A method for screening and reconstructing microorganisms targeting carbon metabolism repression

Abstract

This invention relates to a method for targeted carbon metabolism inhibition microbial screening and microbial community reconstruction, belonging to the field of agricultural disease control. This method uses the carbon metabolism response characteristics of plant pathogenic fungi during host infection as an evaluation index. Through multiple rounds of in vitro screening, strains that can interfere with pathogenic fungi at the nutrient regulation level are obtained from environmental samples. Based on the antagonistic relationships and species complementarity among strains, a microbial ensemble system with synergistic intervention capabilities is constructed. The microbial community constructed by this invention can significantly reduce the adaptability of *Fusarium graminearum* to changes in environmental carbon sources, thereby inhibiting the establishment of its infection-related physiological states. This invention does not rely on traditional antibiotic secretion or direct bactericidal mechanisms, but achieves its inhibitory effect by interfering with the key carbon metabolism regulation processes of pathogenic fungi. It provides a new technical approach for the construction of functional microbial ensemble systems and can serve as a basic technical solution for the development of biocontrol-related products.

Description

Technical Field

[0001] This invention belongs to the field of agricultural disease control, and relates to a bacterial screening method and a functional microbial community construction system based on the nutrient regulation mechanism of plant pathogenic fungi. This invention focuses on the adaptive regulation process of pathogenic fungi to changes in environmental carbon sources, encompassing core technologies such as high-throughput screening of functional microorganisms, construction of multi-species synergistic combinations, and regulation of microbial-pathogen interactions. It is applicable to basic research on the development and application of green control products for crop fungal diseases. Background Technology

[0002] Crop diseases caused by plant pathogenic fungi are a key limiting factor in agricultural production, severely restricting grain yield and quality safety. Currently, field disease control still heavily relies on chemical fungicides, especially in the control of major diseases such as wheat scab, where there are no long-lasting resistant varieties. Agents such as carbendazim, tebuconazole, and cyazofamid are widely used. However, long-term application of chemical agents not only easily leads to drug resistance in pathogens but also poses potential threats to the ecological environment and agricultural product safety. Therefore, developing green, efficient, and stable new disease control technologies is of significant practical importance.

[0003] Current biological control strategies largely rely on antagonistic microorganisms secreting antimicrobial substances or competing for nutrients and ecological niches to directly inhibit pathogen growth. However, in complex field environments, the effectiveness of these methods is easily affected by environmental factors and local microbial communities, resulting in insufficient stability of control efficacy and difficulty in meeting actual production needs. Therefore, developing more precise and controllable intervention methods targeting the key physiological regulatory processes of pathogens has become an important research direction in the field of biological control.

[0004] Plant pathogenic fungi are highly dependent on their ability to sense and adapt to environmental nutrient signals during infection. Carbon repression (CCR), as a conserved global metabolic regulatory mechanism, plays a central role in regulating carbon source utilization, metabolic reprogramming, and infection-related physiological processes. Through CCR regulation, pathogenic fungi can optimize their growth and infection strategies in variable nutrient environments, thereby effectively adapting to the host microenvironment.

[0005] The applicant's previous research has shown that some bacteria from the natural environment can interfere with the pathogenic fungi's response to nutrient signals when interacting with them. For example, Pseudomonas CXZ-8 can interfere with the nutritional adaptation of Fusarium graminearum during the infection stage by influencing CCR-related regulatory processes. However, existing studies have mostly focused on single strains, lacking systematic screening methods for multi-strain synergistic effects and techniques for constructing stable microbial communities.

[0006] In addition, current conventional microbial screening methods mostly rely on phenotypic indicators such as inhibition zones and bactericidal activity, which make it difficult to effectively identify functional microbial resources that can interfere with pathogen carbon metabolism regulation and infection adaptation, and there is also a lack of microbial community construction systems guided by carbon source response regulation.

[0007] Therefore, it is necessary to establish a new method for bacterial screening and microbial community construction. From the perspective of pathogen carbon metabolism regulation and infection adaptability, functional microorganisms with interference effects should be screened, and synergistic communities should be constructed through reasonable combination to weaken the infection-related physiological state of pathogens without directly killing them, thereby providing a new technical path for green prevention and control of agricultural diseases. Summary of the Invention

[0008] To address the aforementioned problems and deficiencies, this invention relates to a method for screening antagonistic bacteria and a microbial community construction system for weakening the infection adaptability of plant pathogenic fungi. This invention focuses on the carbon metabolism regulation mechanism of plant pathogenic fungi in response to changes in environmental nutrient conditions. By screening and combining bacterial strains from different genera, it interferes with the establishment of physiological states related to pathogenic fungal infection, thereby providing a new technological foundation for green prevention and control of agricultural diseases.

[0009] In a first aspect, the present invention provides a method for screening antagonistic bacteria that weaken the infection adaptability of plant pathogenic fungi and a microbial community construction system, comprising the following steps: (1) Using the carbon metabolism response characteristics of plant pathogenic fungi during infection as an evaluation index, bacterial strains isolated from environmental samples were screened in vitro under carbon-deficient conditions to obtain candidate bacterial strains that could interfere with the plant pathogenic fungi at the level of nutrient regulation. (2) Based on the antagonistic relationship and species complementarity among the candidate bacterial strains, the candidate bacterial strains are combined to form a microbial community containing at least three or more bacterial strains. (3) The microbial community is co-inoculated with plant pathogenic fungi to weaken the pathogenic fungi's infection adaptability or the establishment of infection-related physiological states.

[0010] Furthermore, the carbon metabolism response characteristics include changes in the intracellular localization of carbon metabolism repression-related regulatory factors in plant pathogenic fungi under different carbon source conditions. This characteristic can be characterized using a reporter system, which detects the intracellular distribution of the regulatory factors using fluorescent labeling or other detectable signals.

[0011] Furthermore, the species complementarity characteristic refers to a complementary combination of bacterial strains from different species or genera, and there is no significant growth inhibition or competitive exclusion relationship between the candidate bacterial strains. The in vitro screening is a parallel or high-throughput screening of multiple bacterial strains isolated from field or natural environmental samples. The obtained microbial community can influence the infectivity of *Fusarium graminearum*.

[0012] Secondly, the present invention also provides a synergistic microbial community obtained through the above-mentioned screening method and microbial community construction system.

[0013] Furthermore, the synergistic microbial community is a mixed microbial community RMC1-1, which can weaken the infection adaptability or the establishment of infection-related physiological states of the plant pathogenic fungi. Specifically, the mixed microbial community RMC1-1 consists of the following strains: Pseudomonas CXZ-8 Burkholderia CL1, Enterobacter CL2, Bacillus CL3 and Bacillus CL4, and there is no significant mutual growth inhibition or competitive exclusion among the above strains.

[0014] Furthermore, pretreatment of wheat ears with the aforementioned mixed microbial consortium RMC1-1 48 hours before inoculation with Fusarium graminearum significantly inhibited the occurrence of wheat scab. This is in contrast to single-strain inoculation. Pseudomonas Compared to CXZ-8, the mixed bacterial community RMC1-1 exhibits a more stable weakening effect on the pathogenic fungal infection ability. Furthermore, compared to mixed bacterial communities composed of bacterial strains that do not affect the localization of carbon metabolism repression-related regulatory factors in Fusarium graminearum, the RMC1-1 community constructed in this invention shows a more significant and stable inhibitory effect on pathogenic fungal infection.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects: (1) Starting from the infection adaptation and nutritional regulation process of plant pathogenic fungi, it breaks through the traditional control approach that aims to directly inhibit or kill pathogens, and provides a new technical path for green control of agricultural diseases; (2) A screening method for antagonistic bacteria with pathogenic fungal response as the evaluation index was established, which can effectively identify functional microbial resources with the ability to interfere with pathogenic environment adaptation; (3) By introducing a microbial community system that has no antagonistic relationship and is complementary to the species, the stability and controllability of the synergistic effect of multiple bacteria are improved, and the problem of unstable efficacy of single biocontrol bacteria under complex environmental conditions is overcome. (4) The constructed microbial community can play a role without directly inhibiting the survival of pathogens, which helps to reduce selection pressure and delay the development of resistance, and has good ecological compatibility and application potential. Attached Figure Description

[0016] Figure 1 strain Pseudomonas CXZ-8, Burkholderia CL1, Enterobacter CL2, Bacillus CL3 and Bacillus Figure showing the effect of CL4 on the intracellular localization of CreA, a regulatory factor related to carbon metabolism repression in Fusarium graminearum.

[0017] Figure 2 strain Pseudomonas CXZ-8, Burkholderia CL1, Enterobacter CL2, Bacillus CL3 and Bacillus The results of CL4 antagonistic relationship detection under plate conditions.

[0018] Figure 3 The figure shows the effect of mixed microbial communities on wheat spike infection. CK represents the treatment group inoculated only with Fusarium graminearum PH-1, and RMC1-1 represents the treatment group pretreated with mixed microbial community RMC1-1 before PH-1 inoculation.

[0019] Figure 4 This figure shows the comparison between the effects of the mixed bacterial community RMC1-1 and the control mixed bacterial community RMC3-1 on the ability of Fusarium graminearum to infect wheat coleoptiles. The control mixed bacterial community RMC3-1 consisted of strains that did not have the ability to interfere with the intracellular localization of the carbon metabolism repression regulator CreA. Detailed Implementation

[0020] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0021] Example 1 This embodiment provides a method using 4% glucose and bacterial strains. Pseudomonas CXZ-8 is a specific method for screening bacteria that can target Cre, a key factor in the carbon metabolism inhibition of Fusarium graminearum, using CXZ-8 as a reference standard.

[0022] 1.1 Construction of GFP-CreA fusion expression vector Using pKNTG plasmid as a backbone vector, the CreA gene coding sequence (CreA ID: FGSG_09715 in NCBI) was amplified by PCR. After restriction endonuclease digestion, the sequence was ligated into a linearized pKNTG vector to construct a GFP-CreA N-terminal fusion expression vector. The recombinant plasmid was introduced into Fusarium graminearum wild-type strain PH-1 using protoplast-PEG transformation. Transformants were screened and cultured on PDA plates, and genomic DNA was extracted as a template for PCR verification. Positive amplification indicated successful acquisition of a transformed strain expressing the GFP-CreA fusion protein.

[0023] 1.2 Observation of subcellular localization of CreA protein under different treatment conditions The transformed strains were inoculated into carboxymethyl cellulose (CMC) liquid medium (15 g / L Carboxymethyl Cellulose (low viscosity), 1.0 g NH4NO3, 1.0 g KH2PO4, 0.5 g MgSO4·7H2O, 1.0 g Yeast Extract) and cultured for 5 days. Spores were collected and transferred to YEPD medium to induce germination for 12 hours. Germinating hyphae were collected using a sterile filter membrane and washed three times with double-distilled water to thoroughly remove any culture medium residue. The washed hyphae were then placed in suspensions of different field-isolated bacteria without additional carbon sources and co-cultured at 25°C and 175 rpm for 30 minutes (bacterial OD). 600 =1.0 (added at a volume ratio of 1:1000). After treatment, the subcellular localization of CreA protein indicated by GFP signal in each sample was observed and recorded under a fluorescence microscope.

[0024] Group A: Liquid containing 4% glucose; Group B: Blank water control without glucose; Group C: Under carbon-scarce conditions (i.e., without carbon sources available from Fusarium graminearum), add Pseudomonas Aqueous suspensions of CXZ-8 bacteria were co-cultured with pathogenic bacteria; Group D: Under carbon-scarce conditions, an aqueous suspension of Burkholderia CL1 cells was added and co-cultured with the pathogen. Group E: Under conditions of carbon scarcity, add Enterobacter Aqueous suspensions of CL2 bacteria were co-cultured with pathogenic bacteria; Group F: Under conditions of carbon source scarcity, add Bacillus Aqueous suspensions of CL3 bacteria were co-cultured with pathogenic bacteria; Group G: Under conditions of carbon source scarcity, add Bacillus Aqueous suspensions of CL4 bacteria were co-cultured with pathogens.

[0025] The filtering results are as follows Figure 1 As shown, 4% glucose (Group A) or a positive control strain was added to a carbon-deficient environment. Pseudomonas In CXZ-8 (Group C), CreA protein was located in the cell nucleus; in a carbon-deprived environment (negative control) (Group B), CreA underwent significant nucleocytoplasmic translocation; in the DG (four representative strains obtained through screening) treatment groups, the nuclear export of CreA protein was significantly inhibited, and its location was still mainly concentrated in the cell nucleus, indicating that the above strains can interfere with the normal intracellular transport of CreA, a key factor in carbon metabolism repression in Fusarium graminearum.

[0026] Example 2 This embodiment provides a method for detecting the antagonistic relationship between individual strains described in this invention, used to assess their compatibility as a basis for constructing synergistic microbial communities.

[0027] 2.1 To verify the five bacterial strains described in this invention ( Pseudomonas CXZ-8 Burkholderia CL1, Enterobacter CL2, Bacillus CL3 and Bacillus To determine whether significant growth inhibition or competitive exclusion relationships existed among the CL4 strains, the interaction between the strains was detected using a plate confrontation culture method. The five bacterial strains were cultured separately in LB liquid medium at 28°C and 180 rpm with shaking for 12–16 hours, and the bacterial concentration was adjusted to approximately 1 × 10⁻⁶. 8 CFU / mL. Take an LB agar plate and inoculate one side with 5–10 μL of the bacterial suspension of the test strain. After allowing it to air dry, inoculate the remaining bacterial suspensions (5–10 μL each) at equidistant points nearby. Set up three replicates for each strain combination. After inoculation, incubate the plates at 28°C for 24–48 hours, observing and recording the growth of each strain and whether a clear inhibition zone or growth-inhibiting area forms between colonies.

[0028] 2.2 Test results are as follows Figure 2 As shown, all strains grew normally under plate conditions, with clear colony boundaries, and no obvious inhibition zones or growth inhibition were observed between different strains. The results indicate that... Pseudomonas CXZ-8 Burkholderia CL1, Enterobacter CL2, Bacillus CL3 and Bacillus There are no significant antagonistic interactions among the CL4 groups, indicating their compatibility as a basis for the construction of synergistic microbial communities.

[0029] Example 3 This embodiment demonstrates the field control effect of the mixed microbial community described in this invention on the pathogenicity of Fusarium graminearum.

[0030] 3.1 Preparation of Fusarium graminearum spore suspension Wild-type strain PH-1 of *Fusarium graminearum*, cultured on PDA plates for 2-3 days, was inoculated into CMC liquid medium and cultured at 25°C and 175 rpm with shaking for 5 days. Hyphae and spores were collected using a sterile filter membrane, resuspended in sterile water, and the spore concentration was adjusted to 2 × 10⁻⁶ using a hemocytometer. 6 Quantity / mL, for later use.

[0031] 3.2 Preparation of biocontrol bacteria suspension Activation of the five biocontrol bacteria (Pseudomonas CXZ-8, ...) described in this invention from a -80°C glycerol storage tube. Burkholderia CL1, Enterobacter CL2, Bacillus CL3 and Bacillus CL4). Each strain was streaked onto LB agar plates and incubated at 25°C for 2 days. Single colonies were then picked and inoculated into LB liquid medium, and cultured with shaking at 25-30°C and 175 rpm for 12-16 hours. The concentration of each bacterial suspension was adjusted to 1×10⁻⁶ using a spectrophotometer. 8 CFU / mL.

[0032] 3.3 Construction of mixed bacterial suspension and wheat inoculation experiment The five biocontrol bacteria suspensions were mixed in a volume ratio of 1:1:1:1:1 to prepare a solution with a total concentration of 1×10⁻⁶. 8 A mixed bacterial solution of CFU / mL was prepared. Two days before the wheat flowering stage, the mixed bacterial solution was evenly sprayed onto the wheat ears. When the wheat entered the flowering stage, the prepared Fusarium graminearum PH-1 spore suspension (2×10⁻⁶) was applied. 5 Spray the wheat ears with a concentration of 100 cells / mL. Observe and count the disease incidence in the wheat ears 14 days after inoculation.

[0033] like Figure 3 As shown, compared with the treatment group (CK) that was sprayed with only Fusarium graminearum PH-1, the treatment group (RMC1-1) that was sprayed with the mixed microbial community of the present invention in advance had a milder disease, indicating that the mixed microbial community can effectively inhibit the infection and pathogenic process of Fusarium graminearum.

[0034] Example 4 This embodiment provides a comparative verification of the ability of the mixed bacterial community RMC1-1 described in this invention and the control mixed bacterial community to weaken the infectivity of Fusarium graminearum.

[0035] 4.1 To further verify the superior effect of the mixed bacterial community RMC1-1 described in this invention in weakening the infectivity of Fusarium graminearum, a comparative experiment was conducted with a control mixed bacterial community. The control mixed bacterial community (RMC3-1) was composed of five bacterial strains selected in Example 1 that did not possess the ability to interfere with the intracellular localization of CreA, a regulatory factor related to carbon metabolism repression in Fusarium graminearum, mixed in equal volume proportions, with its total bacterial concentration adjusted to be the same as that of RMC1-1.

[0036] 4.2 The mixed bacterial community RMC1-1 and the control mixed bacterial community RMC3-1 were cultured in LB liquid medium to the logarithmic growth phase, and the bacterial concentration was adjusted to OD=0.1. Both mixed bacterial communities were then sprayed evenly onto the surface of wheat coleoptiles, while the control group was sprayed with only an equal volume of sterile water. After pretreatment, a spore suspension (2×10⁻⁶) of the wild-type strain PH-1 of *Fusarium graminearum* was prepared. 5 Coleoptiles in each treatment group were inoculated with cells per mL and cultured under suitable conditions to promote infection. After the specified culture time, the disease incidence in the coleoptiles of each treatment group was observed and statistically analyzed, and the disease index was calculated.

[0037] 4.3 Experimental results are as follows Figure 4 As shown: Compared with the control mixed microbial community treatment group (RMC3-1), the wheat coleoptile disease severity in the treatment group sprayed with mixed microbial community RMC1-1 was significantly reduced, and its disease index was significantly lower than that of the control mixed microbial community treatment group. The results indicate that Pseudomonas CXZ-8, Burkholderia CL1, Enterobacter CL2, Bacillus CL3 and Bacillus The mixed microbial community RMC1-1, composed of CL4, has a superior effect in weakening the infectivity of Fusarium graminearum.

[0038] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A method for screening antagonistic bacteria that weaken the infection adaptability of plant pathogenic fungi and constructing their microbial community, characterized in that, Includes the following steps: (1) Using the carbon source change response characteristics of plant pathogenic fungi as evaluation indicators, in vitro screening was carried out under carbon-deficient conditions to obtain candidate bacterial strains that interfere with their nutritional regulation. (2) Based on the antagonistic relationship and species complementarity among candidate strains, construct a microbial community containing at least three different strains; (3) By co-culturing and screening, mixed bacterial groups that can weaken the infectivity or adaptability of pathogens can be obtained.

2. The method according to claim 1, characterized in that, The aforementioned carbon source change response characteristics refer to the changes in the intracellular localization of the carbon metabolism repressor factor CreA as carbon source conditions change.

3. The method according to claim 2, characterized in that, The response characteristics are characterized by a reporting system that uses fluorescent labeling or other detectable signals to visualize the intracellular distribution of CreA factor.

4. The method according to claim 1, characterized in that, The carbon-deficient condition refers to the absence of a carbon source available to plant pathogenic fungi in the culture system, or a glucose mass fraction of 0%.

5. The method according to claim 1, characterized in that, In the screening in step (1), bacterial strains known to interfere with the nutritional regulation process of plant pathogenic fungi are used as positive controls to determine the screening threshold for interference effects of candidate strains.

6. The method according to claim 1, characterized in that, The in vitro screening in step (1) is a parallel or high-throughput screening of multiple strains of bacteria isolated from natural environmental samples.

7. The method according to claim 1, characterized in that, In step (1), the ability of candidate strains to interfere with the ability of Fusarium graminearum to adapt to changes in environmental carbon sources is used as the screening criterion.

8. The method according to claim 1, characterized in that, The species complementarity specifically refers to the complementary combination formed by strains from multiple species, and there is no significant growth inhibition or competitive exclusion relationship between candidate strains.

9. The microbial community obtained by the method of claim 1.

10. The application of the microbial community according to claim 9, characterized in that, The application includes at least one of the following (1) to (2): (1) Used for developing technologies related to biological control; (2) Used in the preparation of biological control-related products; The targets of the biological control include Fusarium graminearum.

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