Synthetic microbial consortium b2 for preventing and treating banana fusarium wilt and application thereof

Abstract

The present application relates to the field of microbial technology, and specifically provides a synthetic microbial consortium B2 for preventing and treating banana wilt and application thereof. The synthetic microbial consortium B2 is composed of three antagonistic bacteria S-1-56, S-2-42 and 1-119 of Pseudomonas genus isolated from the rhizosphere soil of banana. Foc The synthetic microbial consortium B2 has a significant synergistic inhibitory effect. The synthetic microbial consortium B2 can be applied to the rhizosphere soil of banana by irrigation and the like, effectively prevents and controls the occurrence and spread of banana wilt, has the advantages of environmental friendliness, stable prevention and treatment effect, strong adaptability and the like, and is suitable for the production practice of green and sustainable agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a synthetic microbial group B2 for the prevention and control of banana wilt disease and its application. Background Technology

[0002] Bananas are an important global food crop and economic fruit, and their production has long been severely threatened by Fusarium wilt (also known as Panama disease). This disease is caused by Fusarium oxysporum f. sp. Cuban specific variant (…). Fusarium oxysporum f. sp. Cuban Soil-borne vascular diseases caused by Foc (Functional Orientation of the Banana), among which Tropical Race 4 (TR4) is the most pathogenic and has the widest host range. It can infect almost all banana cultivars (including the currently main Cavendish series), causing plant wilting and death, resulting in devastating losses. Moreover, the pathogen can survive in the soil for a long time, making control extremely difficult.

[0003] Currently, regarding banana wilt disease (… Fire For the control of TR4 (trichlorobenzene 4), there is a lack of highly resistant commercial varieties, and the main approach relies on integrated management measures. Traditionally, chemical control has been the primary method, which involves applying fumigants or fungicides to reduce the number of pathogens in the soil. However, chemical control has significant limitations: firstly, most chemical agents are unable to completely kill pathogens deep in the soil, resulting in limited and short-lasting efficacy; secondly, long-term and excessive use of chemical agents can easily lead to drug resistance in pathogens, while also causing problems such as soil microbial imbalance, environmental pollution, and excessive residues in agricultural products, which does not meet the requirements of sustainable agricultural development.

[0004] In recent years, biological control has become a research hotspot in this field due to its environmentally friendly and sustainable characteristics. Utilizing beneficial microorganisms (biocontrol bacteria) to inhibit pathogen growth, induce systemic resistance in plants, or compete for ecological sites is an effective strategy for achieving green disease control. Current research and applications largely focus on the screening and utilization of single strains, such as certain Bacillus species (…). Bacillus spp.), Pseudomonas ( Pseudomonas spp.) and Trichoderma ( Trichoderma (spp.), etc. Although some single strains show good antagonistic activity in the laboratory or under controlled conditions, they often face problems such as weak colonization ability, poor stability, and fluctuating control effect due to environmental factors (such as soil type, pH, moisture, and indigenous microbial communities) in the actual complex field soil environment, which limits the effectiveness and reliability of their large-scale application.

[0005] Furthermore, the plant rhizosphere micro-ecosystem is a complex microbial network, and the occurrence and development of diseases are manifestations of an overall imbalance in the microbial community's function. The introduction of a single strain is unlikely to simulate or reconstruct the function of a healthy microbial community. In contrast, a "synthetic microbial community" composed of multiple complementary and symbiotic beneficial strains combined in specific proportions may, through synergy and specialization among microorganisms, more stably colonize the rhizosphere, forming a more resilient and inhibitory microecological barrier, thereby achieving more efficient and sustained control of soil-borne pathogens. However, how to screen core strains with synergistic effects from abundant microbial resources and construct targeted... Fire The efficient, stable, and easy-to-produce synthetic microbial preparations of TR4 remain a key issue that urgently needs to be addressed in current biological control technologies.

[0006] Based on this, the present invention aims to overcome the above-mentioned defects of the prior art and provide a synthetic bacterial community composed of a specific strain, which is effective against the pathogen of banana wilt (Fusarium wilt). Fire TR4 has a significant and stable synergistic antagonistic effect, which can effectively control diseases and has good environmental adaptability and application potential. Summary of the Invention

[0007] The purpose of this invention is to provide a synthetic microbial community and its application to overcome existing banana wilt disease (…). Fire Deficiencies exist in TR4 prevention and control technologies.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides a synthetic bacterial consortium B2 for controlling banana wilt disease. Its core feature is that it consists of three antagonistic bacteria isolated, screened, and identified from banana rhizosphere soil: strain S-1-56, strain S-2-42, and strain 1-119. Strain S-1-56 has the accession number GDMCC No:67659, the accession date is January 14, 2026, and the accession classification is named... Pseudomonas The strain S-2-42 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with the telephone number 020-87137633. Its accession number is GDMCC No:67660, the deposit date is January 14, 2026, and the accession classification is named as follows: Pseudomonas The depositary institution for strain 1-119 is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with telephone number 020-87137633; the accession number for strain 1-119 is GDMCC No:67658, the deposit date is January 14, 2026, and the accession classification is named as follows: Pseudomonas sp., deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, Tel: 020-87137633.

[0009] The strains S-1-56, S-2-42, and 1-119 were obtained using the following method: banana rhizosphere soil samples were collected using the root shaking method, separated by dilution plating, purified by LB broth streak plating, and then screened for their resistance to [specific strains] using the plate confrontation method. Fire The TR4 strain exhibited significant antagonistic activity. Sequencing and phylogenetic analysis confirmed the strain's taxonomic position, and compatibility testing verified that there was no antagonistic effect among the three strains, indicating a basis for synergistic action.

[0010] A second aspect of the present invention provides the direct application of the synthetic microbial community B2: the synthetic microbial community B2 can be directly used to inhibit the pathogen of banana wilt disease. Fire TR4 inhibits the growth of banana plants by forming a dominant micro-ecological community in the rhizosphere, competing for ecological sites, and producing antagonistic substances. Fire TR4 reproduction and infection.

[0011] A third aspect of the present invention provides the application of a bacterial solution containing synthetic bacterial group B2: a bacterial solution containing said synthetic bacterial group B2 is provided, the active ingredient of which is the mycelium of strains S-1-56, S-2-42 and S-1-119, and the bacterial solution can be used to inhibit the pathogen of banana wilt disease. Fire TR4 is suitable for disease control in various scenarios, including field and potted plants.

[0012] A fourth aspect of this invention provides a compound microbial agent and its application: the compound microbial agent comprises the aforementioned synthetic microbial group B2, and the compound microbial agent can be used to... Fire TR4 exhibits a specific antagonistic effect, enabling the control of banana wilt disease. It can be prepared into different formulations such as liquid and powder according to actual application needs.

[0013] The fifth aspect of this invention provides a microbial compound fertilizer: the active ingredient of the microbial compound fertilizer includes the synthetic microbial community B2. When this fertilizer is applied to banana planting, it can improve the soil micro-ecological environment while continuously exerting an inhibitory effect on Foc TR4, thus taking into account both fertilizer efficiency and disease control functions.

[0014] The sixth aspect of this invention provides a method for controlling banana wilt disease. The method involves applying a solution of the synthetic microbial community B2 to the rhizosphere soil of banana plants via root irrigation, thereby establishing a co-culture relationship between the synthetic microbial community B2 and the banana plant. This constructs a stable protective barrier around the banana root system, blocking the spread of the disease. FireTR4 infects the vascular bundles of bananas, thus effectively controlling the occurrence and spread of banana wilt disease.

[0015] The beneficial effects of this invention are: (1) Significant and stable control effect: The synthetic microbial community B2 consists of three antagonistic strains with complementary functions and synergistic effects. Compared with a single strain, it has a stronger colonization ability in complex soil environments and can form a more resilient micro-ecological barrier through the division of labor and cooperation among strains. Fire TR4 exhibits a more significant and sustained inhibitory effect, effectively addressing the issue of fluctuating control efficacy of single biocontrol strains.

[0016] (2) Environmentally friendly and safe: The present invention adopts the biological control approach. The synthetic microbial community B2 is derived from the rhizosphere soil of bananas. It has strong environmental adaptability and leaves no chemical pesticide residues after application. It will not cause soil or water pollution, nor will it pose a threat to non-target organisms or human health. It meets the requirements of green and sustainable agricultural development.

[0017] (3) Wide range of applications and easy operation: Synthetic microbial community B2 can be applied in various forms such as preparing bacterial liquid, compound microbial agent, and microbial compound fertilizer, and is suitable for different banana planting modes; it is applied by root irrigation, which is easy to operate and easy to promote on a large scale, reducing the labor and technical threshold in agricultural production.

[0018] (4) It has both practical value and research significance: This synthetic microbial community not only provides a practical and effective technical solution for the prevention and control of banana wilt disease, but also provides an ideal research carrier for studying the interaction mechanism of plant rhizosphere microorganisms, the complementary law of microbial community function and the biological control principle of soil-borne diseases, which helps to promote the in-depth application of microbial technology in the field of agricultural disease prevention and control. Attached Figure Description

[0019] Figure 1 This diagram illustrates the antibacterial activity of antagonistic strains screened using the plate confrontation method. The figure shows the confrontation culture results of pathogen FocTR4 and the tested antagonistic strains on PDA plates. By measuring the diameter of the inhibition zone, the inhibitory ability of each strain against the pathogen is visually reflected, and strains with significant antagonistic activity are screened accordingly.

[0020] Figure 2 To determine the OD of three candidate antagonistic strains (S-1-56, S-2-42, 1-119) and the synthetic strain (B2) after 12 h of culture. 600 After 12 hours of incubation, the OD of the synthetic bacteria B2... 600 The highest is S-1-119, followed by S-2-42 and S-1-56, while the lowest are S-2-42 and S-1-56.

[0021] Figure 3 In the pot experiment of this invention, inoculation FireThe image shows a comparison of the growth status of banana seedlings two weeks after applying TR4 and different treatments (including a water control, a pathogen control, a single-strain treatment, and a synthetic microbial group B2 treatment). Each treatment group had 16 replicates. The bulbs were dissected to observe internal browning, and the roots were examined using a laser confocal microscope. The image shows typical bulbs and root systems with average disease levels from each group.

[0022] Figure 4 This is a comparative image showing the growth status of banana seedlings in different treatment groups during the fourth week of the pot experiment of this invention. It demonstrates that the synthetic microbial community B2 treatment group, compared to the single-strain treatment group and the positive control, maintained plant health and significantly reduced typical symptoms of banana wilt disease in the middle and late stages of disease development. Each treatment group had 16 replicates. The bulbs were dissected to observe internal browning, and the roots were examined using a laser confocal microscope. The images show typical bulb and root images of each group with the average disease level. Detailed Implementation

[0023] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0024] Example 1: Isolation of antagonistic microorganisms Rhizosphere soil samples were collected from different locations using the root shaking method. Bacteria were isolated using the dilution-spread method, specifically as follows: 5 g of rhizosphere soil sample was added to 45 mL of sterile water, thoroughly shaken, and then serially diluted. 10 μL of each sample was then applied to each sample. -2 10 -3 10 -4 100 μL of each dilution solution was evenly spread onto Pseudomonas isolation medium. The inoculation dishes were placed in an incubator at 28 °C and inverted for 2 days. After colony growth, single colonies with different morphological characteristics were picked, streaked onto LB agar plates for purification, and the strains were preserved for subsequent studies.

[0025] Example 2: Identification of antagonistic microorganisms After streaking the strains on LB agar, single colonies were picked and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Sequence alignment was performed in the EzBioCloud database based on the sequencing results. Subsequently, a phylogenetic tree of the strains and related closely related genera and species was constructed using IQ-TREE software to reveal their phylogenetic relationships. Phylogenetic analysis showed that strains S-1-56, S-2-42, and 1-119 all belong to the genus *Pseudomonas*.

[0026] Example 3: Screening of antagonistic microorganisms and construction of synthetic microbial communities The isolated and purified strains were initially and subsequently screened using the plate confrontation method. Fire TR4 was inoculated in the center of PDA medium, with the test strain inoculated 2 cm away from the pathogen. The control group was inoculated with only the pathogen. Each treatment was repeated three times, with three plates per replicate. All plates were incubated at 28 °C until the mycelium in the control group covered the entire plate. The diameter of the colonies in each treatment was measured, and the inhibition rate was calculated. Finally, three strains were selected as the inhibitory agents. Fire TR4 has significant antagonistic effects on the following strains: antagonistic bacteria of the genus Pseudomonas S-1-56, S-2-42 and 1-119.

[0027] The compatibility of the three strains was tested on plates. The results showed that the three strains were compatible and not antagonistic to each other. Further resistance testing was conducted, and a synthetic bacterial community was constructed. The viable cell ratio of each individual strain in the synthetic community was 1:1:1. The effects of individual strains and the synthetic community on each other were calculated. Fire TR4 inhibition rate ( Figure 1 ), and the growth rates of the corresponding single bacteria and synthetic bacterial groups were measured ( Figure 2 The results showed that at a wavelength of 600 nm, the OD of the synthetic bacterial group B2 was... 600 The growth rate was significantly higher than that of strains S-1-56, S-2-42, and 1-119, indicating that the combination of strains promoted growth.

[0028] Example 4: Pot Experiment Verification The pot experiment included six treatments: a water treatment group, a water treatment group, and a water treatment group. Fire The TR4 treatment group, the three most active single bacteria each forming a separate treatment group, and a synthetic bacteria treatment group consisting of three single bacteria, with the viable cell ratio of each single bacteria in the synthetic bacteria being 1:1:1. The treatment was carried out using clean water and... Fire TR4 treatment alone served as negative and positive controls, while all other treatments were administered via inoculation. Fire TR4; vaccination Fire Following TR4, antagonistic single bacteria and synthetic flora were simultaneously inoculated. Each treatment group contained 16 seedlings. The root-wound inoculation method was used. FireTR4 treatment involves the following steps: Select healthy banana tissue culture seedlings with uniform growth at the 3-4 leaf stage, slightly injure the roots, and soak them in... Fire The seedlings were soaked in TR4 spore suspension for 30 minutes, then transplanted into seedling pots. A water treatment was then performed: the banana seedlings were soaked in water for 30 minutes, followed by transplanting into plastic pots filled with soil. The roots were then drenched. Fire TR4 spore solution and cultured antagonistic bacterial solution were applied to the soil around the roots of each banana seedling in the remaining 5 treatment groups. The positive control group was only applied to the soil around the roots. Fire TR4 spore solution. During the pot experiment, banana seedlings in all treatment groups were uniformly placed in a constant temperature plant culture room for management. The disease status of banana seedlings in each treatment group was continuously observed. The specific methods were to observe whether the banana corms turned black and to observe the root infection using laser confocal microscopy.

[0029] Pot experiment verification results: The experimental results show that ( Figure 3 ), during vaccination Fire Two weeks after inoculation with TR4 spore suspension, no significant changes were observed in any of the treatments. Observation continued until 4 weeks post-inoculation. Figure 4 ), Fire The TR4 inoculation group and the single-strain S-1-56 inoculation group exhibited typical symptoms of banana wilt, including leaf yellowing, wilting, and vascular bundle browning. The single-strain S-1-42 inoculation group showed mild disease, while the synthetic strain B2 showed no obvious symptoms. Observation using a laser confocal microscope revealed... Fire Strong GFP fluorescence signals were detected in the root tissue of the TR4-inoculated group, confirming successful pathogen infection; no significant difference was observed in the water control group. This result indicates that the beneficial bacterial combinations isolated in this study have a positive effect on soil environment. Fire TR4 showed a certain inhibitory effect. The antibacterial effect of single strain S-1-56 was significantly less stable than that of its synthetic flora. It is further speculated that single strain S-1-56 may contain a substance that can stimulate other microorganisms to play a role, thereby inhibiting the occurrence of wilt disease.

Claims

1. A synthetic microbial community B2 for controlling banana wilt disease, characterized in that: The synthetic bacterial group B2 consists of three strains, namely Pseudomonas ( ). Pseudomonas sp.) S-1-56, Pseudomonas spp. ( Pseudomonas sp.) S-2-42 and Pseudomonas spp. ( Pseudomonas sp.) 1-119, The Pseudomonas s. S-1-56 has the accession number GDMCC No:67659, the accession date is January 14, 2026, and the accession location is Guangdong Provincial Center for Microbial Culture Collection. The Pseudomonas s. S-2-42 has the accession number GDMCC No:67660, the accession date is January 14, 2026, and the accession location is Guangdong Provincial Center for Microbial Culture Collection. The accession number of the Pseudomonas genus 1-119 is GDMCC No:67658, the accession date is January 14, 2026, and the accession location is Guangdong Provincial Center for Microbial Culture Collection. The pathogen causing banana wilt is Fusarium oxysporum, specifically the Cuban variant (…). Fusarium oxysporum f. sp. cubense Tropical race 4.

2. The application of the synthetic microbial community B2 described in claim 1 in inhibiting the pathogen of banana wilt disease, characterized in that, The pathogen causing banana wilt is Fusarium oxysporum, specifically Tropical Race 4 of the Cuban Specialized Type.

3. The application of a bacterial solution containing the synthetic microbial group B2 as described in claim 1 in inhibiting the pathogen of banana wilt disease, characterized in that, The pathogen causing banana wilt is Fusarium oxysporum, specifically Tropical Race 4 of the Cuban Specialized Type.

4. The application according to claim 3, characterized in that, The effective components of the bacterial solution of the synthetic bacterial group B2 are the hyphae of Pseudomonas S-1-56, Pseudomonas S-2-42 and Pseudomonas 1-119 as described in claim 1.

5. A compound microbial agent, characterized in that, The compound microbial agent comprises the synthetic microbial community B2 as described in claim 1.

6. The application of the compound microbial agent according to claim 5 in inhibiting the pathogen of banana wilt disease, characterized in that, The pathogen causing banana wilt is Fusarium oxysporum, specifically Tropical Race 4 of the Cuban Specialized Type.

7. The application according to claim 6, characterized in that: The application is to produce an antagonistic effect against Fusarium oxysporum Cuban specialized type tropical race 4.

8. A microbial compound fertilizer, characterized in that, The active ingredient of the microbial compound fertilizer includes the synthetic microbial community B2 described in claim 1.

9. A method for controlling banana wilt disease, characterized in that, The method involves drenching banana roots with the bacterial solution of the synthetic microbial community B2 described in claim 1 to achieve co-culture of synthetic microbial community B2 and banana; the pathogen of banana wilt is Fusarium oxysporum, Cuban specialized type tropical race 4.

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