Curtobacterium wilt S5 with efficient ultraviolet resistance and application thereof

Abstract

The invention provides curtobacterium wilt S5 with efficient ultraviolet resistance and application of the curtobacterium wilt S5, and belongs to the technical field of agricultural microorganisms. According to the present invention, the curtobacterium wilting S5 is a natural high ultraviolet resistance bacterium screened from the extreme environment of the Qinghai plateau, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.37175; the strain not only can promote nitrogen fixation of plants, but also can improve the resistance of the plants to ultraviolet rays by absorbing the ultraviolet rays, and has the functions of stress resistance and yield increase. The brevibacterium wilt S5 strain disclosed by the invention can be applied to regions with high ultraviolet radiation to replace chemical protective agents, has no residual pollution, and is suitable for organic agriculture.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a strain of Brassica puerariae S5 with highly efficient UV resistance and its applications. Background Technology

[0002] Ultraviolet (UV) radiation is a crucial component of natural sunlight, comprising long-wave UVA (315–400 nm), medium-wave UVB (280–315 nm), and short-wave UV (200–280 nm). UV radiation has a significant damaging effect on organisms. In agricultural environments, prolonged or high-intensity UV radiation directly impacts crop growth, leading to photo-oxidative damage to leaves, decreased photosynthetic efficiency, and weakened plant resistance. It also indirectly affects the stability of the symbiotic microbial community. The phyllosphere is a vital interface between plants and their external environment, and its surface is home to a large number of microorganisms, collectively known as the phyllosphere microbiome. These microorganisms play a key role in promoting plant growth, enhancing disease resistance, and helping plants adapt to abiotic stresses.

[0003] Currently, common protective measures against ultraviolet (UV) stress mainly include physical shielding, chemical UV absorbers, and genetically modified microorganisms. While physical and chemical methods have some effectiveness, they suffer from high costs, low environmental compatibility, and potential ecological risks. In the area of ​​microbial agents, screening for naturally occurring UV-resistant strains or enhancing the UV tolerance of strains through genetic engineering has become an important research direction in recent years. However, genetically engineered strains still face limitations such as biosafety approval and ecological risks. Therefore, isolating and screening indigenous microorganisms with naturally high UV resistance from natural environments, especially extreme light habitats, is urgently needed.

[0004] Nitrogen is one of the most important limiting nutrients for plant growth. The overuse of chemical nitrogen fertilizers has led to a series of environmental problems, including soil degradation, water pollution, and greenhouse gas emissions. Therefore, utilizing nitrogen-fixing microorganisms to provide plants with green and sustainable nitrogen nutrition is an important direction for developing ecological agriculture. Traditional nitrogen-fixing microorganisms, including rhizobia, mainly reside in the rhizosphere or form specific symbiotic relationships with plants. However, strains capable of surviving and performing nitrogen fixation in the aboveground phyllosphere are relatively scarce, and their application potential has not yet been fully explored.

[0005] Currently, research on agricultural microorganisms largely focuses on their biocontrol characteristics or some beneficial functions, while systematic research on their UV resistance and corresponding foliar colonization capabilities remains lacking. Therefore, isolating and identifying a new strain of bacteria with highly efficient UV resistance and nitrogen-fixing function can provide new microbial resources and technical approaches for developing microbial inoculants suitable for areas with strong sunlight, enhancing crop stress resistance, and promoting eco-friendly agricultural development. Summary of the Invention

[0006] In view of this, the present invention provides a strain of Atrophic pus var. wilt S5 with highly efficient UV resistance and its application, in order to solve the above problems.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a strain of Curtobacterium flaccumfaciens S5, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37175, deposited on December 22, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The present invention also provides a microbial agent, wherein the active ingredient of the agent is the aforementioned *Atrophus brevis* S5.

[0010] Preferably, the bacterial agent is a liquid bacterial agent, and the OD of the liquid bacterial agent is... 600 It ranges from 0.4 to 0.6.

[0011] The present invention also provides a method for preparing the microbial agent, wherein the *Atrophus puerariae* S5 is inoculated into a fermentation medium for fermentation culture to obtain the agent.

[0012] Preferably, the fermentation culture temperature is 26~30℃ and the rotation speed is 180~220rpm.

[0013] The present invention also provides the application of the aforementioned *Atrophicum brevis* S5 in any one or more of the following:

[0014] (1) Application in promoting nitrogen fixation in plants;

[0015] (2) Application in improving plant ultraviolet resistance;

[0016] (3) Application in the preparation of microbial agents that promote nitrogen fixation in plants;

[0017] (4) Application in the preparation of microbial agents that enhance plant UV resistance.

[0018] A method for improving plant resistance to ultraviolet radiation, the method comprising: spraying or drenching the plant with a bacterial solution of the aforementioned *Brucella pulveratum* S5.

[0019] Preferably, the plants include legumes, grasses, sedges, and miscellaneous grasses.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] This invention marks the first time that *Curtobacterium flaccumfaciens* S5, a naturally occurring, highly UV-resistant photosynthetic bacterium, has been screened from the extreme environment of the Qinghai-Tibet Plateau. This strain not only promotes nitrogen fixation in plants but also repairs photosystem II damage by absorbing ultraviolet radiation, exhibiting both stress resistance and yield-increasing functions. The *Curtobacterium flaccumfaciens* S5 strain of this invention can be applied in areas with high ultraviolet radiation, replacing chemical protective agents, leaving no residual pollution, and is suitable for organic agriculture. Attached Figure Description

[0022] Figure 1 The survival rate and colony diagram of the Enterobacter, Bulb, Serratia, Pantotheca, and Leroyella species selected in this invention after ultraviolet irradiation.

[0023] Figure 2 This is a colony characteristic diagram of *Atrophus brevicornu* S5 of the present invention.

[0024] Figure 3 This is a circular genome diagram of the *Atrophus brevis* S5 strain of the present invention.

[0025] Figure 4 This is a diagram showing the growth status of alfalfa plants under different treatments in Example 4 of the present invention.

[0026] Figure 5 This is a diagram showing the growth status of *Leymus chinensis* plants under different treatments in Example 4 of the present invention.

[0027] Biological Preservation Instructions

[0028] The present invention relates to Curtobacterium flaccumfaciens S5, taxonomically named Curtobacterium flaccumfaciens, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37175, on December 22, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1. Isolation of strains

[0031] In June 2024, leaves of *Leymus chinensis*, *Alfalfa*, *Artemisia argyi*, and *Potentilla multifida* were collected from Tongbaoshan, Xihai Town, Qinghai Province (36.92°N, 100.79°E) and placed in sterile sampling bags. The samples were immediately transported to the laboratory using dry ice. 10g of each leaf sample was weighed and placed in a sterile Erlenmeyer flask. 90mL of 0.85% sterile physiological saline was added, and the flask was shaken at 4°C and 240 rpm for 1 h. The supernatant of the washing solution was diluted 1000-fold and evenly inoculated onto LB agar using the plate spread method. After incubating the plates at 28°C for 48 h, the plates were streaked and purified. The purified bacterial strains were preserved in 20% (v / v) glycerol solution.

[0032] The selected strains were inoculated again into LB liquid medium and cultured with shaking at 25°C and 240 rpm for 16 h. After centrifugation, the bacterial cells were collected and stored at 4°C for later use. The isolated strains were amplified by 16S rRNA gene PCR (primers 27F / 1492R) and Sanger sequencing was performed. The obtained sequences were compared with BLASTN in the NCBI database (https: / / www.ncbi.nlm.nih.gov) to complete the taxonomic identification of the strains. A total of 36 strains were identified, belonging to 4 genera, of which 20 were identified as belonging to the genus *Burkholderia*, as shown in Table 1.

[0033] Table 1. Statistical table of isolation and identification of phyllodes bacteria strains

[0034] Serial Number strain Colony count 1 Enterobacteriaceae 3 2 genus Curtobacterium 20 3 Serratia 11 4 Pantoea 1 5 Lelliottia 1

[0035] Example 2. UV resistance treatment of isolated strains

[0036] 1. Screening of S5 strain

[0037] Five genera of bacterial strains selected in Example 1 were used as controls to evaluate their resistance to ultraviolet radiation. The specific method is as follows: Each strain was inoculated onto LB agar plates, and a 365 nm ultraviolet lamp was used at a distance of 20 cm (radiation intensity of 74 mW / cm²). 2 Irradiation was performed for 15 seconds, with unirradiated plates serving as a control. After irradiation, all plates were incubated in a 30°C light incubator for 24 hours, followed by colony counting. Each treatment was performed in triplicate. The survival rate of each strain was calculated by comparing the colony counts in the irradiated and control groups. The results are shown below. Figure 1 As shown in the figure. The results showed that *Bacillus* spp. exhibited the highest survival rate under ultraviolet radiation among the tested strains.

[0038] 2. Identification of Strain S5

[0039] (1) Microbiological characteristics

[0040] The obtained single strain was inoculated onto LB agar plates and incubated at 28°C for 2 days. Colony characteristics were then observed, such as... Figure 2 As shown. The colonies of this strain are nearly circular, about 1.5 to 2.5 mm in diameter, light milky yellow and translucent, with a smooth and viscous surface and slightly serrated edges.

[0041] (2) Molecular biological characteristics

[0042] The genome of this strain was sequenced and assembled, and its complete 16S rRNA sequence is shown in SEQ ID NO.1:

[0043] >Curtobacterium flaccumfaciens

[0044]

[0045] After comparing the sequence obtained above with known sequences in the NCBI database, the results showed that the S5 strain had 98% similarity to Curtobacterium flaccumfaciens, and the strain was named Curtobacterium flaccumfaciens S5.

[0046] Further sequencing analysis of *Brucea brevis* S5 revealed the following whole-genome circular map of this strain: Figure 3 As shown in the figure. The genome size of this strain is 3.65 Mb, and its GC content is 70.77%. Through functional annotation of its genes, it was found that this bacterium possesses key genes including trehalose synthesis, transmembrane transport, polyamine export, and specialized metabolism, which can resist the accumulation of reactive oxygen species (ROS) caused by external stress and protect it from oxidative damage caused by environmental stress.

[0047] Example 2. Preparation of *Bacillus pumilus* S5 bacterial culture

[0048] (1) Take out the frozen tube of *Bacillus wiltii* S5 from -80℃, transfer it to -20℃ for 30 min, and then place it on ice to thaw slowly to obtain a bacterial suspension; use a sterile inoculation loop to dip the rehydrated bacterial suspension, spread it on LB slant agar plate, and incubate at 28℃ for 48 h to obtain slant culture;

[0049] (2) Pick the slant culture and inoculate it into LB liquid medium, and incubate at 28℃ for 24h to obtain seed culture;

[0050] (3) Inoculate the seed culture into the liquid culture medium at an inoculation rate of 2% and incubate at 28°C for 24 hours to obtain the fermentation culture;

[0051] (4) Centrifuge the fermentation broth at 6000 rpm for 5 min, discard the supernatant, add sterile water to suspend the bacterial cells, and centrifuge at 6000 rpm for 5 min. Repeat this step twice. Discard the supernatant and add sterile water to suspend the bacterial cells to prepare a bacterial suspension.

[0052] (5) Prepare bacterial suspensions of two concentrations and adjust their OD values ​​accordingly. 600 =0.2 (low concentration) and 0.6 (high concentration).

[0053] Example 3. *Brucea fulvidracoides* S5 promotes nitrogen fixation in plants.

[0054] Elymus nutans, a dominant grass species in the alpine meadows of the Qinghai-Tibet Plateau, and Medicago sativa, a representative leguminous species, were selected as experimental subjects. Their seeds were collected and stored in a dry environment at room temperature.

[0055] The steps for the aseptic germination experiment of seeds are as follows:

[0056] (1) Sterilize the petri dishes in a clean bench for 15 min. After heating and melting 1 / 2 MS solid culture medium, pour 10 mL into each petri dish, cool and solidify, and then set aside.

[0057] (2) Remove the seed shell and select plump seeds. Wash them three times with 75% ethanol, inverting and mixing, for 1 minute each time.

[0058] (3) Discard the waste liquid and wash with 0.1% HgCl2 by inverting and mixing for 6 min.

[0059] (4) After pouring out the waste liquid in the clean bench, wash with sterile water by inverting and mixing 5 times, each time for 30 seconds.

[0060] (5) Discard the supernatant and remove excess water with a pipette. Sterilize the tweezers with an alcohol lamp. After the tweezers have cooled, place the seeds in a petri dish containing 1 / 2 MS medium and incubate in a constant temperature incubator (20℃, 16 h light, 8 h dark) for 7 days.

[0061] 2. Soil sterilization

[0062] Sift the soil through a sieve to remove large clods, then moisten it with water and pack it into sterilization bags, approximately 200g per bag. Sterilize in a quick-opening pressure autoclave at 121℃ for 15 minutes, repeating the sterilization process 5 times. Before use, conduct a sterile cultivation test to check for contamination. If contamination is found, sterilize again until the soil is completely free of bacterial contamination before proceeding to the next step.

[0063] 3. Inoculate seedlings under aseptic conditions

[0064] Three treatments were set up: a high-dose group, a low-dose group, and a control group.

[0065] (1) Inoculation with strains: Select sterile seedlings that have grown for 7 days and have basically the same growth rate. The high-dose group is inoculated with a high concentration of bacterial suspension of Bacillus wiltus S5 (OD200). 600 =0.6) soaked for 3 h, the low dose group was treated with a low concentration of Bacillus wiltii S5 bacterial solution (OD 600 =0.2) Soaked for 3 h, while the control group was soaked in sterile water for 3 h.

[0066] (3) Add 200 g of sterile soil to the plastic box, transplant the seedlings into the plastic box, transplant 20 seedlings into each plastic box, and finally place them in the greenhouse to grow for 14 days.

[0067] 4. Nitrogen application treatment

[0068] pass 15N-labeled (NH4)2SO4 (30%) 15 Nitrogen source (N atom abundance, purchased from Shanghai Chemical Industry Research Institute) was provided. The fertilizer was dissolved in sterile deionized water to prepare a solution, providing 7.5 mg of N per pot of soil (approximately 35 mg (NH4)2SO4 / box). This solution was then evenly applied to a box containing 200 g of sterile soil before transplanting.

[0069] 5. Indicator Measurement and Calculation

[0070] At 15 After 14 days of treatment with N-labeled (NH4)2SO4, five plants were randomly selected from each treatment, and the treatment was repeated three times. After rinsing three times with distilled water, the plants were blotted dry with filter paper, and the underground and aboveground parts were separated. The plant height, root length, underground fresh weight / aboveground fresh weight, and underground dry weight / aboveground dry weight were measured separately. The measurement methods are as follows:

[0071] Plant height: Measure the length from the embryo to the tip of the longest leaf using a ruler.

[0072] Root length: Measure the length (mm) of each plant from the seed embryo to the tip of the main root using a ruler.

[0073] Underground fresh weight / above-ground fresh weight: The selected plant roots / above-ground parts are placed on an electronic balance and weighed.

[0074] Underground dry weight / above-ground dry weight: The selected plant roots / above-ground parts were placed in an oven at 105℃ for 30 minutes to kill the green, and then dried at 65℃ to constant weight, and the dry weight was measured.

[0075] At the same time, the nitrogen isotope ratios of the aboveground and underground samples of the plants in the treatment and control groups were measured. 15 N / 14 N), and calculate δ 15 The N value (relative ratio of nitrogen stable isotope abundance) was determined by isotope ratio in plants using an isotope mass spectrometer (DeltaV Advantage, Germany).

[0076]

[0077] The formula for calculating %Ndfa (the percentage of nitrogen from the atmosphere) is: %Ndfa = [1 - (δ...] 15 N treatment group / δ 15 [N control group)]* 100%.

[0078] After 14 days of greenhouse cultivation, inoculation with *Brachys pumilus* S5 significantly promoted the growth of various plant seedlings (Table 1). The overall trend was: higher dose of bacterial solution > lower dose of bacterial solution > control group (sterile water).

[0079] Table 1. Growth indicators and biomass of various plant seedlings under different treatments

[0080]

[0081] Note: Different lowercase letters after the data in the same column indicate that the differences between treatments in the same column are significant at the p<0.05 level.

[0082] The results in Table 1 show that, in legumes and grasses, high-dose inoculation significantly promoted all growth indicators better than low-dose inoculation, exhibiting a clear dose-dependent relationship.

[0083] Table 2. δ values ​​of different plant parts after inoculation with strain S5 15 N value, atmospheric nitrogen contribution rate (%Ndfa)

[0084]

[0085] Note: Different lowercase letters after the data in the same column indicate that the differences between treatments in the same column are significant at the p<0.05 level. δ 15 The N value is expressed in per mille (‰).

[0086] The results in Table 2 show the δ values ​​for all vaccination treatment groups. 15 The N values ​​were significantly lower than those of the control group of the same family and genus, proving that biological nitrogen fixation occurred; and the %Ndfa in the aboveground parts of the plants treated with low and high doses was generally higher than that in the roots, indicating that nitrogen fixation products are preferentially transported to the aboveground parts to support photosynthesis and growth.

[0087] Example 4. Determination of the UV resistance of plants enhanced by *Brachystomia pumilus* S5.

[0088] Grass seeds of alfalfa and Leymus chinensis were selected from the grass seeds collected from the Qinghai alpine meadow experimental platform as experimental materials. After labeling the grass seed names, they were stored in a dry environment at room temperature and brought back to the laboratory for sowing as soon as possible.

[0089] 1. Aseptic germination of seeds

[0090] (1) Sterilize the petri dishes in a clean bench for 15 min. After heating and melting 1 / 2 MS solid culture medium, pour 10 mL into each petri dish, cool and solidify, and then set aside.

[0091] (2) Remove the seed shell and select plump seeds. Wash them three times with 75% ethanol, inverting and mixing, for 1 minute each time.

[0092] (3) Discard the waste liquid and wash with 0.1% HgCl2 by inverting and mixing for 6 min.

[0093] (4) After pouring out the waste liquid in the clean bench, wash with sterile water by inverting and mixing 5 times, each time for 30 seconds.

[0094] (5) Discard the supernatant and remove excess water with a pipette. Sterilize the tweezers with an alcohol lamp. After the tweezers have cooled, place the seeds in a petri dish containing 1 / 2 MS medium and incubate in a constant temperature incubator (20℃, 16 h light, 8 h dark) for 7 days.

[0095] 2. Soil sterilization

[0096] Sift the soil through a sieve to remove large clods, then moisten it with water and pack it into sterilization bags, approximately 100g per bag. Sterilize in a quick-opening pressure autoclave at 121℃ for 90 minutes, repeating the sterilization process five times. Before use, conduct a sterile cultivation test to check for contamination. If contamination is found, sterilize again until the soil is completely free of bacterial contamination before proceeding to the next step.

[0097] 3. Seedling inoculation experiment under aseptic conditions

[0098] (1) Seedling preparation and transplanting

[0099] Select sterile seedlings that have grown for 7 days and are of uniform growth, and transplant them into plastic boxes containing 200 g of sterile soil (5 seedlings per box), and then place them in a greenhouse environment for 14 days.

[0100] (2) Inoculation treatment of strains

[0101] The bacterial suspensions of *Pantotheca agglutinosa* and *Brachys wiltii* S5 selected in Example 1 were prepared, and the bacterial cells were collected by centrifugation. The bacterial cells were resuspended in sterile distilled water to prepare a bacterial suspension, and the OD was adjusted. 600 The value was set to 0.2. The experiment was divided into three groups. In Experiment 1, 2 mL of *Brucella wiltii* S5 bacterial suspension was sprayed evenly on the plant leaves daily. In Experiment 2, 2 mL of *Pantotheca cumulus* bacterial suspension was sprayed evenly on the plant leaves daily. Plants sprayed with an equal amount of sterile distilled water were used as controls. The treatment was carried out for 3 consecutive days.

[0102] 4. Ultraviolet treatment

[0103] After continuous treatment with the bacterial suspension for 3 days, the plant leaves were irradiated with a 365 nm ultraviolet lamp. Different treatment conditions were set according to the differences in plant tolerance to ultraviolet radiation: alfalfa plants were placed 20 cm away from the ultraviolet lamp (radiation intensity 74 mW / cm²). 2 Irradiation for 2 hours; the *Leymus chinensis* plants were placed 5 cm away from the ultraviolet light source (radiation intensity 482 mW / cm²). 2 Irradiate for 1 hour.

[0104] The results are as follows Figure 4and Figure 5 As shown, after ultraviolet treatment, the leaves of alfalfa seedlings in the control group turned yellow and withered significantly; alfalfa seedlings treated with Pantothecin showed only slight yellowing, demonstrating a certain degree of resistance to ultraviolet radiation; and alfalfa seedlings treated with Brassica pumilus S5 showed almost no obvious yellowing, exhibiting the most outstanding resistance to ultraviolet stress.

[0105] After ultraviolet treatment, the above-ground parts of the control group of *Leymus chinensis* plants withered significantly and the stems became thin. The plants treated with *Pantotheca acuminata* still had some green tissue after ultraviolet treatment, but their overall growth was generally poor.

[0106] Plants treated with *Brucella pustulosa* S5 retained relatively bright green above-ground parts, had robust stems, and exhibited the strongest resistance to adverse conditions after ultraviolet treatment.

[0107] The above results indicate that both Pantotheca cum Cliniculata and Bacillus pumilus S5 strain can enhance the UV resistance of Leymus chinensis, with Bacillus pumilus S5 showing a more significant effect.

[0108] As can be seen from the above embodiments, the present invention provides a strain of *Brachys pumilus* S5 with high UV resistance and its application. The *Brachys pumilus* S5 of the present invention can absorb ultraviolet light and reduce the damage caused by high UV radiation to plants, possibly because it has a highly efficient DNA photorepair or excision repair system. It can be used to prepare UV-resistant microbial preparations and provides valuable resources for the study of DNA damage repair mechanisms.

[0109] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of *Atrophus brevicornu* ( Curtobacterium flaccumfaciens S5, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37175, on December 22, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial inoculant, characterized in that, The active ingredient of the bacterial agent is *Atrophus brevis* S5 as described in claim 1.

3. The microbial agent according to claim 2, wherein the agent is a liquid agent, and the OD of the liquid agent is... 600 The value is 0.2~0.

6.

4. The method for preparing the microbial inoculant according to claim 3, characterized in that, The *Atrophus brevicornu* S5 was inoculated into a fermentation medium for fermentation culture to obtain the bacterial agent.

5. The preparation method according to claim 3, characterized in that, The fermentation culture temperature is 26~30℃, and the rotation speed is 180~220rpm.

6. The use of *Atrophicum brevis* S5 according to claim 1 in any one or more of the following: (1) Application in promoting nitrogen fixation in plants; (2) Application in improving plant ultraviolet resistance; (3) Application in the preparation of microbial agents that promote nitrogen fixation in plants; (4) Application in the preparation of microbial agents that enhance plant UV resistance.

7. A method for improving plant resistance to ultraviolet radiation, characterized in that, The plant was sprayed or drenched with the bacterial solution of the aforementioned *Brucella pulveratum* S5.

8. The method according to claim 7, characterized in that, The plants mentioned include legumes, grasses, sedges, and miscellaneous grasses.

Images