ and 180 rpm until 20% of the spores detached, at which point fermentation was terminated, yielding the BJ699 inoculum fermentation broth. The spore count of the BJ699 inoculum fermentation broth was determined to be 12 billion CFU / mL.

[0051] The shake-flask fermentation medium consists of the following components: sweet potato flour 14.5 g / L, yeast extract 17 g / L, corn steep liquor 4 g / L, and magnesium sulfate 1 g / L.

[0052] The experiment in this embodiment was conducted in a strawberry greenhouse in Changping District, Beijing, using the 'Hongyan' strawberry variety. The main pathogen of this strawberry variety is Fusarium oxysporum. The BJ699 inoculant fermentation liquid was diluted 200 times to obtain a root drenching solution. After the strawberry seedlings had recovered from transplanting, the root drenching solution was applied three times, with each application 15 days apart. The amount of water used for each treatment was 200 mL per plant. The blank control was treated with water. All treatment plots were randomly arranged. Each treatment and blank control had three replicates, with 30 plants randomly selected from each replicate.

[0053] After 50% of the control plants showed symptoms, statistics were compiled, and the number of diseased plants at each disease level was recorded.

[0054] The severity grading (see VESTBERG M, KUKKONEN S, SAARI K, et al. Microbialinoculation for improving the growth and health of micropropagated strawberry[J]. Appl Soil Ecol, 2004, 27(3): 243-258.) is as follows:

[0055] Level 1: Root disease incidence ≤ 30%, leaves normal; Level 2: 30% < root disease incidence ≤ 60%, leaves normal; Level 3: 60% < root disease incidence ≤ 80%, leaves yellowing; Level 4: Root disease incidence ≥ 80%, leaves withered; Level 5: Entire plant dead, leaves withered. Disease index = ∑(Number of plants at each disease level × Level) / (Total number of plants surveyed × Highest representative level) × 100. The highest representative level is 5.

[0056] Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0057] The experimental results showed that BJ699 inoculant had a good control effect on strawberry root rot, with a field control efficacy of 80.20% (as shown in Table 3).

[0058] Table 3. Field control efficacy of BJ699 inoculant against strawberry root rot

[0059]

[0060] Example 4: The colonization ability of BJ699 on strawberry roots

[0061] BJ699 was induced to develop resistance in a medium containing rifampicin at a concentration of 300 μg / mL. -1 LB plates containing rifampicin can grow normally.

[0062] Strawberry ('Hongyan' variety) was cultured in seedling pots until it reached the 6-leaf stage. The BJ699 strain, after resistance induction, was fermented according to Example 3. The resulting fermentation broth was diluted 5 times and used as a root drench (10 mL / plant). After 14 days, the plants were removed, and the roots were rinsed three times with sterile water. 1 g of the roots was cut off, ground, diluted, and then spread onto a substrate containing 300 μg / mL of the fermentation broth. -1 The colonization ability of BJ699 on strawberry roots was determined by counting rifampicin on LB plates at 37°C for 24 hours.

[0063] The results showed that BJ699 could establish itself well at the strawberry roots (establishment rate of 9.44 × 10⁻⁶). 6 (cfu / g), this colonization ability is particularly helpful in controlling soil-borne diseases of strawberries.

[0064] Example 5: Degradation of pyraclostrobin by BJ699

[0065] Topsoil (0-20cm) from strawberry greenhouses where no pyraclostrobin was applied was collected as soil samples. After collection, the soil samples were first air-dried in a cool, ventilated place, then ground and passed through a 2mm sieve. A certain amount of pyraclostrobin was dissolved in dimethyl sulfoxide (DMSO), and then used to soak diatomaceous earth to ensure complete adsorption of the pyraclostrobin. The soaked diatomaceous earth was dried in a fume hood and then mixed into the soil to achieve a pyraclostrobin concentration of 5 mg / kg. 500g of each soil sample was incubated at 30℃ in a constant temperature incubator. A 10% inoculum of the BJ699 bacterial agent fermentation broth prepared in Example 3 was added, with a treatment using distilled water as a control. The soil moisture content was maintained at 40% during the incubation process. After 10 days of continuous incubation, samples were taken periodically, and the pyraclostrobin residue was determined by HPLC, and the degradation rate was calculated.

[0066] Degradation rate = (Residual amount in control sample - Residual amount in treated sample) / Residual amount in control sample × 100%

[0067] The results showed that BJ699 had a very good degradation effect on pyraclostrobin, with a degradation rate of 97.67% after 10 days (as shown in Table 4).

[0068] Table 4. Degradation effect of BJ699 on pyraclostrobin

[0069]

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Bacillus curvatureus ( Bacillus flexus strain BJ699, characterized in that, The accession number of strain BJ699 at the China Center for Type Culture Collection is CCTCC NO: M20252013.

2. A microbial inoculant, characterized in that, The microbial agent comprises Bacillus curvatureis as described in claim 1. Bacillus flexus BJ699 strain is used as the active ingredient.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is in the form of fermentation broth.

4. The microbial agent according to claim 3, characterized in that: The culture medium used for the fermentation broth is NB medium, or The culture medium used in the fermentation broth has the following formula: sweet potato powder 14-16 g / L, yeast extract 16-19 g / L, corn steep liquor 3-4 g / L, magnesium sulfate 0.8-1.2 g / L, pH 6.5-7.

5. The Bacillus curvatureus according to claim 1 ( Bacillus flexus The application of strain BJ699 or the microbial agent according to any one of claims 2 to 4 in the preparation of a microbial agent for the control of strawberry root rot and / or degradation of pyraclostrobin; wherein the strawberry root rot is caused by one or more pathogens selected from Phytophthora cactorum, Fusarium oxysporum, Rhizoctonia solani, and Colletotrichum gloeosporioides.

6. The application according to claim 5, characterized in that, The microbial agent is in the form of a fermentation liquid and is applied by root irrigation or spraying.

7. The application according to claim 5, characterized in that, The pyraclostrobin is the form of pyraclostrobin that remains in the soil of strawberry growing areas.

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