Sphingomonas paucimobilis-like bacteria YH4 and its application in controlling tobacco bacterial wilt

Abstract

The present invention discloses a Sphingomonas paucimobilis-like bacterium YH4 and its use in preventing and controlling tobacco bacterial wilt, belonging to the technical field of tobacco bacterial wilt prevention and control. The Sphingomonas paucimobilis-like bacterium YH4 was screened from cigar tobacco leaves and deposited with the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242381. Field trials have shown that applying this strain during tobacco seedling transplanting can significantly reduce the incidence and disease index of tobacco plants. Sixty-two days after transplanting, compared with the control group, the incidence was reduced by 29.6% and the disease index was reduced by 61.96%. Therefore, the present invention provides a new approach for field prevention and control of tobacco bacterial wilt.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco bacterial wilt prevention and control, and particularly relates to a Sphingomonas paucimobilis-like bacterium YH4 and an application thereof in preventing and controlling tobacco bacterial wilt. Background Art

[0002] Ralstonia solanacearum ( Ralstonia solanacearum ), commonly known as bacterial wilt, is a soil-borne bacterial disease caused by this fungus that seriously affects the yield of economic crops such as the Solanaceae family. R. solanacearum has strong stress resistance and a wide range of transmission routes. It invades the plant body through the roots and colonizes the vascular system, multiplies in large numbers in the xylem vessels and produces exopolysaccharides (EPS), which leads to blockage and dysfunction of the xylem vessels, and ultimately causes plant wilt and death. R. solanacearum has high genetic diversity, rapid mutation, and a wide host range. Currently, there is a lack of effective disease-resistant varieties in production, which poses a challenge to the prevention and control of bacterial wilt. Biological control has attracted much attention as a new green and safe prevention and control strategy involving multifaceted interactions between antagonistic bacteria, pathogens and plant hosts.

[0003] Currently, my country primarily controls tobacco bacterial wilt through cultivating disease-resistant varieties, improving farming systems, and employing chemical control measures. However, the cultivation of disease-resistant varieties requires a long cycle and is susceptible to environmental influences, resulting in less than ideal results. Improving farming systems, such as crop rotation, is currently an effective measure to combat the disease, but in practice, it impacts economic returns. While chemical pesticides can mitigate the damage caused by tobacco bacterial wilt to a certain extent, they are affected by a variety of factors, including the characteristics of the chemical pesticides themselves, the characteristics of the pathogens, environmental factors, and application techniques, leading to subsequent problems such as increased pathogen resistance and environmental pollution. Biological control, with its advantages of being non-toxic, leaving no residue, and being readily available in abundant resources, has become an effective approach to combating soil-borne diseases.

[0004] Bacterial wilt biocontrol agents have been extensively studied in many crops, including tomato, tobacco, potato, and pepper, all members of the Solanaceae family. Microorganisms that can control bacterial wilt outbreaks primarily come from the genera Streptomyces, Bacillus, Pseudomonas, Sphingomonas, Flavobacterium, mycorrhizal fungi, and a few actinomycetes. However, not all species within these genera are capable of inhibiting R. solanacearum, making the identification of species-level biocontrol agents that inhibit R. solanacearum crucial. Furthermore, currently reported biocontrol agents are mostly limited to laboratory settings. Furthermore, existing studies have shown that R. solanacearum exhibits high genetic diversity and rapid mutation, with strains from different geographic regions and hosts exhibiting significant genetic variation and divergence, and its pathogenic pathways exhibit diverse host specificity. Currently, R. solanacearum is divided into four phylotypes, five races, and six biovars. While research and biocontrol agents targeting tomato bacterial wilt are relatively extensive, biocontrol agents for tobacco bacterial wilt are lacking. Summary of the Invention

[0005] In view of this, the present invention aims to discover a biocontrol bacterium that can effectively prevent tobacco bacterial wilt in the field, providing a new approach for field prevention and control of tobacco bacterial wilt.

[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a strain of Sphingomonas paucimobilis ( Sphingomonas paramotorcycle ) YH4, which was isolated from cigar tobacco leaves; the strain was deposited in the China Center for Type Culture Collection on October 30, 2024, with the deposit number CCTCC NO: M 20242381, and the deposit address is Wuhan University, Wuhan, China.

[0008] In a second aspect, the present invention provides a use of the above-mentioned Sphingomonas paucimobilis-like bacteria YH4, comprising at least the following:

[0009] a) preparing a microbial agent comprising Sphingomonas paucimobilis YH4 as one of the active ingredients;

[0010] b) preparing an agent for antagonizing tobacco solanacearum;

[0011] c) Control tobacco bacterial wilt in the field.

[0012] In the aforementioned application, when using Sphingomonas paucimobilis YH4 to control tobacco bacterial wilt in the field, Sphingomonas paucimobilis YH4 can be applied to the roots of tobacco seedlings, allowing it to colonize in the tobacco, thereby enhancing the tobacco's resistance to bacterial wilt. It is understood that methods for applying Sphingomonas paucimobilis YH4 to the roots of tobacco seedlings include, but are not limited to, spraying a bacterial solution containing Sphingomonas YH4 directly onto the roots of tobacco seedlings, or applying the bacterial solution containing Sphingomonas YH4 to the soil at the roots of tobacco seedlings.

[0013] In a third aspect, based on the Sphingomonas paucimobilis-like bacteria YH4 provided by the present invention, the present invention provides a method for controlling tobacco bacterial wilt in the field, comprising the following steps:

[0014] S1. Cultivating Sphingomonas paucimobilis YH4 to obtain a bacterial solution;

[0015] S2. Before transplanting tobacco seedlings, spray the bacterial solution on the roots of the tobacco seedlings;

[0016] S3. 13-17 days after transplanting, irrigate the roots with bacterial solution.

[0017] Preferably, in the above method, step S1 specifically comprises: activating Sphingomonas paucimobilis YH4 and culturing the culture medium until the OD600 The value is 0.8-1.0.

[0018] Preferably, the above method further comprises: S4, field management until harvesting.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The strain provided by the present invention that can be used to prevent and treat tobacco bacterial wilt belongs to the genus Sphingomonas ( Sphingomonas ) strain, identified as Sphingomonas paucimobilis ( Sphingomonas parapaucimobilis ), and currently no strain of this species has been reported to be able to prevent and control tobacco bacterial wilt.

[0021] The Sphingomonas paucimobilis-like bacterium YH4 provided by the present invention was isolated and screened from healthy tobacco leaves. It can colonize tobacco plants, thereby regulating and enhancing tobacco's resistance to bacterial wilt. Moreover, this strain can colonize in the soil for a long time, making it effective for field bacterial wilt control. Experimental data show that the use of this biocontrol bacterium in field trials has a significant effect on the prevention and control of bacterial wilt. At the same time, the experimental data also show that many species or strains of the genus Sphingomonas do not have the ability to antagonize Ralstonia solanacearum in tobacco, indicating that the strains of this genus that control bacterial wilt have species or strain differences.

[0022] The present invention provides a new approach for preventing and controlling tobacco bacterial wilt in the field. By utilizing beneficial microorganisms, the method has the characteristics of being green, economical, and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a staining diagram of the Sphingomonas paucimobilis YH4 of the present invention;

[0024] Figure 2 This is the phylogenetic tree of Sphingomonas paucimobilis-like YH4 constructed based on the genome in Example 1 of the present invention;

[0025] Figure 3 A growth curve diagram of the Sphingomonas paucimobilis-like bacteria YH4 provided by the present invention;

[0026] Figure 4 This is a graph showing the antagonistic ability of Sphingomonas paucimobilis YH4 and other Sphingomonas bacteria against Ralstonia solanacearum in Example 2 of the present invention;

[0027] Figure 5 The survey results of the incidence rate (A) and disease index (B) of cigar tobacco in the field after application of Sphingomonas paucimobilis YH4 in Example 3 of the present invention, as well as a schematic diagram of the grading standard for cigar tobacco bacterial wilt disease (C) are shown. DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The term "comprise" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions.

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0030] If no specific techniques or conditions are specified in the following examples, the procedures were carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0031] Example 1

[0032] This example provides the isolation and identification process of Sphingomonas paucimobilis YH4, which specifically includes the following steps:

[0033] (1) Isolation and purification of strain YH4.

[0034] The strain YH4 provided by the present invention was specifically isolated from healthy tobacco leaf samples collected from the tobacco-growing area of ​​Laifeng County, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province in June 2023. The specific separation and purification operation is as follows: first, the leaves were disinfected with alcohol, then cut into pieces, soaked in a triangular flask, placed in a shaker at 28°C for half an hour, and then 1 mL was added to 9 mL of sterile saline to gradually dilute to 10 -4 , 10 -5 , 10 -6 and 10 -7 0.1 mL of each solution was spread onto solid culture medium, with three plates spread for each gradient. Culture was incubated at 28°C for one week, and single colonies were streaked and purified. After identification by 16S rRNA gene sequencing of bacteria belonging to the genus Sphingomonas, the bacteria were stored in screw-cap tubes at a 1:1 volume ratio of 50% glycerol to bacterial suspension at -80°C.

[0035] (2) Culture and morphological characteristics of strain YH4.

[0036] like Figure 1 As shown, the cells were rod-shaped and cultured on R2A agar plate at 28°C for 24 h to form yellow colonies that were round, convex, and had complete and smooth edges.

[0037] (3) Classification and identification of strain YH4.

[0038] The 16S rRNA gene of strain YH4 was sequenced and its phylogenetic tree was constructed. Combined with its physiological and biochemical characteristics, the strain was identified as Sphingomonas Genus.

[0039] The whole genome of strain YH4 was sequenced and its phylogenetic tree was constructed, as shown in Figure 2 As shown, it shows that YH4 and Sphingomonas parapaucimobilis NBRC 15100 is the closest relative, while other species of the genus are more distantly related, such as Sphingomonas yabuuchiae DSM 14562 and Sphingomonas fuzhouensis SGZ-02, etc. Secondly, a comprehensive judgment is made by combining the average nucleotide similarity (ANI) and DNA-DNA molecular hybridization (dDDH). When the ANI value between the strain to be identified and the known species is greater than 95-96% and the dDDH value is greater than 70%, it is determined that the strain to be identified and the known species are the same species. Sphingomonas parapaucimobilis The ANI value of the standard strain NBRC 15100 was 97.71% and the dDDH value was 79.6% (Table 1), indicating that the two belong to the same species. Based on the comparison of genetic homology such as phylogenetic tree, ANI value, and dDDH value, strain YH4 was identified as Sphingomonas parapaucimobilis .

[0040] Table 1 Comparison of genetic homology between strain YH4 and known species

[0041]

[0042] (4) Growth curve of strain YH4.

[0043] The strain was added to R2A medium at a 1% inoculum volume (with 3 replicates) and cultured at 28°C for 10 days. 2 ml of the sample was taken every 12 hours and the bacterial growth concentration (OD) was measured using a UV spectrophotometer. 600 The results are as follows: Figure 3 As shown, the strain grew well in R2A medium, providing a basis for the long-term colonization of the bacteria in the soil in field experiments.

[0044] Example 2

[0045] This study verified the antagonistic effect of Sphingomonas paucimobilis YH4 against Ralstonia solanacearum in a laboratory setting. The specific process was as follows:

[0046] The strain YH4 isolated in Example 1 and other Sphingomonas bacteria also isolated from tobacco leaves were cultured in R2A liquid medium at 28°C and 150 rpm overnight to obtain antagonistic bacterial solutions. Ralstonia solanacearum) Culture overnight in BG liquid medium at 28°C and 150 rpm to obtain a bacterial wilt solution. Spread 0.2 mL of R. solanacearum onto a disposable plate to create a pathogen-containing plate, air dry, and cool. Pipette 5.0 μL of the antagonistic bacterial solution onto a sterile filter paper sheet, place it on the pathogen-containing plate, and incubate in a 28°C incubator for 48 hours. Record the presence of a clear zone of inhibition.

[0047] The results are as follows Figure 4 As shown, 1-3 correspond to Sphingomonas sp. YH1; Sphingomonas paramotorcycle YH4; Sphingomonas melonis DAPP-PG 224. From Figure 4 It can be seen that the present invention provides Sphingomonas parapaucimobilis YH4 has the ability to antagonize Ralstonia solanacearum, while other isolated and purified Sphingomonas species have no antagonistic ability to Ralstonia solanacearum.

[0048] Example 3

[0049] In this case, a field experiment was conducted in a tobacco field where bacterial wilt has frequently occurred in recent years to verify the field control effect of strain YH4 on tobacco bacterial wilt. The specific steps are as follows:

[0050] (1) Prepare a 1L bottle of sterilized R2A culture medium, inoculate it with 1% YH4 bacterial solution in the logarithmic phase (OD value is around 1), culture it in a shaking incubator at 28℃ for 48h, then centrifuge it, collect it in a sterilized centrifuge tube (leave a small amount of culture medium), and store it in a 4℃ refrigerator.

[0051] (2) An experimental group and a control group were set up. The control group did not take any measures to prevent and treat bacterial wilt, while the experimental group only used strain YH4. In this example, the specific application method of strain YH4 was as follows: before transplanting the tobacco seedlings, the bacterial precipitate obtained in step (1) was diluted with 5L of water and evenly sprayed on the roots of approximately 100 tobacco seedlings; approximately 15 days after transplanting, the cigar tobacco seedlings were irrigated with the same method to supplement the bacteria. The control group was sprayed with a corresponding amount of clean water at the same time as the experimental group was applied with the bacterial solution.

[0052] (3) Field management until 62 days after transplanting (at the time of harvesting). The incidence rate and disease index of the experimental group and the control group were observed and recorded on the 0th, 12th, 27th, 51st and 62nd days respectively. Disease index = ∑ (number of diseased plants at each level × representative value at each level) / (total number of leaves surveyed × highest representative value), and the representative values ​​and judgment criteria of each level are shown in Table 2 and Figure 5 As shown in C.

[0053] Table 2 Disease classification of tobacco bacterial wilt

[0054]

[0055] Specifically, the incidence and disease index of the experimental and control groups from transplanting to harvest are shown in Table 3 and Figure 5 As shown in A and 5B.

[0056] Table 3 Effects of strain YH4 on cigar tobacco morbidity and disease index

[0057]

[0058] The above results indicate that the addition of strain YH4 significantly reduced both the incidence and disease index. Specifically, compared to the control group, the YH4 strain reduced the incidence by 29.6% and the disease index by 61.96%, demonstrating that strain YH4 can be used as a biocontrol agent for the prevention of tobacco bacterial wilt. It should also be noted that this experiment was conducted in a tobacco field where bacterial wilt has frequently occurred in recent years. The control group, which did not implement any control measures for bacterial wilt, had a relatively high incidence.

[0059] In summary, the Sphingomonas paucimobilis-like bacteria YH4 provided by the present invention can effectively prevent and control tobacco bacterial wilt disease in the field and has a good prevention and control effect. The present invention provides a new approach for field prevention and control of tobacco bacterial wilt disease.

[0060] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solutions of the present invention rather than to limit them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A Sphingomonas paucimobilis-like bacterium YH4, characterized in that The Sphingomonas paucimobilis-like bacteria ( Sphingomonas parapaucimobilis ) YH4 strain was deposited in China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20242381.

2. A microbial agent, characterized in that: The Sphingomonas paucimobilis YH4 described in claim 1 is one of the active ingredients.

3. Use of the Sphingomonas paucimobilis-like bacterium YH4 according to claim 1 in the preparation of an agent for antagonizing tobacco solanacearum.

4. Use of the Sphingomonas paucimobilis-like bacterium YH4 according to claim 1 or the microbial agent according to claim 2 in controlling tobacco bacterial wilt in the field.

5. The use according to claim 4, characterized in that The Sphingomonas paucimobilis-like bacteria YH4 were applied to the roots of tobacco seedlings.

6. The use according to claim 5, characterized in that The dosage of the Sphingomonas paucimobilis YH4 is 1×10 8 CFU-1×10 10 CFU / strain tobacco seedlings.

7. A method for preventing and controlling tobacco bacterial wilt in the field, characterized in that: Field control is carried out using the Sphingomonas paucimobilis YH4 described in claim 1 as a biocontrol bacterium.

8. The method for controlling tobacco bacterial wilt in the field according to claim 7, characterized in that: The following steps are involved: S1. Cultivating Sphingomonas paucimobilis YH4 to obtain a bacterial solution; S2. Before transplanting tobacco seedlings, spray the bacterial solution on the roots of the tobacco seedlings; S3. Fifteen days after transplanting, irrigate the roots with bacterial solution.

9. The method for controlling tobacco bacterial wilt in the field according to claim 8, characterized in that: Step S1 specifically includes: activating Sphingomonas paucimobilis YH4 and culturing it to an OD of 600 The value is 0.8-1.

0.

10. The method for controlling tobacco bacterial wilt in the field according to claim 8, characterized in that: The following steps are also included: S4. Field management until harvest.

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