Composition of cupriavidus necator cup3, cupriavidus necator cup3 and raoultella ornithinolytica rao1 and application thereof in preventing and controlling crop bacterial wilt

Abstract

The application discloses cupriavidus CUP3, a combination of cupriavidus CUP3 and raoultella ornithinolytica RAO1 and application of the combination in prevention and control of crop bacterial wilt. Cupriavidus necator Cupriavidus pinatubonensis Cupri The application discloses cupriavidus CUP3, a combination of cupriavidus CUP3 and raoultella ornithinolytica RAO1 and application of the combination in prevention and control of crop bacterial wilt. The application discloses cupriavidus CUP3, a combination of cupriavidus CUP3 and raoultella ornithinolytica RAO1 and application of the combination in prevention and control of crop bacterial wilt. The application discloses cupriavidus CUP3, a combination of cupriavidus CUP3 and raoultella ornithinolytica RAO1 and application of the combination in prevention and control of crop bacterial wilt.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to a combination of Cupriavidus yeoncheonensis CUP3, Cupriavidus yeoncheonensis CUP3 and Raoultella ornithinolytica RAO1, and the application of CUP3 and RAO1 in combination for the control of soil-borne diseases of crops, especially the control of bacterial wilt of tomatoes. Background Technology

[0002] Bacterial wilt caused by Ralstonia solanacearum is widespread, has diverse hosts, causes severe economic losses, and poses a serious threat to sustainable agricultural development. [1] Beneficial rhizosphere bacteria inhibit pathogen growth and protect plant health by competing with pathogens for nutrients and secreting antagonistic substances. [2] However, introducing a single beneficial bacterial strain into the complex soil micro-ecosystem often faces significant challenges. Fluctuations in soil pH, salinity (Ec), and other physicochemical factors, as well as competition and repulsion among native microbial communities, frequently result in low colonization success rates and unstable population sizes of exogenous beneficial bacteria, making it difficult to sustainably and effectively exert their antibacterial function. [3] Studies have shown that constructing functionally complementary microbial assemblages is an effective way to overcome this bottleneck. These strain combinations enhance their colonization ability and disease-suppressing effect in the rhizosphere through metabolic complementarity, increased nutrient competition, and the production of more antagonistic substances against pathogens. [4] Therefore, it is necessary to screen for strain combinations that can efficiently promote the colonization of beneficial bacteria and enhance their effectiveness, thereby strengthening the ability of beneficial bacteria to control soil-borne bacterial wilt in tomatoes. Developing a highly efficient and stable compound microbial agent will provide a new technical solution for the green control of soil-borne bacterial wilt in tomatoes. Currently, there is a lack of research on the use of copper-loving bacteria or its combination with ornithine-releasing Raoultella for the control of bacterial wilt. Summary of the Invention

[0003] Using tomato as a model crop and soil-borne bacterial wilt as a prevalent disease, the inventors investigated the inhibitory effects of combinations of *Bacillus thuringiensis* CUP3, *Bacillus thuringiensis* CUP3, and *Raoultella ornithine-releasing* RAO1 on soil-borne bacterial wilt. The study found that all combinations of *Bacillus thuringiensis* CUP3, *Bacillus thuringiensis* CUP3, and *Raoultella ornithine-releasing* RAO1 could reduce the incidence of tomato bacterial wilt, especially the combination of *Bacillus thuringiensis* CUP3 and *Raoultella ornithine-releasing* RAO1, which significantly reduced the incidence of tomato bacterial wilt.

[0004] The purpose of this invention is to provide a copper-loving bacterium CUP3, a composition of copper-loving bacterium CUP3 and ornithine-releasing bacterium RAO1, and the application of the composition in the control of soil-borne bacterial wilt of tomato.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The copper-loving bacterium CUP3, classified as *Cupriavidus yeoncheonensis*, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 22, 2025, with accession number GDMCC NO:67512.

[0007] Raoultella ornithine-soluble Raoultella RAO1, classified as Raoultella ornithine-soluble Raoultella, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 22, 2025, with accession number GDMCC NO:67513.

[0008] Another objective of this invention is to provide the application of copper-loving bacteria CUP3 in the control of soil-borne bacterial wilt in crops.

[0009] Preferably, the application is the use of copper-loving bacteria CUP3 in the preparation of reagents for controlling soil-borne bacterial wilt in crops.

[0010] Another object of the present invention is to provide a bacterial composition containing *Cup3*, a copper-loving bacterium, and *Rauvolfia ornithine-releasing*, or a fermentation broth containing *Cup3*, a copper-loving bacterium, and *Rauvolfia ornithine-releasing*, or ...

[0011] Preferably, the bacterial combination consists of copper-loving bacteria CUP3 and ornithine-releasing Raoultella ovalis RAO1, or it consists of the fermentation broth of copper-loving bacteria and ornithine-releasing Raoultella ovalis RAO1.

[0012] Another object of the present invention is a method for preparing the bacterial composition described above, comprising: activating both *Cup3* (copper-loving bacterium) and *RAO1* (ornithine-releasing rauwolfia) using TSA medium; picking single colonies of *Cup3* on TSB medium, incubating overnight at 28–30°C and 150–190 rpm with shaking; collecting fresh bacterial culture, centrifuging, collecting bacterial cells, washing the bacterial cells with sterile water to remove the culture medium, and adjusting the bacterial concentration to 1 × 10⁻⁶ with sterile water. 7 ~1×10 9 CFU / mL was used to obtain a suspension of *Cup3*, a copper-loving bacterium; a single colony of *Rauvolfia ornithine-releasing* RAO1 was picked and cultured on TSB medium at 28–30°C and 150–190 rpm overnight with shaking. Fresh bacterial suspension was collected by centrifugation, and the bacterial cells were washed with sterile water to remove the culture medium. The bacterial concentration was adjusted to 1 × 10⁻⁶ CFU / mL with sterile water. 7 ~1×10 9CFU / mL was used to obtain a suspension of *Rauvolfia ornithine-releasing* RAO1. Equal volumes of *CUP3* suspension and *Rauvolfia ornithine-releasing* RAO1 suspension were mixed to obtain the bacterial composition. Alternatively, *Rauvolfia ornithine-releasing* RAO1 suspension was mixed with OS basal inorganic salt medium and carbon source mixture, and cultured at 28–30°C and 150–190 rpm for 48 h with shaking. After centrifugation, the supernatant was filtered to obtain *Rauvolfia ornithine-releasing* RAO1 fermentation broth. The *CUP3* suspension and *Rauvolfia ornithine-releasing* RAO1 fermentation broth were then mixed to obtain the bacterial composition.

[0013] When preparing the copper-loving bacterium CUP3 suspension, the centrifugation temperature is room temperature, the centrifugation speed is 4000-6000 rpm, and the centrifugation time is 5-10 min.

[0014] When preparing the ornithine-releasing Raoulbacterium RAO1 suspension, the centrifugation temperature is room temperature, the centrifugation speed is 4000-6000 rpm, and the centrifugation time is 5-10 min.

[0015] Preferably, when the bacterial composition contains copper-loving bacterium CUP3 and ornithine-lysinic rauwolfia RAO1, the total concentration of the bacterial composition is 1×10⁻⁶. 8 CFU / mL.

[0016] Preferably, the volume ratio of the ornithine-soluble Raoulbacterium RAO1 suspension to the OS basic inorganic salt culture medium and carbon source mixture is 1:2:17.

[0017] When preparing the ornithine-releasing RAO1 fermentation broth, the centrifugation temperature is room temperature, the centrifugation speed is 4000-6000 rpm, and the centrifugation time is 5-10 min.

[0018] Preferably, the supernatant is filtered using a 0.22 μm 96-well plate filter membrane.

[0019] Preferably, the volume ratio of the copper-loving bacterium CUP3 suspension to the ornithine-releasing bacterium RAO1 fermentation broth is 1:2.

[0020] Another object of the present invention is to provide the application of the bacterial composition described herein in the control of soil-borne bacterial wilt in crops.

[0021] Preferably, the application is the use of the bacterial composition in the preparation of reagents for controlling soil-borne bacterial wilt in crops.

[0022] The combination of copper-loving bacterium CUP3 and ornithine-degrading Raoulbacterium RAO1 can enhance the ability to inhibit bacterial wilt and reduce the incidence of bacterial wilt in tomatoes.

[0023] Another objective of this invention is to provide a method for controlling soil-borne bacterial wilt of crops by combining *Cup3* (copper-loving bacterium) and *Rauvolfia ornithine-releasing* (ornithine-releasing rauvolfia ROO1), comprising: adding a bacterial composition to the rhizosphere soil of the crop 7-10 days after transplanting the crop seedlings by root irrigation; wherein, when the bacterial composition contains *Cup3* (copper-loving bacterium) and *Rauvolfia ornithine-releasing* (ornithine-releasing rauvolfia ROO1), 10 mL of a 1×10⁻⁶ solution is added per crop seedling. 7 ~1×10 9 A bacterial composition of CFU / mL.

[0024] The aforementioned soil-borne bacterial wilt disease is a soil-borne bacterial wilt disease of Solanaceae crops, preferably soil-borne bacterial wilt disease of tomato.

[0025] Preferably, each crop seedling is inoculated with 10 mL of a 1×10⁻⁶ solution. 8 A bacterial composition of CFU / mL.

[0026] The crop seedlings mentioned are tomato seedlings with 3 to 5 true leaves.

[0027] The beneficial effects of this invention are:

[0028] The combination of copper-loving bacteria CUP3, copper-loving bacteria CUP3 and ornithine-releasing bacteria RAO1, and the combination of copper-loving bacteria CUP3 and ornithine-releasing bacteria RAO1 fermentation broth can all reduce the occurrence of bacterial wilt in tomatoes. In particular, the combination of copper-loving bacteria CUP3 and ornithine-releasing bacteria RAO1 can significantly reduce the occurrence of bacterial wilt in tomatoes. Attached Figure Description

[0029] Figure 1 The biomass of the fermentation broths of copper-loving bacterium CUP3 and ornithine-releasing bacterium RAO1 was cross-cultured.

[0030] Figure 2 The combined effect of copper-loving bacterium CUP3 and ornithine-releasing bacterium RAO1 on inhibiting the growth of Ralstonia solanacearum.

[0031] Figure 3 The combined effect of *Bacillus thuringiensis* CUP3 and *Rauvolfia ornithine-degrading* RAO1 in reducing the incidence of bacterial wilt was investigated.

[0032] Biological Preservation Information:

[0033] The copper-loving bacterium CUP3, classified as Cupriavidus yeoncheonensis, was deposited on December 22, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China, with accession number GDMCC NO:67512.

[0034] Raoultella ornithinolytica (RAO1) was deposited on December 22, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China, with accession number GDMCC NO:67513. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0036] Test culture medium:

[0037] Nutrient Broth (NB) medium: 3 g / L beef extract, 0.5 g / L yeast extract, 5 g / L tryptone, 10 g / L glucose, deionized water to a final volume of 1 L, adjust pH to 7.2-7.4, autoclave at 115℃ for 30 min.

[0038] Nutrient Agar (NA) medium: NB medium with 2% agar powder.

[0039] Tryptic Soy Broth (TSB) medium: 15 g / L peptone, 5 g / L soybean peptone, 5 g / L sodium chloride, deionized water to a final volume of 1 L, adjust pH to 7.2-7.4, autoclave at 115℃ for 30 min.

[0040] Tryptic Soy Agar (TSA) medium: TSB medium with 2% agar powder.

[0041] OS basic inorganic salt culture medium: disodium hydrogen phosphate 7.01 g, potassium dihydrogen phosphate 6.8 g, magnesium sulfate heptahydrate 1.1 g, ammonium sulfate 1.2 g, calcium chloride heptahydrate 88 mg, ferrous sulfate heptahydrate 7 mg, ammonium molybdate tetrahydrate 0.2 mg, sodium EDTA-2.5 mg, zinc sulfate heptahydrate 1.11 g, manganese sulfate hexahydrate 1.54 mg, copper sulfate pentahydrate 0.39 mg, cobalt nitrate hexahydrate 0.25 mg, sodium borate decahydrate 0.18 mg, nickel chloride hexahydrate 1.3 mg, deionized water 1 L, adjust pH to 7.2-7.4, autoclave at 121℃ for 20 min.

[0042] A mixture of 48 carbon sources: L-alanine, β-alanine, L-arginine, L-asparagine, γ-butyric acid, citrulline, glutamine, L-glycine, L-histidine, isoleucine, L-lysine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-valine, acetic acid, ascorbic acid, citric acid, formic acid, galacturonic acid, Glutaric acid, glycolic acid, lactic acid, maleic acid, malic acid, propylene glycol, 2-ketoglutaric acid, pyruvic acid, succinic acid, tartaric acid, oxalic acid, fructose, glucose, sucrose, maltose, L-arabinose, D-galactose, D-mannose, D-xylose, D-ribose, ethanolamine, inositol, D-mannitol, and inosine were prepared into 100 mM stock solutions using deionized water. After filtration and sterilization, 0.2 mL of each of the 48 carbon source stock solutions was added to 10.4 mL of sterile water, resulting in a final concentration of 0.1 mM for each carbon source.

[0043] Beneficial bacteria tested: *Bacillus thuringiensis* CUP3; *Rauvolobacterium ornithine-lysing* RAO1. Both strains were isolated from rhizosphere soil of healthy tomatoes in Qilin Town, Nanjing, Jiangsu Province (118°57' E, 32°03' N). 1 g of soil sample was weighed and added to 9 mL of sterile SM buffer. The mixture was shaken at 30°C and 170 rpm for 30 min to prepare a soil suspension. The soil suspension was then serially diluted with sterile water, and the appropriate dilution (10⁻⁶) was obtained. -5 ~10 -6100 μL of soil suspension was spread onto 1 / 10 TSA solid medium plates and incubated at 30°C in the dark for 48 h. After incubation, single colonies were picked based on colony morphology and repeatedly streaked on TSA plates for purification, yielding two strains, designated CUP3 and RAO1, respectively. Both purified strains were inoculated into TSB liquid medium for activation and culture, and glycerol was added to prepare a bacterial culture preservation solution, which was stored at -80°C. The rhizosphere bacterial strains preserved in glycerol were then streaked onto TSA solid medium plates for activation and cultured at 30°C for 48 h. Single colonies were picked and inoculated into 96-well plates containing TSB medium, and cultured at 30°C and 170 rpm for 24 h with shaking. Subsequently, using the bacterial culture as a template, PCR amplification was performed using the universal primers 27F (5'-AGAGTTTGATCATGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3') for the bacterial 16S rRNA gene. The PCR reaction system consisted of 25 μL: 12.5 μL of 2× Master Mix, 1 μL each of forward and reverse primers, 2 μL of bacterial template, and 8.5 μL of deionized water. The PCR amplification conditions were: 95 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 58 ℃ annealing for 30 s, and 72 ℃ extension for 90 s, for a total of 30 cycles; and a final extension at 72 ℃ for 60 s. After sequencing the amplified products, the 16S rRNA gene sequence was compared and analyzed using the NCBI and RDP databases to identify the taxonomic position of the strain. *Cupriavidus yeoncheonensis* CUP3, taxonomically named *Cupriavidus yeoncheonensis*, was deposited on December 22, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, with accession number GDMCC NO:67512. Raoultella ornithinolytica RAO1, classified as Raoultella ornithinolytica, was deposited on December 22, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China, with accession number GDMCC NO:67513.

[0044] Preparation of *CUP3* bacterial suspension: Take the *CUP3* strain glycerol tube stored at -80℃, streak it onto TSA medium, and incubate at 30℃ until single colonies appear. Pick a single colony from the plate and transfer it to TSB medium, incubate overnight at 30℃ and 170 rpm. Take fresh bacterial suspension, centrifuge at room temperature and 6000 rpm for 5 min, collect the bacterial cells, wash the cells three times with sterile water, remove the medium, and adjust the OD with sterile water to obtain the OD.600 It is 0.5 (corresponding to 1×10). 8 A suspension of *C. coli* CUP3 (CFU / mL).

[0045] Preparation of *Rauvolfia ornithine-releasing* strain RAO1 suspension: A glycerol tube containing *Rauvolfia ornithine-releasing* strain RAO1, stored at -80℃, was streaked onto TSA medium and incubated at 30℃ until single colonies appeared. Single colonies were picked from the plate and transferred to TSB medium, incubated overnight at 30℃ and 170 rpm. Fresh bacterial suspension was collected, centrifuged at room temperature and 6000 rpm for 5 min, and the bacterial cells were washed three times with sterile water. The medium was removed, and the suspension was adjusted with sterile water to obtain OD. 600 It is 0.5 (corresponding to 1×10). 8 Ornithine-neutralizing Raoulbacterium RAO1 suspension (CFU / mL).

[0046] Preparation of mixed bacterial suspension: Mix the copper-loving bacterium CUP3 suspension and the ornithine-releasing bacterium RAO1 suspension at a volume ratio of 1:1 to obtain a mixed bacterial suspension for later use.

[0047] Preparation of aseptic fermentation broth of *Copper-Gnaphalium* strain CUP3: Take the glycerol tube of *Copper-Gnaphalium* strain CUP3 stored at -80℃, streak it on TSA medium, and incubate at 30℃ until single colonies grow. Pick a single colony from the plate and transfer it to TSB medium, incubate overnight at 30℃ and 170 rpm. Take fresh bacterial culture, centrifuge at room temperature and 6000 rpm for 5 min, collect the bacterial cells, wash the bacterial cells three times with sterile water, remove the medium, adjust with sterile water, and obtain OD. 600 It is 0.5 (corresponding to 1×10). 8 A suspension of *Bacillus cereus* CUP3 (CFU / mL) was prepared. 170 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, and 10 μL of LOD were added to each well of a 96-well plate. 600 The 0.5% suspension of *CUP3* bacteria was placed in a 96-well plate and continuously shaken for 48 hours in a constant temperature shaker at 30 ℃ and 170 rpm. The bacterial suspension was centrifuged at 4000 rpm for 5 min using an enzyme-labeled plate centrifuge. The supernatant was filtered through a 96-well plate filter membrane (0.22 μm) to obtain the sterile fermentation broth of *CUP3* bacteria.

[0048] Preparation of aseptic fermentation broth for *Rauvolobacterium ornithine solubilizer* RAO1: A glycerol tube containing *Rauvolobacterium ornithine solubilizer* strain RAO1, stored at -80℃, was streaked onto TSA medium and incubated at 30℃ until single colonies appeared. Single colonies were picked from the plate and transferred to TSB medium, incubated overnight at 30℃ and 170 rpm. Fresh bacterial culture was collected, centrifuged at room temperature and 6000 rpm for 5 min, and the cells were washed three times with sterile water. The culture medium was removed, and the culture was adjusted with sterile water to obtain the OD. 600 It is 0.5 (corresponding to 1×10). 8 A suspension of *Rauvolfia ravellatus* RAO1 (CFU / mL) that can solubilize ornithine was added to each well of a 96-well plate. 170 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, and 10 μL of OD were added to each well. 600 A 0.5 μL suspension of *Rauvolfia ornithine-releasing* RAO1 was prepared and cultured in a 96-well plate at 30 °C and 170 rpm for 48 h using a constant temperature shaker. The bacterial suspension was then centrifuged at 4000 rpm for 5 min using an ELISA plate centrifuge. The supernatant was filtered through a 96-well plate filter membrane (0.22 μm) to obtain the sterile fermentation broth of *Rauvolfia ornithine-releasing* RAO1.

[0049] The tested Ralstonia solanacearum strain was wild-type Ralstonia solanacearum QL-Rs1115 (GenBank accession GU390462, hereinafter referred to as Ralstonia solanacearum), isolated from Qilin Town, Nanjing, Jiangsu Province (118°57' E, 32°03' N). Its red fluorescent protein-labeled strain QL-RFP is the model invasive pathogen of this invention. This strain was created by transforming the red fluorescent protein gene mCherry into Ralstonia solanacearum QL-Rs1115 using plasmid pYC12, and it can grow on NA medium plates containing 30 µg / mL gentamicin. [5] .

[0050] Preparation of Ralstonia solanacearum QL-RFP bacterial suspension: Ralstonia solanacearum QL-RFP was streaked onto NA medium containing 30 μg / mL gentamicin and incubated at 30℃ until single colonies appeared. Single colonies from the plates were transferred to NB medium and incubated overnight at 30℃ and 170 rpm. Fresh bacterial suspension was centrifuged at room temperature and 6000 rpm for 5 min to collect the bacterial cells. The cells were washed three times with sterile water, the medium was removed, and the OD of the bacterial suspension was adjusted with sterile water. 600 Dilute to 0.5 and then 100 times to obtain a Ralstonia solanacearum QL-RFP suspension (1×10⁻⁶). 6 (CFU / mL), for later use.

[0051] Example 1

[0052] The biomass of the fermentation broths of copper-loving bacteria CUP3 and ornithine-releasing bacteria RAO1 were cross-cultured.

[0053] The experiment included four treatments: two CUP3 groups (CUP3 treatment group with RAO1 sterile fermentation broth and CUP3 bacterial suspension, and CUP3 control group with sterile water and CUP3 bacterial suspension); and two RAO1 groups (RAO1 treatment group with CUP3 sterile fermentation broth and RAO1 bacterial suspension, and RAO1 control group with sterile water and RAO1 bacterial suspension). Each treatment was repeated six times. The CUP3 treatment group consisted of 150 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, 20 μL of RAO1 sterile fermentation broth, and 10 μL of OD medium added to each well of a 96-well plate. 600 The control group consisted of a 0.5 μL suspension of *Copper-Loving Bacteria* CUP3; the control group consisted of 150 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, 20 μL of sterile water, and 10 μL of LOD added to each well of a 96-well plate. 600 The concentration of *Copper-Grease Bacterium* CUP3 suspension was 0.5 μL; RAO1 treatment group: 150 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, 20 μL of sterile CUP3 fermentation broth, and 10 μL of OD were added to each well of a 96-well plate. 600 The first control group consisted of 0.5 μL of ornithine-lysing Raoulbacterium RAO1 suspension; the second control group consisted of 150 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, 20 μL of sterile water, and 10 μL of OD medium added to each well of a 96-well plate. 600 The concentration of *Rauvolfia ulmoides* RAO1 suspension was 0.5 μL. 96-well plates were incubated at 30 °C and 170 rpm in a constant-temperature shaker for 48 h. OD values ​​for each treatment were measured using a microplate reader. 600 value.

[0054] As shown in Table 1 and Figure 1 As shown, the aseptic fermentation broth of *Rauvolfia ornithine-releasing* RAO1 significantly promoted the growth of *Ralstonia solanacearum* CUP3 (P<0.05), which may enhance the competitive ability of *Ralstonia solanacearum* CUP3 against *Ralstonia solanacearum*, thereby inhibiting the growth of *Ralstonia solanacearum*.

[0055] Table 1. Biomass of the fermentation broths of *Cup3* (copper-loving bacterium) and *RAO1* (ornithine-releasing bacterium) cross-cultured.

[0056]

[0057] Note: Different letters in the same column in the table indicate differences that reach a significance level of 0.05.

[0058] Example 2

[0059] The indoor effect of the combination of copper-loving bacteria CUP3 and ornithine-releasing Raoulbacterium arabinose inhibiting the growth of Ralstonia solanacearum.

[0060] The experiment was set up with four treatments: control group (only Ralstonia solanacearum was added), CUP3 group (CUP3 and Ralstonia solanacearum were added), RAO1 group (RAO1 and Ralstonia solanacearum were added), and CUP3+RAO1 group (CUP3, RAO1 and Ralstonia solanacearum were added), with four replicates for each treatment.

[0061] Group CUP3: 160 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, and 10 μL of OD were added to each well of a 96-well plate. 600 The concentrations were 0.5 μL of *Ralstonia solanacearum* CUP3 bacterial suspension and 10 μL of *Ralstonia solanacearum* QL-RFP bacterial suspension.

[0062] RAO1 group: 160 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, and 10 μL of OD were added to each well of a 96-well plate. 600 The concentrations were 0.5 μL of *Ralstonia solubilizing* RAO1 bacterial suspension and 10 μL of *Ralstonia solubilizing* QL-RFP bacterial suspension.

[0063] CUP3+RAO1 group: 160 μL of OS basal inorganic salt medium, 20 μL of a mixture of 48 carbon sources, and 10 μL of OD were added to each well of a 96-well plate. 600 The mixture consisted of 0.5 μL of a mixed bacterial suspension of *Ralstonia solubilizer* RAO1 and *Ralstonia pulveratum* CUP3 and 10 μL of *Ralstonia solubilizer* QL-RFP.

[0064] Control group: The copper-loving bacteria CUP3 suspension in the CUP3 group was replaced with an equal volume of sterile water.

[0065] The 96-well plates were incubated at 30 °C and 170 rpm in a constant-temperature shaker for 48 h. The OD values ​​of each treatment were measured using a microplate reader. 600 The values ​​of fluorescence and RFP (excitation light: 580 nm, absorption light: 610 nm) were calculated. The relative mCherry Fluorescence Unit (RFU) of Ralstonia solanacearum was calculated.

[0066] Calculation formula: RFU=lg(RFP / OD) 600 ).

[0067] See results Figure 2As shown in Table 2, compared with the control group, the relative biomass of Ralstonia solanacearum decreased in the CUP3 group, RAO1 group and CUP3+RAO1 group. In particular, the combination of copper-loving bacterium CUP3 and ornithine-releasing bacterium RAO1 significantly reduced the relative biomass of Ralstonia solanacearum (P<0.05) and significantly inhibited the growth of bacterial wilt.

[0068] Table 2. Indoor effect of the combination of copper-loving bacteria CUP3 and ornithine-releasing Raoulbacterium arabinose in inhibiting the growth of Ralstonia solanacearum.

[0069]

[0070] Note: Different letters in the same column in the table indicate differences that reach a significance level of 0.05.

[0071] Example 3

[0072] The potted plant effect of the combination of copper-loving bacterium CUP3 and ornithine-degrading rauwolfia RAO1 in reducing the incidence of bacterial wilt.

[0073] Tomato tested: Red dwarf tomato.

[0074] Tomato seedling cultivation: Tomato seeds are first soaked in sterile water and vernalized overnight in a 4℃ refrigerator. They are then soaked in 75% alcohol for 1 minute, washed three times with sterile water, and then soaked in 3% sodium hypochlorite (NaClO) solution for 5 minutes, followed by rinsing six times with sterile water. The sterilized tomato seeds are then placed on sterile plates lined with filter paper moistened with sterile water, spread evenly, and placed in a 30℃ constant temperature incubator for 48 hours to germinate. Well-germinating seedlings are then selected and sown in seedling trays.

[0075] When seedlings reached the three-leaf stage, seedlings of uniform growth were transplanted into six-cell seedling trays (each cell containing 100 g of soil). Four treatments were set up: control group, CUP3 group, RAO1 group, and CUP3+RAO1 group, with 12 replicates for each treatment and one tomato plant per replicate, for a total of 48 tomato plants. One week after transplanting, each tomato plant in the CUP3, RAO1, and CUP3+RAO1 groups was inoculated with 10 mL of OD245 solution. 600 It is 0.5 (corresponding to 1×10). 8 The control group was inoculated with 10 mL of 1×10⁻⁶ CFU / mL copper-loving bacterium CUP3 suspension, ornithine-lysing Raoultella RAO1 suspension, and a mixed suspension. An equal volume of deionized water was added to each tomato plant. One week later, each tomato plant was inoculated with 10 mL of 1×10⁻⁶ CFU / mL copper-loving bacterium CUP3 suspension, ornit 6 CFU / mL of Ralstonia solanacearum QL-RFP suspension. All plants were grown in a greenhouse with a natural temperature of 25–30℃ and a humidity of 60–80%RH, with the pots being moved twice a week and watered regularly. Plant disease indices were monitored daily until the end of the experiment.

[0076] Tomato disease incidence statistics: Ralstonia solanacearum disease severity is classified into 5 levels: Level 0: No wilting of leaves; Level 1: 1%-25% of leaves wilting; Level 2: 26%-50% of leaves wilting; Level 3: 51%-75% of leaves wilting; Level 4: 76%-100% of leaves wilting or dying. The disease index curve is plotted using the "audpc" function from the "agricolae" package in R.

[0077] See results Figure 3 According to Table 3, both the application of copper-loving bacterium CUP3 and the application of ornithine-releasing bacterium RAO1 alone can reduce the incidence of soil-borne bacterial wilt in tomatoes. Compared with the CUP3 group and the RAO1 group, the combination of copper-loving bacterium CUP3 and ornithine-releasing bacterium RAO1 can significantly reduce the incidence of soil-borne bacterial wilt in tomatoes (P<0.05) and significantly reduce the occurrence of bacterial wilt.

[0078] Table 3. Potted plant effects of the combination of *Bacillus thuringiensis* CUP3 and *Rauvolfia ornithine-degrading* RAO1 in reducing the incidence of bacterial wilt.

[0079]

[0080] Note: Different letters in the same column in the table indicate differences that reach a significance level of 0.05.

[0081] References

[0082] [1] Jiang G, Wei Z, Xu J, Chen H, Zhang Y, She X, Macho AP, Ding W, Liao B. Bacterial Wilt in China: History, Current Status, and FuturePerspectives. Front Plant Sci. 2017, 8: 1549.

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Claims

1. Cupriavidus yeoncheonensis, classified as Cupriavidus yeoncheonensis, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 22, 2025, with accession number GDMCC NO:67512.

2. The copper-loving bacterium CUP3 described in claim 1 in controlling the bacterial wilt caused by Ralstonia solanacearum Ralstonia solanacearum Applications that cause soil-borne bacterial wilt in crops.

3. A bacterial composition, characterized in that: The product contains the copper-loving bacteria CUP3 and ornithine-releasing Raoultella RAO1 as described in claim 1, or the fermentation broth containing the copper-loving bacteria CUP3 and ornithine-releasing Raoultella RAO1 as described in claim 1. Among them, Raoultella ornithinolytica RAO1, classified as Raoultella ornithinolytica, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 22, 2025, with accession number GDMCC NO:67513.

4. A method for preparing the bacterial composition according to claim 3, characterized in that: include: Both *Cup3*, a copper-loving bacterium, and *RAO1*, an ornithine-lysing bacterium, were activated using TSA medium. Single colonies of *Cup3* were picked and cultured overnight at 28–30°C with shaking at 150–190 rpm on TSB medium. Fresh bacterial culture was collected, centrifuged, and the cells were washed with sterile water to remove the culture medium. The bacterial concentration was adjusted to 1 × 10⁻⁶ with sterile water. 7 ~1×10 9 CFU / mL was used to obtain a suspension of *Cup3*, a copper-loving bacterium; a single colony of *Rauvolfia ornithine-releasing* RAO1 was picked and cultured on TSB medium at 28–30°C and 150–190 rpm overnight with shaking. Fresh bacterial suspension was collected by centrifugation, and the bacterial cells were washed with sterile water to remove the culture medium. The bacterial concentration was adjusted to 1 × 10⁻⁶ CFU / mL with sterile water. 7 ~1×10 9 CFU / mL was used to obtain a suspension of *Rauvolfia ornithine-releasing* RAO1. Equal volumes of *CUP3* suspension and *Rauvolfia ornithine-releasing* RAO1 suspension were mixed to obtain the bacterial composition. Alternatively, *Rauvolfia ornithine-releasing* RAO1 suspension was mixed with OS basal inorganic salt medium and carbon source mixture, cultured at 28–30°C and 150–190 rpm for 48 h with shaking, centrifuged, and the supernatant was filtered to obtain *Rauvolfia ornithine-releasing* RAO1 fermentation broth. The *CUP3* suspension and *Rauvolfia ornithine-releasing* RAO1 fermentation broth were then mixed to obtain the bacterial composition.

5. The method for preparing the bacterial composition according to claim 4, characterized in that: When the bacterial composition contains copper-loving bacterium CUP3 and ornithine-releasing rauwolfia RAO1, the total concentration of the bacterial composition is 1×10⁻⁶. 8 CFU / mL.

6. The method for preparing the bacterial composition according to claim 4, characterized in that: The volume ratio of the ornithine-releasing Raoulbacterium RAO1 suspension to the OS basic inorganic salt medium and carbon source mixture is 1:2:17; the volume ratio of the copper-loving bacterium CUP3 suspension to the ornithine-releasing Raoulbacterium RAO1 fermentation broth is 1:

2.

7. The bacterial composition of claim 3 in controlling Ralstonia solanacearum Ralstonia solanacearum Applications that cause soil-borne bacterial wilt in crops.

8. The application according to claim 7, characterized in that: The soil-borne bacterial wilt disease mentioned refers to the soil-borne bacterial wilt disease of Solanaceae crops.

9. A method for controlling soil-borne bacterial wilt of crops by combining copper-loving bacterium CUP3 and ornithine-releasing bacterium RAO1, characterized in that: include: Seven to ten days after transplanting crop seedlings, the bacterial composition described in claim 3 is added to the rhizosphere soil of the crop by root irrigation; wherein, when the bacterial composition contains copper-loving bacterium CUP3 and ornithine-releasing rhabditis elegans RAO1, 10 mL of a 1×10⁻⁶ concentration is added to each crop seedling. 7 ~1×10 9 A bacterial composition of CFU / mL.

10. The method according to claim 9, characterized in that: The soil-borne bacterial wilt disease mentioned refers to the soil-borne bacterial wilt disease of Solanaceae crops.

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