Lactobacillus hilakii with antibacterial activity and application thereof

Abstract

The application discloses a lactobacillus hilardi with antibacterial activity and application thereof, and belongs to the technical field of crop disease biological control. Lactobacillus hilgardii The lactobacillus hilardi (CNZD-007) is preserved in the China General Microbiological Culture Collection Center on September 9, 2022, and the preservation number is CGMCC No. 25670. The lactobacillus hilardi CNZD-007 strain is isolated from corn, and it is proved through a plate confrontation test that the lactobacillus hilardi CNZD-007 strain can effectively antagonize various corn pathogenic fungi, the screened strain CNZD-007 has broad-spectrum antibacterial property, and it is proved through a potting test that the strain can effectively control corn stem rot, and safe and reliable biological products are provided for corn disease control.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for crop diseases, and in particular to a strain of Lactobacillus sheari with antibacterial activity and its applications. Background Technology

[0002] Corn is an important food and cash crop in my country's agricultural production, with a wide range of applications and significant economic benefits. However, soil-borne diseases have always existed during the planting and growth of corn. Among them, corn stalk rot is a serious soil-borne disease that threatens corn production, causing severe lodging and yield loss. It is widely distributed and causes severe damage. Currently, the main methods for controlling this disease are chemical seed dressing agents and increased application of potassium fertilizer, but the control efficacy is generally poor, with most showing only a 30% effect. Furthermore, the long-term use of chemical seed dressing agents and fertilizers has had varying degrees of adverse effects on the soil ecological environment.

[0003] Biological control, with its environmentally friendly, safe, reliable, cost-effective, and efficient characteristics, has become a research hotspot in recent years, with a very broad application prospect. Therefore, this invention screened and obtained a new strain with broad-spectrum antibacterial activity, providing a new approach for the control of soil-borne diseases in maize. Summary of the Invention

[0004] The purpose of this invention is to provide a *Lactobacillus sheari* with antibacterial activity and its application, in order to solve the problems existing in the prior art. The *Lactobacillus sheari* CNZD-007 provided by this invention can effectively antagonize a variety of maize pathogenic fungi and effectively control maize stalk rot, providing a safe and reliable biological product for the prevention and control of maize diseases.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a Lactobacillus sherry ( Lactobacillus hilgardii CNZD-007 was deposited on September 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25670.

[0007] The present invention also provides a microbial agent comprising the Lactobacillus hesseri CNZD-007 strain or its fermentation broth.

[0008] Furthermore, the concentration of Lactobacillus hessler CNZD-007 in the fermentation broth is 1×10⁻⁶. 8 ~1×10 10 cfu / mL.

[0009] The present invention also provides the application of the aforementioned Lactobacillus hesseri CNZD-007 or the aforementioned inoculum in inhibiting the growth of maize pathogenic fungi.

[0010] Furthermore, the maize pathogenic fungi include *Monosporium zeylanum*, *Fusarium zeylanum*, *Fusarium graminearum*, *Fusarium moniliforme*, *Pythium*, *Colletotrichum graminearum*, and *Cephalosporium zeylanum*.

[0011] The present invention also provides the application of the aforementioned Lactobacillus hessler CNZD-007 or the aforementioned bacterial agent in the preparation of a drug for preventing and controlling corn stalk rot.

[0012] The present invention also provides a method for preventing and controlling corn stalk rot, which involves applying the Lactobacillus hessei CNZD-007 or the bacterial agent to the corn planting soil.

[0013] The present invention also provides a drug for preventing and controlling corn stalk rot, comprising the aforementioned Lactobacillus hesseri CNZD-007 or the aforementioned inoculum.

[0014] The present invention discloses the following technical effects:

[0015] This invention isolates a strain of Lactobacillus hesseri from corn. Lactobacillus hilgardii The CNZD-007 strain was demonstrated through plate confrontation tests to effectively antagonize a variety of maize pathogenic fungi. The selected strain CNZD-007 exhibits broad-spectrum antibacterial activity. Combined with pot experiments, it was confirmed that this strain can effectively control maize stalk rot, providing a safe and reliable biological product for the prevention and control of maize diseases. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results show the inhibition rate of Lactobacillus hessler strain CNZD-007 against seven maize pathogenic fungi. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1: Isolation, identification, and preservation of strain CNZD-007

[0024] 1. Isolation and identification of strains

[0025] Place the cleaned corn kernels into a sterilized beaker, add 150 mL of sterile water, and shake at 100 rpm for 2 hours. Dilute the suspension to 10... -1 10 -2 10 -3 Then, 150 μL was spread onto an MRS solid plate and incubated statically at 37°C upside down for 72 h. Single colonies were picked and streaked onto new MRS solid plates for a second streaking culture. The obtained monoclonal strain was obtained by a second streaking culture to obtain pure strain CNZD-007. After growing on MRS medium at 37°C for 3 days, the colonies formed by this strain were small, white, round, moist, with a smooth surface and short rod-shaped cells.

[0026] The method of directly amplifying 16S rDNA from bacterial cells was adopted. Universal bacterial primers for 16S rDNA amplification were used, specifically 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 2) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 3). The 25 μL PCR reaction system contained: 1 μL DNA template, 3 μL dNTPs (10 mM), 1 μL each of primers (10 mM), 7 μL 10x PCR Buffer, 2 μL MgSO4 (50 mM), and 5 U Taq enzyme. -1 0.5 μL of amplified DNA and 9.5 μL of ultrapure water were added. PCR reaction conditions: pre-denaturation at 95℃ for 5 min, followed by thermal cycling: denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 90 s, for 30 cycles; final extension at 72℃ for 10 min. After amplification, 5 μL of the amplified sample was electrophoresed on a 1% agarose gel at 60 V. The target DNA fragment was recovered and sent to a sequencing company for sequencing. The sequencing results are as follows (SEQ ID No. 1):

[0027]

[0028] The assembled sequence was compared with the NCBI database using BLAST, and combined with morphological identification results, strain CNZD-007 was identified as Lactobacillus hesseri. Lactobacillus hilgardii ).

[0029] 2. Preservation of strain CNZD-007

[0030] The strain CNZD-007 is classified as Lactobacillus heathi ( Lactobacillus hilgardii It was deposited on September 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25670.

[0031] Example 2: Antibacterial effect of Lactobacillus hessler strain CNZD-007 against pathogenic fungi.

[0032] Preparation of Lactobacillus hessler CNZD-007 bacterial suspension: After activating the screened Lactobacillus hessler in MRS medium, the OD of the bacterial suspension was adjusted. 600nm =0.5, and inoculated at a rate of 2% into 100 mL of MRS medium for expansion culture. The culture was incubated at 37℃ for 2 days to obtain fresh bacterial suspension. Then, the CNZD-007 bacterial suspension was centrifuged at 4000 r / min for 20 min, the waste liquid was discarded, the precipitate was resuspended in sterile water, centrifuged again at 4000 r / min for 20 min, the waste liquid was discarded, sterile water was added, and the OD of the fermentation broth was... 600 Adjust the value to OD 600 =1 is reserved.

[0033] CNZD-007 strain was subjected to plate confrontation culture with various maize pathogenic fungi (Cephalosporium chrysogenum). Diplodia maydis Fusarium truncatum specialized type ( Fusarium roseum f.sp.cerealis Fusarium graminearum ( ), F. graminearum Fusarium moniliforme () F. moniliforme ), Pythium ( Pythium spp. ), *Colletotrichum gracilis* ( Colletotrichum graminicolum ) and Cephalosporium maize ( Cephalosporium maydisAntagonistic experiments were conducted using samples from the biological laboratory of Zhongdao Ecological Environment Engineering (Huimin) Co., Ltd. Activated corn pathogenic fungal mycelial cakes (d=6 mm) were inoculated at the center of MRS medium. Two perpendicular lines were drawn with the center of the medium as the intersection point. A position 20 mm from the intersection point was marked on each line, and a sterile filter paper disc was placed on top. 10 μL of CNZD-007 strain fermentation broth was added to the sterile filter paper disc. After standing for 30 min, the discs were incubated upside down at 28℃. Each treatment was repeated three times. After 4 days of incubation, the colony diameter of the pathogenic fungus in the treatment and control groups was measured to determine the antagonistic effect of Lactobacillus hesseri CNZD-007 strain. Results are shown below. Figure 1 See Table 1.

[0034] Table 1. Inhibition rate of CNZD-007 strain against seven maize pathogenic fungi

[0035]

[0036] Note: The formula is: inhibition rate (%) = (colon radius of control group - colony radius of treatment group) / colony radius of control group × 100;

[0037] The values ​​in the table are the mean ± standard deviation;

[0038] The results, as indicated by the t-test, showed a significant difference between the treatment group and the control group at the 1% level.

[0039] Example 3: Efficacy of Lactobacillus hessler strain CNZD-007 against maize stalk rot

[0040] 1. Preparation of Lactobacillus schlegelii inoculum and pathogens

[0041] The Lactobacillus hesitant CNZD-007 bacterial culture prepared in Example 2 was concentrated, freeze-dried, and pulverized to obtain a bacterial count of 1×10⁻⁶. 8 Solid bacterial agent with cfu / g; Preparation of pathogen inoculum: Soak corn kernels overnight, cook them in water, crush the corn seed coat, dispense into Erlenmeyer flasks, and inoculate Fusarium graminearum into PDA liquid medium at a ratio of 0.5% (v / w). Incubate in a light incubator for 7 days to obtain the inoculum - Fusarium graminearum corn kernel culture.

[0042] 2. Pot experiment

[0043] A pot experiment was conducted on Zhengdan 958 maize seedlings. Before planting, 5g of Lactobacillus hesperidin CNZD-007 inoculant was applied to the soil holes per pot. A control without inoculant was used. Five plants were planted per pot, and five pots were used for each treatment. The emergence rate was recorded when the average emergence rate reached 50%. Seedling height was measured at the 3-leaf stage. All pots were inoculated with 10g of Fusarium maize kernel culture using the root-damaging method. Disease incidence was recorded after 7 days, and the disease index and control efficacy were calculated. Disease incidence (%) = (Number of diseased plants / Total number of plants surveyed) × 100; Disease index = (Number of diseased plants × Representative value for that disease level) / (Total number of plants × Representative value for the most severe disease level) × 100; Control efficacy = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0044] Seedling stem rot disease severity assessment criteria: Grade 0: No disease; Grade 1: Normal growth of above-ground and underground parts, with a few lesions visible on the roots, covering less than 1 / 4 of the total root surface area, and the root system is whitish-brown in color; Grade 2: Significantly inhibited growth of above-ground and underground parts, pale leaves, few and short lateral roots, no fibrous roots, lesions confluent, covering 1 / 4 to 1 / 2 of the total root surface area, and the root system is predominantly whitish-brown in color; Grade 3: Extremely abnormal growth of above-ground and underground parts, with wilting and yellowing of the above-ground parts, very small lateral roots, lesions covering 1 / 2 to 3 / 4 of the total root surface area, and the root system is brownish-white in color; Grade 4: Sprouting but failing to emerge, almost suffocating and dying, lesions covering more than 3 / 4 of the total root surface area, and the roots are brown.

[0045] Table 2. Control efficacy of Lactobacillus hesperidin strain CNZD-007 against maize stalk rot.

[0046]

[0047] As shown in Table 2, Lactobacillus hessler CNZD-007 can effectively reduce the incidence and disease index of corn stalk rot caused by Fusarium graminearum, and has a significant biocontrol effect.

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Lactobacillus histriense ( Lactobacillus hilgardii CNZD-007, characterized in that, It was deposited on September 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25670.

2. A microbial agent, characterized in that, It contains the Lactobacillus hesseri CNZD-007 strain or its fermentation broth as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The concentration of Lactobacillus hesitant CNZD-007 in the fermentation broth was 1×10⁻⁶. 8 ~1×10 10 cfu / mL.

4. The application of *Lactobacillus schlegelii* CNZD-007 as described in claim 1 or the inoculum agent as described in any one of claims 2-3 in inhibiting the growth of maize pathogenic fungi, characterized in that, The pathogenic fungus of maize is *Monosporium cuspidatum* (also known as *Cephalospora maize*). Diplodia maydis Fusarium truncatum specialized type ( Fusarium roseumf.sp.cerealis Fusarium graminearum () F. graminearum Fusarium moniliforme () F.moniliforme ), Pythium ( Pythium spp ), Colchicum gracilis ( Coletotrichum graminicolum ) and Cephalosporium maize ( Cephalosporium maydis ).

5. The use of Lactobacillus hesseri CNZD-007 as described in claim 1 or the bacterial agent as described in any one of claims 2-3 in the preparation of a drug for controlling corn stalk rot.

6. A method for preventing and controlling corn stalk rot, characterized in that, The Lactobacillus hesseri CNZD-007 of claim 1 or the inoculum of any one of claims 2-3 is applied to the soil in which corn is planted.

7. A drug for preventing and controlling corn stalk rot, characterized in that, It contains the Lactobacillus hesseri CNZD-007 as described in claim 1 or the bacterial agent as described in any one of claims 2-3.

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