Sphingomonas paucimobilis YH4 and application thereof in prevention and treatment of tobacco bacterial wilt

Abstract

The invention discloses sphingomonas paucimobilis YH4 and application thereof in prevention and treatment of tobacco bacterial wilt, and belongs to the technical field of tobacco bacterial wilt prevention and treatment, the sphingomonas paucimobilis YH4 is screened from cigar tobacco leaves and preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20242381. Field tests show that the morbidity and the disease index of tobacco plants can be remarkably reduced by applying the strain when tobacco seedlings are transplanted, and the morbidity is reduced by 29.6% and the disease index is reduced by 61.96% when the strain is compared with a control group 62 days after transplanting. Therefore, the invention provides a new way for field prevention and treatment of tobacco bacterial wilt.

Description

Technical Field

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

[0002] Ralstonia solanacearum Ralstonia solanacearum ), commonly known as Ralstonia solanacearum, causes soil-borne bacterial diseases that seriously affect the yield of economic crops such as Solanaceae. Ralstonia solanacearum has strong stress resistance and a wide range of transmission routes. It invades the plant body through the roots and colonizes in the vascular system, multiplies in large numbers in the xylem vessels and produces extracellular polysaccharides (EPS), which lead to xylem vessel obstruction and dysfunction, and ultimately cause plant wilt and death. Ralstonia 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] At present, my country mainly controls tobacco bacterial wilt by cultivating disease-resistant varieties, improving farming systems, and adopting chemical control measures. However, the cultivation of disease-resistant varieties has a long cycle and is easily affected by the environment, so the effect is not ideal; improving farming systems such as crop rotation is an effective measure to combat the disease, but it will affect economic benefits in actual production; although chemical pesticide control can reduce the harm of tobacco bacterial wilt to a certain extent, it is affected by many factors such as the characteristics of chemical pesticides themselves, the characteristics of pathogens, environmental factors, and pesticide application technology, which will lead to subsequent problems such as increased resistance of pathogens and environmental pollution. Biological control has the advantages of being non-toxic, residue-free, and rich in available resources, and has become an effective way to solve soil-borne diseases.

[0004] Bacteria that control bacterial wilt have been widely studied in many crops, including tomatoes, tobacco, potatoes, peppers and other Solanaceae crops. Microorganisms that can prevent and control bacterial wilt outbreaks mainly come from Streptomyces, Bacillus, Pseudomonas, Sphingomonas, Flavobacterium, mycorrhizal fungi and a few actinomycetes, but not all species of the above genera can inhibit Ralstonia solanacearum, so it is crucial to screen species-level biocontrol bacteria that can inhibit Ralstonia solanacearum. Moreover, the biocontrol bacteria reported so far are mostly limited to laboratory environments. In addition, existing studies have shown that Ralstonia solanacearum has high genetic diversity and rapid mutation. Strains in different geographical regions or different hosts show significant genetic variation and differentiation, and its pathogenic pathways show diversified and host-specific characteristics. At present, Ralstonia solanacearum is divided into 4 phylotypes, 5 physiological races and 6 biovars. There are relatively more studies and biocontrol bacteria for tomato bacterial wilt, but there is a lack of biocontrol bacteria for tobacco bacterial wilt. 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, and provide 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: In a 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.

[0007] In a second aspect, the present invention provides an application of the above-mentioned Sphingomonas paucimobilis YH4, which at least comprises the following: a) preparing a microbial agent, wherein the microbial agent has Sphingomonas paucimobilis YH4 as one of the active ingredients; b) preparing an agent for antagonizing tobacco solanacearum; c) Control tobacco bacterial wilt in the field.

[0008] In the above 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 so that it can enter and colonize in tobacco, thereby enhancing tobacco's resistance to bacterial wilt. It is understandable that the method of applying Sphingomonas paucimobilis YH4 to the roots of tobacco seedlings includes but is not limited to: spraying a bacterial solution containing Sphingomonas YH4 directly to the roots of tobacco seedlings, or applying a bacterial solution containing Sphingomonas YH4 to the soil at the roots of tobacco seedlings.

[0009] In a third aspect, based on the Sphingomonas paucimobilis YH4 provided by the present invention, the present invention provides a method for controlling tobacco bacterial wilt in the field, which comprises the following steps: S1. culturing Sphingomonas paucimobilis YH4 to obtain a bacterial liquid; S2. Spray the bacterial solution on the roots of tobacco seedlings before transplanting them; S3. 13-17 days after transplanting, irrigate the roots with bacterial solution.

[0010] Preferably, in the above method, step S1 specifically comprises: activating Sphingomonas paucimobilis YH4 and culturing the culture medium until the OD 600 The value is 0.8-1.0.

[0011] Preferably, the above method further comprises: S4, field management until harvesting.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 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.

[0013] The paucimobile-like Sphingomonas YH4 provided by the present invention is isolated and screened from healthy tobacco leaves, and can colonize in tobacco plants, thereby regulating and enhancing tobacco's resistance to bacterial wilt disease; and the strain can also colonize in the soil for a long time, so that it can be effectively used for field bacterial wilt disease prevention and control. Experimental data show that the use of the biocontrol bacteria in field trials has a significant effect on the prevention and control of bacterial wilt diseases. 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 tobacco solanacearum, which shows that the strains of this genus for preventing and controlling bacterial wilt have species or strain differences.

[0014] The invention provides a new approach for preventing and controlling tobacco bacterial wilt in the field, and by utilizing beneficial microorganisms, the method has the characteristics of being green, economical, and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a staining diagram of the Sphingomonas paucimobilis YH4 of the present invention; Figure 2 The phylogenetic tree of Sphingomonas paucimobilis YH4 constructed based on the genome in Example 1 of the present invention; Figure 3 A growth curve diagram of the Sphingomonas paucimobilis YH4 provided by the present invention; Figure 4 This is a graph showing the antagonistic ability of Sphingomonas paucimobilis YH4 and other bacteria of the genus Sphingomonas to Ralstonia solanacearum in Example 2 of the present invention; Figure 5 The results of the investigation on the incidence rate (A) and disease index (B) of cigar tobacco in the field after the application of Sphingomonas paucimobilis YH4 in Example 3 of the present invention and the schematic diagram of the grading standard of cigar tobacco bacterial wilt disease (C) are shown. DETAILED DESCRIPTION

[0016] 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 belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0017] The technical solution of the present invention will be described clearly and completely 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 used to limit the present invention.

[0018] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0019] Example 1 This example provides the isolation and identification process of Sphingomonas paucimobilis YH4, which specifically includes the following steps: (1) Isolation and purification of strain YH4.

[0020] The strain YH4 provided by the present invention is 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 separation and purification operation is specifically as follows: first disinfect the leaves with alcohol, then cut them into pieces, soak them in a triangular flask, shake them in a shaker at 28°C for half an hour, then take 1 mL and add them to 9 mL of sterile saline to gradually dilute to 10 -4 , 10 -5 , 10 -6 and 10 -7 , take 0.1mL and spread it on solid culture medium, and spread 3 plates for each gradient. Incubate at 28℃ for one week, pick single colonies and purify by streaking. After identification by 16S rRNA gene sequencing, the bacteria belonging to the genus Sphingomonas were confirmed, and then stored in screw-capped tubes at 50% glycerol: bacterial solution (volume ratio 1:1) and stored at -80℃.

[0021] (2) Culture and morphological characteristics of strain YH4.

[0022] like Figure 1 As shown, the cells were rod-shaped and cultured on R2A agar plate medium at 28°C for 24 h to form yellow colonies that were round, convex, and had complete and smooth edges.

[0023] (3) Classification and identification of strain YH4.

[0024] 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 Belong to.

[0025] The whole genome of strain YH4 was sequenced and its phylogenetic tree was constructed, such as 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 yabuuchiaeDSM 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. Combining the comparison of genetic homology such as phylogenetic tree, ANI value, and dDDH value, strain YH4 was identified as Sphingomonas parapaucimobilis .

[0026] Table 1 Comparison of genetic homology between strain YH4 and known species

[0027] (4) Growth curve of strain YH4.

[0028] The strain was added to R2A medium at a 1% inoculum (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, which provided a basis for the long-term colonization of the bacteria in the soil in field experiments.

[0029] Example 2 In this case, the antagonism of Sphingomonas paucimobilis YH4 against Ralstonia solanacearum was verified in a laboratory environment. The specific process is as follows: The strain YH4 isolated in Example 1 and other bacteria of the genus Sphingomonas isolated from tobacco leaves were cultured in R2A liquid culture medium at 28°C and 150 r / min overnight to obtain antagonistic bacterial liquid. Ralstonia solanacearum ) Culture overnight in BG liquid medium at 28℃ and 150r / min to obtain bacterial solution of Ralstonia solanacearum. Spread 0.2mL of Ralstonia solanacearum on a disposable plate to make a plate containing pathogens, blow dry and cool. Pipette 5.0μL of antagonistic bacteria solution and drop it on a sterilized filter paper and place it on the plate containing pathogens. Culture it in a 28℃ incubator for 48h and record whether an obvious inhibition zone is produced.

[0030] The results are as follows Figure 4 As shown, 1-3 correspond to Sphingomonas sp. YH1; Sphingomonas paramotorcycle YH4; Sphingomonas melonis DAPP-PG 224. 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 species of Sphingomonas do not have the ability to antagonize Ralstonia solanacearum.

[0031] Example 3 In this case, a tobacco field with frequent bacterial wilt in recent years was selected for field experiment to verify the field control effect of strain YH4 on tobacco bacterial wilt. The specific steps are as follows: (1) Prepare a 1L bottle of sterilized R2A medium and inoculate it with 1% YH4 bacterial solution in the logarithmic phase (OD value is around 1). Incubate in a shaking incubator at 28℃ for 48h and then centrifuge. Collect and store the culture in a sterilized centrifuge tube (leave a small amount of culture medium) and place in a 4℃ refrigerator.

[0032] (2) An experimental group and a control group were set up, wherein the control group did not take any measures to prevent and treat bacterial wilt, and the experimental group only used strain YH4. In this example, the specific application method of strain YH4 was as follows: before transplanting tobacco seedlings, the bacterial precipitate obtained in step (1) was diluted with 5L of water and evenly sprayed on the roots of about 100 tobacco seedlings; about 15 days after transplanting, the cigar tobacco seedlings were irrigated with roots to supplement the bacteria in the same way. The control group was sprayed with a corresponding amount of clean water at the same time as the bacterial solution was applied to the experimental group.

[0033] (3) Field management until 62 days after transplanting (when 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. 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.

[0034] Table 2 Disease classification of tobacco bacterial wilt

[0035] Specifically, the incidence and disease index of the experimental group and the control group from transplanting to harvest are shown in Table 3 and Figure 5 A, as shown in 5B.

[0036] Table 3 Effects of strain YH4 on cigar tobacco morbidity and disease index

[0037] From the above results, it can be seen that the addition of strain YH4 can significantly reduce the incidence and disease index; specifically, compared with the control group, after using strain YH4, the incidence was relatively reduced by 29.6%, and the disease index was relatively reduced by 61.96%. It can be seen that strain YH4 can be used as a biological control bacteria for the prevention of tobacco bacterial wilt. In addition, it should be noted that this experiment was conducted in a tobacco field where bacterial wilt has frequently occurred in recent years. The control group had a relatively high incidence rate because no measures were taken to prevent and control bacterial wilt.

[0038] In summary, the Sphingomonas paucimobilis 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.

[0039] 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 solution of the present invention rather than to limit it. 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 YH4, characterized in that: The Sphingomonas paucimobilis ( Sphingomonas parapaucimobilis ) The YH4 strain is 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 YH4 as claimed in claim 1 in the preparation of an agent for antagonizing tobacco solanacearum.

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

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 was 1×10 8 CFU-1×10 10 CFU / strain tobacco seedling.

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

8. The method for controlling tobacco bacterial wilt in the field according to claim 7, characterized in that: The following steps are involved: S1. culturing Sphingomonas paucimobilis YH4 to obtain a bacterial liquid; S2. Spray the bacterial solution on the roots of tobacco seedlings before transplanting them; 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 harvesting.

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