A strain of walnut bacterial wilt biocontrol bacteria and its application

Abstract

The present application relates to the field of microbiological technology, and particularly relates to a walnut decay disease biocontrol bacterium Achromobacter insuavis P52 and application thereof.The Achromobacter insuavis is Achromobacter insuavis P52, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.28849.A strain of Achromobacter insuavis is isolated from a pot seedling of Populus trichocarpa, and the strain has a significant antagonistic effect on a walnut decay disease pathogenic fungus Cytospora chrysosperma, with an inhibition rate of 62.4%, and an in-vitro prevention and treatment effect on walnut branch decay disease of 76%.The Achromobacter insuavis in the present application has a significant prevention and treatment effect on walnut decay disease, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a biocontrol bacterium for walnut rot, Achromobacterium tumefaciens P52, and its applications. Background Technology

[0002] Walnut rot, also known as bark rot or black water disease, is a relatively common fungal disease, generally believed to be caused by fungi of the genus *Cytospora*, with *Cytosporachrysosperma* being the most common causative species. Walnut rot occurs in all walnut-producing areas of China. This disease primarily affects the branches, trunk, and bark of walnut trees. Symptoms vary depending on the age of the tree and the affected area. In young trees, the lesions penetrate deep into the xylem, with callus tissue appearing around them. These appear as dark gray, rhomboid spots, water-soaked, and release a liquid with a fermented odor when pressed. In later stages, the lesions sink, developing black spots, which then crack longitudinally, exuding a large amount of black liquid. In mature trees, due to the thicker bark, there are no obvious external symptoms. When black liquid is observed oozing from the bark, large ulcers have already formed beneath the bark. In major walnut-growing areas and regions with poor management and weak tree vigor, the incidence rate of this disease can reach over 80% when it is severe, leading to the death of large branches or even the entire tree, seriously threatening walnut production.

[0003] Currently, the main control methods for walnut rot include chemical control and physical control. While chemical control is faster, it threatens ecological security and human and animal health, and excessive application of chemical agents can easily lead to drug resistance in pathogens, increasing the difficulty of control. Physical control mainly uses tools such as scrapers to remove rot lesions, which can reduce the damage of rot to walnut trees, but it cannot eradicate the disease and recurrence is common. Biological control has become one of the important measures for the green and efficient control of walnut rot.

[0004] Biological control of walnut rot mainly utilizes pathogen-controlling microorganisms to inhibit the occurrence of the disease. This method has advantages such as no residual toxins, no pollution, no harm to humans and animals, low likelihood of developing drug resistance, and long-lasting efficacy. Therefore, the exploration and efficient application of highly effective biocontrol microbial strains for walnut rot have attracted widespread attention.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a biocontrol bacterium against walnut rot, *Achromobacterium tumefaciens* P52, and its applications. *Achromobacterium tumefaciens* was isolated from potted *Populus tomentosa* seedlings. *Achromobacterium tumefaciens* and its microbial preparations exhibit significant antagonistic effects against *Cytospora* sp. fungi, demonstrating good control efficacy against walnut rot and showing broad application prospects.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] In one aspect, the present invention provides an Achromobacter insuavis strain, namely Achromobacter insuavis P52, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 28849.

[0009] This invention isolated a strain of *Achromobacterium tumefaciens* from potted *Populus tomentosa* seedlings. This strain exhibits significant antagonistic effects against *Cytospora* sp. fungi, achieving an inhibition rate of 62.4% on PDA plates. Under in vitro conditions, it demonstrates a 76% control effect on walnut branch rot. The *Achromobacterium tumefaciens* strain and the microbial preparations prepared from it show significant control effects on walnut rot and have promising application prospects.

[0010] In some specific embodiments, the sequence of the 16S rRNA gene fragment of Achromobacter insuavis P52 described in this invention is shown in SEQ ID No. 1.

[0011] In another aspect, the present invention provides a biocontrol microbial preparation, the active ingredient of which comprises the Achromobacter insuavis P52.

[0012] The biocontrol microbial agent of this invention contains Achromobacter insuavis P52 isolated from poplar bonsai seedlings. This strain has a significant antagonistic effect on Cytospora sp. fungi. The biocontrol microbial agent provided by this invention has a significant effect on the prevention and control of walnut rot disease and has good application prospects.

[0013] In some specific embodiments, the biocontrol microbial preparation includes a fermentation culture of the Achromobacter insuavis P52, such as, but not limited to, strain cultures, fermentation broths, or filtrates of fermentation broths.

[0014] In some specific embodiments, the content of Achromobacter insuavis P52 in the biocontrol microbial preparation is 1×10⁻⁶. 6 CFU / mL or higher.

[0015] In some specific embodiments, the content of *Achromobacter insuavis* P52 in the biocontrol microbial preparation is preferably 1 × 10⁻⁶. 6 CFU / mL to 1×10 9 CFU / mL.

[0016] In some specific embodiments, the biocontrol microbial preparation is obtained by fermenting the aforementioned Achromobacter insuavis P52 in a liquid culture medium, collecting the culture, and then collecting the culture.

[0017] In this invention, the biocontrol microbial preparation can also be prepared together with other adjuvants or excipients to form a biocontrol microbial preparation.

[0018] In another aspect, the present invention provides the use of the aforementioned Achromobacterium tumefaciens or the aforementioned biocontrol microbial agents in the prevention and control of plant diseases.

[0019] In some specific embodiments, the application includes inoculating the achromobacterium tumefaciens into a liquid culture medium, culturing it at 180-200 rpm and 28-32°C for more than 24 hours, and using the fermentation broth, the supernatant obtained after centrifuging the fermentation broth, the sterile filtrate obtained by filtering the supernatant with a sterile filter membrane, and the fermentation mixture to control or prevent plant diseases.

[0020] In some specific embodiments, the plant includes, but is not limited to, at least one of walnut, poplar, or willow.

[0021] In some specific embodiments, the plant is a walnut.

[0022] In some specific embodiments, the plant diseases include diseases caused by fungi of the genus *Cyclophorus*.

[0023] In some specific embodiments, the plant disease includes, but is not limited to, rot.

[0024] In some specific embodiments, the plant disease includes, but is not limited to, walnut rot.

[0025] In another aspect, the present invention provides a method for preventing and controlling walnut rot, the method comprising applying the aforementioned Achromobacterium tumefaciens or a biocontrol microbial preparation containing it as an active ingredient to the walnut.

[0026] In some specific embodiments, the method involves preparing the anaphylactic colorless bacillus into a bacterial suspension and then applying it to the diseased parts of the walnut plant.

[0027] In some specific embodiments, the achromobacterium tumefaciens is prepared into a bacterial suspension and then applied to walnut branches.

[0028] Biological sample preservation information: Achromobacter insuavis P52, this strain was deposited on November 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 28849; deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Postcode: 100101. The sample was confirmed as a viable strain by the preservation center on November 3, 2023.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) The Achromobacter insuavis P52 provided by this invention is a novel strain. This invention is the first to discover its effect in controlling plant rot. This strain has good biocontrol potential, especially against walnut rot. Under in vitro conditions, the control effect on walnut branch rot reaches 76%.

[0031] (2) The Achromobacter insuavis P52 strain used in this invention is highly safe, low-cost, residue-free, environmentally friendly, and efficient. The results of plate antagonism and in vitro walnut branch disease control experiments show that strain P52 in this invention has a significant control effect on walnut rot disease, providing a new and effective approach for the control of rot diseases in woody plants such as walnut and poplar, and has good application prospects. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 The inhibitory effect of *C. chrysospermae* P52 on the growth of *C. chrysospermae* was defined as follows: A represents the plate antagonistic effect of strain P52 on *C. chrysospermae* (C. chrysospermae was the control group, inoculated with *C. chrysospermae*; C. chrysospermae + P52 was the treatment group, inoculated with both *C. chrysospermae* and strain P52); B represents the colony diameter of *C. chrysospermae*; C represents the inhibition rate of strain P52 on *C. chrysospermae*.

[0034] Figure 2 Colony morphology of Achromobacterium tumefaciens P52 (scale bar: 5mm);

[0035] Figure 3 A phylogenetic tree constructed based on the 16S rRNA gene fragment sequence of Achromobacter p52;

[0036] Figure 4 The severity level of detached walnut branch rot (scale bar: 1cm);

[0037] Figure 5 The study aimed to assess the control effect of *C. chrysosperma* P52 on detached walnut branch rot. A represents walnut branches one day after treatment [*C. chrysosperma+LB* was the positive control group, inoculated with *C. chrysosperma* and Luria-Bertani (LB) medium; *C. chrysosperma+P52* was the treatment group, inoculated with both *C. chrysosperma* and strain P52; scale bar: 1 cm]; B represents walnut branches seven days after treatment; C represents peeled walnut branches seven days after treatment; D represents the disease index of detached walnut branch rot; E represents the control effect of strain P52 on detached walnut branch rot. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0039] Example 1: Isolation and Screening of Strains

[0040] Isolation: The strain P52 in this invention was isolated from poplar seedlings.

[0041] Screening: A 6 mm diameter Cytospora chrysosperma mycelial cake was inoculated in the center of a potato dextrose agar (PDA medium: potato 200 g / L, glucose 20 g / L, agar 20 g / L) plate.

[0042] Take 2 μL of bacterial suspension of strain P52 (OD) 600=1) Add the solution dropwise to a plate inoculated with the pathogenic fungus, using Luria-Bertani medium (LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7.0) as a control. Incubate at 28°C for 5 days, record the colony diameter of the pathogenic fungus, and calculate the inhibition rate. Inhibition rate calculation formula:

[0043] Inhibition rate = (Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group) / Coronavirus colony diameter in control group × 100%.

[0044] After 5 days of constant temperature incubation, the colony diameter of *C. chrysosperma* in the control group was 8.5 cm, while that in the treatment group was 3.2 cm, significantly lower than that in the control group, with an inhibition rate of 62.4%. This indicates that strain P52 has a significant antagonistic effect on *C. chrysosperma*. Figure 1 ).

[0045] Example 2: Strain Identification

[0046] (1) Morphological characteristics of colonies

[0047] After incubating at 30℃ for 24 hours, the colony morphology of strain P52 was observed. Figure 2 The colonies of strain P52 are milky white, round, with neat edges, slightly convex surface, and are moist and glossy.

[0048] (2) Physiological and biochemical characteristics of the strain

[0049] The results of physiological and biochemical tests showed that strain P52 was a Gram-negative bacterium. Its results for starch hydrolysis, fat hydrolysis, protein hydrolysis, cellulose hydrolysis, VP test, MR test, and hydrogen sulfide test were all negative, while the hydrogen peroxide test was positive. The test results are shown in Table 1.

[0050] Table 1. Physiological and biochemical test results of strain P52

[0051]

[0052] (3) Molecular biological identification of the strain

[0053] Genomic DNA was extracted from strain P52, and the 16S rRNA gene fragment was amplified by PCR. After electrophoresis detection, purification and sequencing analysis of the amplification product, the 16S rRNA gene fragment sequence of strain P52 as shown in SEQ ID No. 1 was obtained.

[0054] SEQ ID No. 1:

[0055]

[0056] The above sequences were submitted to the National Center for Biotechnology Information (NCBI) for BLAST comparison. The results showed that the sequence with the highest similarity to the 16S rRNA gene fragment of strain P52 was Achromobacter insuavis LMG 26845, with a similarity of 99.64% and a coverage of 100%. Thus, strain P52 was identified as Achromobacter insuavis.

[0057] A phylogenetic tree was constructed using 16S rRNA gene fragment sequences from six strains of the genus *Achromobacter* (*Achromobacter pulmonis* R-16442T, *Achromobacter aegrifaciens* LMG 26852, *Achromobacter aloeverae* AVA-1, *Achromobacter cycloclastes* IAM1013, *Achromobacter insuavis* LMG 26845, and *Achromobacter ruhlandii* EY3918) and strain P52. Figure 3 The results showed that strain P52 was most closely related to Achromobacter insuavis, and therefore strain P52 could be identified as Achromobacter insuavis and named Achromobacter insuavis P52.

[0058] (4) Preservation of strains

[0059] Biological sample preservation information: Achromobacter insuavis P52, this strain was deposited on November 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 28849; deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Postcode: 100101. The sample was confirmed as a viable strain by the preservation center on November 3, 2023.

[0060] Example 3: Disease Prevention Experiment Using Isolated Walnut Branches

[0061] The bacterial suspension of Achromobacterium tumefaciens P52 in this invention was adjusted to OD. 600=0.2, walnut branches approximately 8 cm long were soaked in the bacterial suspension for 1 hour, then dried. The branches were then scalded, and *Cyclocarya paliurus* mycelial cakes approximately 6 mm in diameter were inoculated at the wound sites as the treatment group. Walnut branches approximately 8 cm long were soaked in LB liquid medium for 1 hour, then dried. The branches were then scalded, and *Cyclocarya paliurus* mycelial cakes approximately 6 mm in diameter were inoculated at the wound sites as the blank control and positive control, respectively. The walnut branches were kept moist and incubated at a constant temperature of 25℃. Disease incidence was observed, and the rot disease was graded according to the percentage of lesion length to the branch length. The disease index and disease control effect were calculated. Figure 4 The severity levels of detached walnut branch rot are shown on a scale bar of 1 cm. Level 0: No lesions on the branch; Level 1: Lesions less than 20% of the branch length; Level 2: Lesions between 20% and 40% of the branch length; Level 3: Lesions between 40% and 60% of the branch length; Level 4: Lesions between 60% and 80% of the branch length; Level 5: Lesions exceeding 80% of the branch length.

[0062] Formula for calculating the disease index:

[0063] Disease index = (number of branches at each disease level × disease level) / (total number of branches × 5) × 100.

[0064] Formula for calculating prevention and control effect:

[0065] Prevention and control effect = (disease index of positive control group - disease index of treatment group) / disease index of positive control group × 100%.

[0066] like Figure 5 As shown, 7 days after inoculation with *Cyclocarya paliurus*, the walnut branches in the positive control group had turned completely red and appeared dehydrated, with the branches after peeling off the bark being reddish-brown; while the walnut branches inoculated with strain P52 and *Cyclocarya paliurus* only turned slightly red, and the branches after peeling off the bark were white.

[0067] By measuring the length of walnut branches and the length of lesions 7 days after inoculation with *C. chrysosperma*, the average length of lesions in the positive control group (*C. chrysosperma* + LB) was equal to the average length of the branches, with a disease index of 100. In the treatment group (*C. chrysosperma* + strain P52), the average length of lesions was only 18.4% of the average branch length, with a disease index of 24. Strain P52 achieved a 76% control effect against detached walnut branch rot, demonstrating good disease control efficacy.

[0068] In summary, this invention is the first to propose that Achromobacter insuavis P52 has the function of preventing and controlling walnut rot, and its control effect on detached walnut branch rot reaches 76%, showing good application prospects.

[0069] The Achromobacter insuavis P52 strain used in this invention is highly safe, low-cost, residue-free, environmentally friendly, and efficient. Results from plate antagonism and in vitro walnut branch disease control experiments show that strain P52 has a significant control effect on walnut rot disease, providing a new and effective approach for the control of rot diseases in woody plants such as walnut and poplar, and has promising application prospects.

[0070] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. A biocontrol bacterium for walnut rot disease is Achromobacterium tumefaciens ( ) Achromobacter insuavis P52, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 28849.

2. A biocontrol microbial preparation, characterized in that, The active ingredient of the biocontrol microbial preparation comprises the anaphylactobacillus anaphylactii described in claim 1. Achromobacter insuavis P52.

3. The biocontrol microbial preparation according to claim 2, characterized in that, The biocontrol microbial preparation includes the anaphylactobacillus ( ). Achromobacter insuavis Fermentation culture of P52.

4. The biocontrol microbial preparation according to claim 3, characterized in that, In the biocontrol microbial preparation, the anaphylactobacillus ( Achromobacter insuavis The content of P52 is 1×10 6 CFU / mL or higher.

5. The biocontrol microbial preparation according to claim 4, characterized in that, By culturing the nasty colorless bacillus in liquid culture medium ( Achromobacter insuavis P52, collect the fermentation culture to obtain the biocontrol microbial preparation.

6. The biocontrol microbial preparation according to claim 1 or any one of claims 2-5 for controlling *Achromobacterium tumefaciens* (… Cytospora chrysosperma Applications of this technology in plant diseases caused by ( ).

7. The application according to claim 6, characterized in that, The plant disease mentioned is walnut rot.

8. A method for preventing and controlling walnut rot disease, characterized in that: The method includes applying the anaphylactic achromobacterium of claim 1 or any of the biocontrol microbial agents of claims 2-5 to the walnut.

9. The method according to claim 8, characterized in that, The method involves preparing the pesky colorless bacillus into a bacterial suspension and then applying it to the diseased parts of the walnut plant.

10. The method according to claim 9, characterized in that, The method involves preparing the anaphylactic colorless bacillus into a bacterial suspension and then applying it to walnut branches.

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