Stenotrophomonas maltophilia and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to stenotrophomonas maltophilia and application thereof. The stenotrophomonas maltophilia is characterized in that the scientific name of the strain is Shenotrophomonas maltophilia DZ4, the preservation number of the strain is CCTCC M 20231664, the preservation number of the strain is CCTCC M 20231664, the preservation number of the strain is CCTCC M 20231664, and the preservation number of the strain is CCTCC M 20231664. The stenotrophomonas maltophilia is preserved in the China Center for Type Culture Collection on September 11, 2023, and the address of the preservation unit is No. 299 on eight road in Wuchang District, Wuhan City, Hubei Province. The natural microorganism for preventing and treating the fusarium root rot is obtained through screening, the natural microorganism has a degradation effect on the pathogen fusarium of the tobacco fusarium root rot, the degradation rate reaches 80% or above after 24-hour co-culture, the biological diversity cannot be damaged, and it is avoided that plants generate drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a Stenotrophomonas maltophilia and its applications. Background Art

[0002] Tobacco Fusarium root rot is a disease caused by a series of Fusarium species, such as *Fusarium incarnatum-equiseti*, a newly discovered Fusarium complex species isolated from the field. It belongs to the Deuteromycetes, Order Gastrodiaales, Family Lepidaceae, and Genus *Fusarium*. This disease causes root lesions, sometimes affecting only one side, resulting in the top bending to one side, small leaves, and root rot. Dissection of the diseased stems and roots reveals browning of the xylem, disrupting the tobacco's vascular system and hindering water and nutrient absorption in the underground parts. Ultimately, this leads to plant death, severely impacting leaf quality and posing a significant threat to tobacco production.

[0003] The prior art (Wang Yunming, Gao Yuguang, Zhao Xiaolu, et al. Screening, identification and degradation characteristics of fusaric acid degrading strain ND6-2 [J]. Journal of Jilin Agricultural University [2025-07-18].) discloses a strain ND6-2 that can degrade fusaric acid (FA), with a degradation rate of 77.27% ± 0.91%.

[0004] The prior art CN107858316B discloses a Stenotrophomonas maltophilia antagonistic to tobacco blackleg fungus. This antagonistic strain has been deposited at the China Center for Type Culture Collection, Wuhan University, China, accession number CCTCC NO: M2017673.

[0005] Prior art CN118909826B relates to a Stenotrophomonas maltophilia strain with preventive and therapeutic effects against pepper mild mottle virus (PMMoV) and its application. This Stenotrophomonas maltophilia DZ4 has been deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2023, with accession number CCTCC NO: M20231664. Its average inhibition rate against PMMoV reached 93.0%.

[0006] However, there are currently no effective reagents to control tobacco Fusarium root rot. Summary of the Invention

[0007] The purpose of this invention is to provide a Stenotrophomonas maltophiliae that can effectively degrade Fusarium mycelium and to apply it to the prevention and control of Fusarium root rot in tobacco.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] A strain of Stenotrophomonas maltophilia, scientifically named Stenotrophomonas maltophilia DZ4, with accession number CCTCC M 20231664, was deposited on September 11, 2023, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0010] In one preferred embodiment, the strain characteristics of Stenotrophomonas maltophilia on NA solid medium are: milky white round colonies with neat edges, smooth surface, and raised opaque surface; Gram staining confirms that the bacterium is a Gram-negative bacterium.

[0011] In one preferred embodiment, the primers for amplifying the DNA of Stenotrophomonas maltophilia are F-P0: GAGAGGTTTGATCCTGGCTCAG (SEQ ID NO.1); R-P6: CTACGGCTACCTTGTTACGA (SEQ ID NO.2).

[0012] Based on the same inventive concept, the present invention also claims protection for the application of the Stenotrophomonas maltophilia in the prevention and control of Fusarium root rot in tobacco.

[0013] In one preferred embodiment, the Stenotrophomonas maltophiliae can degrade the hyphae of Fusarium root rot pathogens.

[0014] In one preferred embodiment, the pathogen causing tobacco root rot is Fusarium incarnatum-equiseti.

[0015] Based on the same inventive concept, this invention also claims protection for a method for preventing and controlling Fusarium root rot in tobacco, which involves drenching the plant with the Stenotrophomonas maltophiliae.

[0016] In one preferred embodiment, each plant is drenched with 10-50 mL of Stenotrophomonas maltophilia inoculum.

[0017] In one preferred embodiment, the OD600 value of Stenotrophomonas maltophilia bacterial suspension is 0.5-1.0.

[0018] The beneficial effects of this invention are as follows: Currently, there are no effective agents in production for the complete control of Fusarium root rot in tobacco. This invention screens a natural microorganism for the prevention and control of Fusarium root rot, which has a degradation effect on Fusarium incarnatum-equiseti, the pathogen of Fusarium root rot in tobacco. The degradation rate reaches 80% after 24 hours of co-culture, significantly higher than other Stenotrophomonas maltophilia disclosed in the prior art. Furthermore, DZ4 is a screened natural antagonistic bacterium that does not pollute the environment, does not damage biodiversity, meets the national green pest control development needs, reduces the pollution of soil and environment by chemical pesticides, and avoids the development of pesticide resistance in plants. Attached Figure Description

[0019] Figure 1 Microscopic observation of the hyphae of DZ4 and Fusarium incarnatum-equiseti after co-culture, among which Figure 1 A represents the untreated Fusarium incarnatum-equiseti hyphae as a control. Figure 1 B represents Fusarium mycelium after 48 hours of treatment with DZ4. Figure 1 C represents Fusarium mycelium after 24 hours of treatment with DZ4.

[0020] Figure 2 This is a plate diagram of the antagonistic test between DZ4 and Fusarium co-culture.

[0021] Figure 3 The culture morphology is based on DZ4 plate culture.

[0022] Figure 4 The image shows the effect of a laboratory experiment on the prevention and treatment of tobacco in potted plants using DZ4.

[0023] Figure 5 The image shows the clustering analysis results of the DZ4 16S rDNA sequence.

[0024] Figure 6 This is a scanning electron microscope image of DZ4. DETAILED DESCRIPTION

[0025] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0026] Example 1

[0027] Acquisition of strains and identification of strains

[0028] 1.1 Strains Isolation

[0029] 1.1.1 Sampling: In tobacco fields where Fusarium root rot has occurred, select still healthy tobacco plants and collect soil samples from the root zone of the tobacco plants at a depth of 20 cm for later use.

[0030] 1.1.2 Preparation of coating solution: Place 10g of prepared soil in a sterilized Erlenmeyer flask, add 20ml of sterile water, shake until the liquid becomes turbid, let stand at room temperature for 10 minutes, and set aside.

[0031] 1.1.3 Purification: Using a sterile pipette tip, aspirate 200 μL of the supernatant from the settled liquid and spread it on NA medium using the plating method. Incubate at 28°C for 24 h. After colonies grow on the isolation plates, based on the characteristics of the microbial colonies, such as morphology, size, surface structure, edge structure, texture, gloss, transparency, color, and soluble pigments produced, use an inoculation loop to pick out isolated single colonies one by one. Select single colonies with different morphologies and streak them on the corresponding blank NA medium plates for purification. Incubate upside down in a 28°C incubator for 24 h to obtain purified strains for later use.

[0032] 1.1.4 Screening: Single colonies of bacteria were selected and placed in sterilized 100ml Erlenmeyer flasks containing 20ml of culture medium. The flasks were shaken and cultured (28℃, 200r / min) for 24 hours to ensure a sufficiently high bacterial concentration (OD600 = 1) for use as fermentation stock. Fusarium incarnatum-equiseti was selected as the target. Pure cultures of the isolated bacteria were co-cultured with Fusarium in liquid PDA medium. The bacteria were inoculated at 10 μL, and the fungus was cultured into 5mm agar plates. After 24 hours of co-culture, the hyphae were observed under a microscope. Fusarium hyphae without bacteria served as a control. After three repeated experiments, bacteria exhibiting degradation activity against Fusarium hyphae were selected; the blank control showed no degradation.

[0033] Bacteria exhibiting degradative activity against Fusarium hyphae were re-screened using the same method, and the bacterium with the best degradation effect was identified and named DZ4 for further research. Hyphae from liquid culture medium co-cultured with DZ4 and Fusarium incarnatum-equiseti were observed under a microscope, and the results are as follows: Figure 1 As shown, where Figure 1 A represents the untreated Fusarium incarnatum-equiseti hyphae as a control. Figure 1 B represents Fusarium mycelium after 48 hours of treatment with DZ4. Figure 1C represents Fusarium mycelium treated with DZ4 for 24 hours. The results showed that the antagonistic strain DZ4 had a degradation effect on Fusarium incarnatum-equiseti, the pathogen of tobacco Fusarium root rot, with a degradation rate of over 80% after 24 hours of co-culture and over 96% after 48 hours of co-culture.

[0034] The DZ4 strain was inoculated onto NA solid medium plates and streaked. After 48 hours of incubation, the colony color and single colony morphology were observed and Gram staining was performed.

[0035] The results showed that the characteristics of the strain after 48 hours of culture on NA solid medium were: milky white, round colonies with neat edges, smooth surface, and opaque raised areas. Figure 3 As shown, the bacteria turn pink after Gram staining, proving that they are Gram-negative bacteria.

[0036] The strains were physiologically and biochemically identified according to the methods in the "Handbook for Identification of Common Bacteria" and subjected to scanning electron microscopy.

[0037] The results showed that, under scanning electron microscopy, the strain was rod-shaped with a single polar flagellum (…). Figure 6 Physiological and biochemical identification results showed negative reactions for VP assay, starch hydrolysis, phenylalanine deaminase, and gelatin liquefaction, but positive reactions for nitrate reduction, nitrite reduction, fluorescent pigment production, and motility. Based on the physiological and biochemical characteristics, it was preliminarily identified as a Stenotrophomonas bacterium.

[0038] 1.2 Strain Identification

[0039] The DZ4 test tube strain was selected, and its DNA was extracted using the Plant Genome Extraction Kit (NO.CW0553) produced by Beijing Kangwei Century Biotechnology Co., Ltd.

[0040] The specific extraction method is as follows:

[0041] Step 1: Scrape about 100mg of DZ4 bacteria into a sterilized mortar and grind thoroughly with liquid nitrogen;

[0042] Step 2: Collect the ground powder into a centrifuge tube, add 700 μL of Buffer GP1 preheated at 65℃, quickly invert and mix, then place the centrifuge tube in a 65℃ water bath for 20 minutes, inverting the centrifuge tube 3 times during the water bath to mix the sample.

[0043] Step 3: Add 700 μL of chloroform, mix thoroughly, and centrifuge at 13000 rpm for 5 minutes. Carefully transfer the upper aqueous phase to a new centrifuge tube, add 700 μL of Buffer GP2, and mix thoroughly.

[0044] Step 4: Add all the obtained solution to the Spin Column DM, centrifuge at 13000 rpm for 30 seconds, and discard the waste liquid. Place the Spin Column DM back into the Collection Tube.

[0045] Step 5: Add 700 μL of Buffer GW1 to the Spin Column DM, centrifuge at 13000 rpm for 30 seconds, and discard the waste liquid. Place the Spin Column DM back into the Collection Tube.

[0046] Step 6: Add 500 μL of Buffer GW2 to the Spin Column DM, centrifuge at 13000 rpm for 30 seconds, and discard the waste liquid. Place the Spin Column DM back into the Collection Tube.

[0047] Step 7: After repeating step 6, centrifuge at 13000 rpm for 2 minutes and discard the waste liquid. Place the Spin Column DM at room temperature for several minutes to thoroughly dry any residual Buffer GW2 in the adsorbent material.

[0048] Step 8: Place the Spin Column DM into a new centrifuge tube, add 100 μL of Buffer GE, incubate at room temperature for 5 minutes, centrifuge at 13000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃.

[0049] The extracted DZ4 DNA was used with universal bacterial primers.

[0050] F-P0: GAGAGTTTGATCCTGGCTCAG (SEQ ID NO. 1); R-P6: CTACGGCTACCTTGTTACGA (SEQ ID NO. 2).

[0051] Perform PCR amplification.

[0052] PCR amplification reaction system

[0053] Reaction components Reaction volume (10 μL) <![CDATA[ddH20(μL)]]> 3.05 <![CDATA[10×Buffer (without Mg 2+ )]]> 1.00 <![CDATA[MgCl2(mmo I / L)]]> 0.50 dNTPs (mmol / L) 0.20 Tag enzyme U / μL 0.25 Primer (μmol / L) 2.00 DNA (ng / L) 3.00

[0054] The PCR amplification procedure is as follows:

[0055] Preheat at 95℃ for 5 minutes;

[0056] 94℃ denaturation for 30s; 55℃ annealing for 30s; 72℃ extension for 30s; 30 cycles;

[0057] Extend at 72℃ for 5 minutes;

[0058] After PCR amplification, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence of the 16s rDNA is as follows:

[0059] CTCACCCCAGTCATCGGCCACACCGTGGCAAGCGCCCTCCCGAAGGTTAAGCTACCTGCTTCTGGTGCAACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTGAGATAGGGTTTCTGGGATTGGCTTACCGTCGCCGGCTTGCAGCCCTCTGTCCCTACCATTGTAGTACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCGGTCTCCTTAGAGTTCCCACCATTACGTGCTGGCAACTAAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCGACATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGCGAACTTAACGCGTTAGCTTCGATACTGCGTGCCAAATTGCACCCAACATCCAGTTCGCATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCCTCAGTGTCAGTGTTGGTCCAGGTAGCTGCCTTCGCCATGGATGTTCCTCCCGATCTCTACGCATTTCACTGCTACACCGGGAATTCCGCTACCCTCTACCACACTCTAGTTGTCCAGTTTCCACTGCAGTTCCAGGTTGAGCCCAGGGCTTTCACA(SEQ IDNO.3).

[0060] The sequencing results were aligned using BLAST on the National Center for Biotechnology Information website. The results showed that the DZ4 strain sequence had a Query coverage of 100% and a Percent identity of 99.89% with Stenotrophomonasindicatrix strain D763 (CP079106.1) on NCBI.

[0061] On the NCBI website, sequences with high similarity after alignment were selected and phylogenetic trees were constructed using the Neighbor-Joining (NJ) method in the MEGA 6.0 phylogenetic analysis software. The results are shown below. Figure 5 .from Figure 5 It can be seen that DZ4 has a short genetic distance and a close evolutionary relationship with Stenotrophomonas maltophilia, thus confirming that the isolated strain DZ4 is Stenotrophomonas maltophilia.

[0062] It has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20231664. This Stenotrophomonas maltophilia was deposited at the China Center for Type Culture Collection on September 11, 2023. The address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0063] Example 2

[0064] Plate co-culture experiment

[0065] Fermentation was carried out with 10 mL of Stenotrophomonas maltophilia DZ4 broth for 24 h. Fermentation was stopped when OD600 = 1. The fermentation broth was centrifuged to collect the cells. The cell precipitate was dissolved in sterile water by pipetting, yielding 1 mL of bacterial solution. This 1 mL of dissolved cells was poured into PDA medium at a suitable temperature (1 mL of bacterial solution per 100 mL of medium), and plated. A control plate without added cells was used. After the plates were dried, Fusarium incarnatum-equiseti plates were punched with a 5 mm punch to create mycelial discs of the same size. These mycelial discs were inoculated onto the DZ4-treated and control PDA plates and incubated upside down for 5 days. The results are as follows: Figure 2 As shown, observations revealed that Fusarium cultured on PDA with added DZ4 cells grew slowly, and the hyphal diameter was significantly smaller than that of Fusarium cultured on control PDA. Figure 2 This indicates that Stenotrophomonas maltophilia DZ4 can significantly inhibit the growth of Fusarium incarnatum-equiseti.

[0066] Example 3

[0067] Indoor potted plant experiment

[0068] Tobacco seedlings of Yunyan 87 were cultivated and prepared for inoculation experiments when they reached the 4-6 leaf stage. The experiment included a healthy control group, a positive control group, a protection group, a treatment group, and a DZ4 group. The protection group was first drenched with 10 mL of DZ4 bacterial solution (OD600 = 1), and 24 hours later, the rootstock was inoculated with 5 mL of Fusarium incarnatum-equiseti bacterial solution (OD600 = 1). The inoculation method involved using a sterile syringe needle to make a small incision in the tobacco rootstock to allow the bacterial solution to contact the incision. The treatment group was first inoculated with Fusarium incarnatum-equiseti bacterial solution using the same method as the prevention group, and 24 hours later, the roots were drenched with 10 mL of DZ4 bacterial solution (OD600 = 1). The healthy control group received no treatment. The positive control group was only inoculated with Fusarium solution. The DZ4 group was only sprayed with DZ4 bacterial solution, without inoculation with Fusarium solution. Each group was replicated with 5 plants. Results were observed and statistically analyzed after 5 days. Some results are as follows Figure 4 As shown, the results indicated that the incidence of disease in the DZ4 prevention group was lower than that in the treatment group. The incidence rate in the DZ4 prevention group was 40%, while the incidence rate in the DZ4 treatment group reached 90% (e.g., Figure 4 Meanwhile, plants in the DZ4 treatment group showed obvious wilting and no new leaf growth. In the DZ4 prevention group, disease-free plants and plants in the control group maintained good growth performance, with new leaf formation in both. The positive control group had a 100% incidence rate, and the plants showed the most obvious wilting. The DZ4 group had a 0% incidence rate, and the plants exhibited the best growth characteristics. Through repeated verification and cross-comparison of a large amount of experimental data, the following significant trend can be clearly observed: Stenotrophomonas maltophiliae DZ4 can prevent the occurrence and development of Fusarium incarnatum-equiseti.

[0069] Using the same method, an indoor pot experiment was conducted on the Stenotrophomonas maltophilia strain GZU-Stm01, which was deposited at the China Center for Type Culture Collection on January 21, 2019, with accession number CCTCC NO: M2019059. The results showed that the incidence rate in the GZU-Stm01 prevention group was 70%, while the incidence rate in the DZ4 treatment group reached 90%.

[0070] Using the same method, an indoor pot experiment was conducted on Stenotrophomonas maltophilia NUT011, which is preserved at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC NO: 62368. The results showed that the incidence rate in the GZU-Stm01 prevention group was 80%, and the incidence rate in the DZ4 treatment group reached 90%.

[0071] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A Stenotrophomonas maltophilia, characterized in that, The scientific name of this strain is Stenotrophomonas maltophilia DZ4, and its accession number is CCTCC M 20231664. This Stenotrophomonas maltophilia was deposited at the China Center for Type Culture Collection on September 11, 2023. The address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The Stenotrophomonas maltophilia according to claim 1, characterized in that, Primers for amplifying the DNA of Stenotrophomonas maltophilia are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The application of Stenotrophomonas maltophilia according to claim 1 in the control of Fusarium root rot in tobacco.

4. The application according to claim 1, characterized in that, The Stenotrophomonas maltophiliae can degrade the hyphae of Fusarium root rot pathogens in tobacco.

5. The application according to claim 1, characterized in that, The Stenotrophomonas maltophiliae can degrade the hyphae of Fusarium root rot pathogens in tobacco.

6. A method for controlling tobacco Fusarium root rot, characterized in that, For: Drenching the roots of the plant with the Stenotrophomonas maltophiliae.

7. The method according to claim 6, characterized in that, Drench each plant with 10-50 mL of Stenotrophomonas maltophilia bacterial solution.

8. The method according to claim 6, characterized in that, The OD600 value of Stenotrophomonas maltophilia bacterial suspension is 0.5-1.0.

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