Panacoglobosum pan-64 and application thereof

Abstract

The present application belongs to the field of microbial technology. More particularly, it relates to a Pantoea agglomerans Pan-64 and its application. The present application provides a Pantoea agglomerans Pan-64 which has high-efficiency nitrogen fixation, phosphorus and potassium solubilization and siderophore production capabilities, can significantly promote plant growth and improve the nutritional status. The Pan-64 strain has strong antagonistic effect on the mulberry bacterial wilt pathogen, and can achieve systemic colonization in plant roots, stems and leaves, and has significant effect on the prevention and control of mulberry bacterial wilt. In addition, the Pan-64 strain has very high biological safety, no hemolytic activity, and the silkworm feeding experiment proves that it has no adverse effect on the survival and cocoon production of silkworm, and can be safely used in mulberry garden. The strain of the present application integrates multiple growth promotion, efficient biological control and high safety, and can be widely used for mulberry bacterial wilt prevention and control, poor soil improvement and crop growth promotion, can effectively reduce the use of chemical fertilizers and pesticides, and has great application value in the sustainable development of sericulture and green agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology. More specifically, it relates to a clustered pantothecin strain, Pan-64, and its applications. Background Technology

[0002] Mulberry trees are the material foundation of the sericulture industry, and their yield and quality directly affect the sustainable development of the silk industry. However, mulberry trees are often attacked by various diseases during their growth, among which bacterial wilt (mainly caused by *Laurella* fungus of the Solanaceae family) is a serious threat. Ralstonia pseudosolanacearum Ralstonia solanacearum (Ralstonia solanacearum) is a devastating soil-borne vascular disease. After infecting the roots of mulberry trees, this pathogen rapidly blocks the vascular bundles and secretes toxins, leading to acute wilting and even the death of the entire tree. Because Ralstonia solanacearum survives for a long time in the soil, spreads rapidly, and easily develops resistance, traditional chemical pesticide control is not only ineffective but also prone to causing soil microecological damage and pesticide residue problems. Therefore, finding safe, efficient, and environmentally friendly biological control strategies has become a major urgent need for the sericulture industry.

[0003] Utilizing microorganisms for biocontrol and growth promotion is currently a hot topic in green agriculture research. Pantoea is a group of Gram-negative bacteria widely distributed in the natural environment, belonging to the Enterobacteriaceae family, and exhibits high ecological adaptability and diversity. Studies have shown that some Pantoea strains can promote root development by secreting plant hormones, demonstrating great potential in promoting plant growth.

[0004] However, despite the broad application prospects of Pantotheca in agricultural biotechnology, existing research and applications still have the following significant shortcomings: For example, Chinese patent CN117050915A discloses a Pantotheca MD10 strain that can produce IAA and siderophores to alleviate copper ion stress, but it lacks the ability to solubilize phosphorus, potassium, and nitrogen, thus having limited growth-promoting effects on plants in poor soil environments and failing to inhibit mulberry bacterial wilt. Furthermore, some biocontrol strains may possess potential pathogenicity, hemolysis, or carry transferable drug resistance genes, and their safety against non-target organisms (such as the extremely sensitive silkworm in the sericulture industry) has not been systematically verified, posing significant safety risks to their practical application in mulberry orchards and agricultural environments.

[0005] Therefore, there is an urgent need to screen out a novel plant growth-promoting bacterium from healthy mulberry plants that has multiple growth-promoting functions (nitrogen fixation, phosphorus solubilization, potassium solubilization, and iron carrier production), has a highly effective antagonistic effect against mulberry bacterial wilt, can colonize well in plants, and is highly safe for non-target organisms such as silkworms. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of the prior art and provide a strain of pantothecin that clumps together ( Pantoea agglomeransPan-64 and its applications.

[0007] The first objective of this invention is to provide a strain of pantothecin (Pantothecinus clumps). Pantoea agglomerans Pan-64.

[0008] A second objective of this invention is to provide a microbial preparation.

[0009] A third objective of this invention is to provide the application of the aforementioned Pan-64 agglomerates or microbial preparations.

[0010] The fourth objective of this invention is to provide a method for preventing and controlling bacterial wilt of mulberry trees.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of pantothecin ( ) Pantoea agglomerans Pan-64, the Pantotheca cumulus strain Pan-64, was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 14, 2026, with accession number GDMCC No: 68283.

[0012] The present invention provides a microbial preparation containing the above-mentioned Pan-64 agglomerates or its fermentation broth.

[0013] The present invention's clustering pantothecin ( Pantoea agglomerans Pan-64 not only possesses highly efficient biological nitrogen fixation, phosphorus solubilization, and potassium solubilization capabilities, but also secretes siderophores and exhibits strong antagonistic effects against bacterial wilt pathogens of mulberry trees, effectively controlling bacterial wilt. Therefore, this invention also claims protection for the following applications: The application of the above-mentioned Pan-64 or the above-mentioned microbial preparations in inhibiting Ralstonia solanacearum.

[0014] The application of the above-mentioned Pan-64 or the above-mentioned microbial preparations in the preparation of products that inhibit Ralstonia solanacearum.

[0015] Specifically, the *Ralstonia solanacearum* is *Ralstonia laurentii* (of the Solanaceae family). Ralstonia pseudosolanacearum ).

[0016] Application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in the prevention and control of bacterial wilt of mulberry.

[0017] The application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in the preparation of products for the prevention and control of bacterial wilt of mulberry trees.

[0018] The application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in promoting plant growth.

[0019] The application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in the preparation of products that promote plant growth.

[0020] The application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in nitrogen fixation.

[0021] The application of the above-mentioned Pan-64 agglomerates or the above-mentioned microbial preparations in the preparation of products with nitrogen-fixing capabilities.

[0022] Application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in phosphorus solubilization.

[0023] The application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in the preparation of products with phosphate-solubilizing capabilities.

[0024] The application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in potassium solubilization.

[0025] The application of the above-mentioned Pan-64 clumps or the above-mentioned microbial preparations in the preparation of products with potassium solubilizing ability.

[0026] The application of the above-mentioned Pan-64 agglomerates or the above-mentioned microbial preparations in the production of siderophores.

[0027] The application of the above-mentioned Pan-64 agglomerates or the above-mentioned microbial preparations in the preparation of products that produce siderophores.

[0028] The application of the above-mentioned Pan-64 agglomerates or the above-mentioned microbial preparations in the preparation of products containing iron carriers.

[0029] The present invention also provides a method for preventing and controlling bacterial wilt of mulberry trees, which involves treating mulberry trees with the above-mentioned Pan-64 clumps of bacteria or the above-mentioned microbial preparations.

[0030] Specifically, the treatment includes root irrigation or spraying.

[0031] The present invention has the following beneficial effects: The present invention provides a strain of pantothecin that clumps together ( Pantoea agglomerans Pan-64 possesses comprehensive and excellent plant growth-promoting properties. This strain not only exhibits highly efficient biological nitrogen fixation, phosphorus solubilization, and potassium release capabilities, but also secretes siderophores, providing plants with abundant nitrogen, phosphorus, potassium, and iron nutrients. Pot experiments showed that the Pan-64 strain significantly promoted the growth of mulberry seedlings, increasing seedling height, root length, fresh weight, dry weight, and chlorophyll content. It effectively improved plant nutrition and enhanced the crop's resistance and growth potential in poor soil environments. The Pan-64 strain of this invention exhibits excellent control efficacy against bacterial wilt of mulberry and possesses a strong ability for systemic colonization within plants. The Pan-64 strain strongly inhibits *Raylordia pseudosporidis*, the pathogen causing bacterial wilt in mulberry. Fluorescent labeling and tracking experiments show that the Pan-64 strain can be effectively absorbed by plant roots and transported upwards, achieving systemic colonization in the root, stem, and leaf tissues of mulberry seedlings. Potted plant control experiments further confirm that the incidence of bacterial wilt in mulberry trees treated with Pan-64 was significantly reduced, and the disease index decreased markedly.

[0032] The Pan-64 strain of this invention exhibits extremely high biosafety. Experiments involving silkworms feeding on mulberry leaves have confirmed that the Pan-64 strain does not cause silkworm mortality and has no adverse effects on economic indicators such as survival rate, cocooning rate, and cocoon weight. Therefore, it can be safely used in mulberry orchards. Furthermore, hemolytic tests show that this strain has no hemolytic activity, meeting the biosafety guidelines for microbial fertilizers.

[0033] In summary, the Pan-64 strain of Pantotheca acuminata of the present invention can be widely used for the biological control of bacterial wilt of mulberry trees, the improvement of barren soil, and the promotion of crop growth. Its multi-effect characteristics can effectively reduce the use of chemical pesticides and fertilizers, protect the agricultural micro-ecological environment, and have extremely high application value in the sustainable development of the sericulture industry and green agriculture. Attached Figure Description

[0034] Figure 1 Figure 1 shows the results of nitrogen fixation detection for strain Pan-64; Figure 2 shows the results of streak inoculation on combined nitrogen fixation medium; Figure 3 shows the results of spot inoculation on combined nitrogen fixation medium; Figure 4 shows the results of perforated inoculation on combined nitrogen fixation medium; Figure 5 shows the results of streak inoculation on nitrogen-free medium; Figure 6 shows the results of spot inoculation on nitrogen-free medium; Figure 7 shows the results of perforated inoculation on nitrogen-free medium; Figure 8 shows the results of streak inoculation of nitrogen-fixing bacteria on Assumption medium; Figure 9 shows the results of spot inoculation of nitrogen-fixing bacteria on Assumption medium; Figure 10 shows the results of perforated inoculation of nitrogen-fixing bacteria on Assumption medium.

[0035] Figure 2 The results show the phosphorus solubility of Pan-64 strain; Figure a shows the results of streak inoculation on inorganic phosphorus bacteria culture medium, Figure b shows the results of spot inoculation on inorganic phosphorus bacteria culture medium, Figure c shows the results of streak inoculation on organic phosphorus bacteria culture medium, and Figure d shows the results of spot inoculation on organic phosphorus bacteria culture medium.

[0036] Figure 3 The results show the potassium solubilization activity of strain Pan-64; Figure a shows the results of streak inoculation on silicate bacteria medium, Figure b shows the results of spot inoculation on silicate bacteria medium, and Figure c shows the results of perforation inoculation on silicate bacteria medium.

[0037] Figure 4The results of the siderophore production detection of Pan-64 strain are shown in Figure a; Figure a shows the inoculation results on the CAS detection medium, and Figure b shows the inoculation results on the CAS detection medium through perforation.

[0038] Figure 5 The results show the antagonistic effect of Pan-64 strain against Ralstonia solanacearum.

[0039] Figure 6 This image shows the colony morphology of strain Pan-64 on NA medium plates.

[0040] Figure 7 Gram staining results for Pan-64 strain.

[0041] Figure 8 The plant phenotypes in a pot experiment of controlling mulberry bacterial wilt with Pan-64 strain are shown. Figure A shows mulberry seedlings treated with group A, Figure B shows mulberry seedlings treated with group B, Figure C shows mulberry seedlings treated with group C, and Figure D shows mulberry seedlings treated with group D.

[0042] Figure 9 Results of a pot experiment on the control of mulberry bacterial wilt by strain Pan-64.

[0043] Figure 10 The results are from the hemolytic activity test of Pan-64 strain.

[0044] Figure 11 Results of a feeding experiment on silkworms with Pan-64 strain.

[0045] Figure 12 Figure 1 shows the antibiotic susceptibility test results for strain Pan-64. Figure 2 shows the antibiotic susceptibility test results: First row (from left to right): cefoperazone, ceftriaxone, ceftazidime; Second row: cefuroxime sodium, cefazolin, cefalexin; Third row: piperacillin, ampicillin, oxacillin. Figure 3 shows the antibiotic susceptibility test results: First row: penicillin, ciprofloxacin, norfloxacin; Second row: azithromycin, erythromycin, minocycline; Third row: tetracycline, streptomycin, kanamycin. Figure 4 shows the antibiotic susceptibility test results: First row: gentamicin, amikacin, florfenicol; Second row: doxycycline, imipenem, levofloxacin; Third row: clindamycin, chloramphenicol, polymyxin B. Figure 5 shows the whole genome circle of strain Pan-64.

[0046] Figure 13 The results of the in vitro co-culture experiment between Pan-64 strain and the pathogen of *Rhizoctonia solani* are presented.

[0047] Figure 14 The results of the colonization experiment of Pan-64 strain in mulberry trees.

[0048] Figure 15The results of the phylogenetic tree analysis of Pan-5 strain are shown.

[0049] Figure 16 The results of the phylogenetic tree analysis of Pan-6 strain are shown.

[0050] Figure 17 The results of the phylogenetic tree analysis of Pan-8 strain.

[0051] Figure 18 The results of the phylogenetic tree analysis of Pan-12 strain.

[0052] Figure 19 The results of the phylogenetic tree analysis of strain Pan-14.

[0053] Figure 20 The results of the phylogenetic tree analysis of Pan-15 strain.

[0054] Figure 21 The results of the phylogenetic tree analysis of Pan-17 strain.

[0055] Figure 22 The results show the antagonistic effects of different Pantotheca bacteria against Ralstonia solanacearum.

[0056] Figure 23 The results show the antagonistic effects of Pan-17 and Pan-64 strains against Ralstonia solanacearum. Detailed Implementation

[0057] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0058] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0059] Sample collection: Leaves of healthy Kangqing 283×Kangqing 10 were collected from the Asia-Pacific Sericulture Training Center of South China Agricultural University in Guangzhou.

[0060] Gram staining kit, purchased from Beijing Solarbio Science & Technology Co., Ltd., product number G1060.

[0061] TE buffer (pH 8.0) was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B548401-0500.

[0062] The sterile deionized water was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number E607017-0500.

[0063] 2×Taq HiFi PCR mix (with blue dye) were purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B639295-0005.

[0064] LB agar (LB solid medium), LB broth (with sugar), LB broth (without sugar), beef extract peptone (NA) medium, and NB medium were all purchased from Guangdong Huankai Microbial Technology Co., Ltd., with product numbers 028330, 028320, 028334, 022032, and 022010, respectively.

[0065] Nitrogen-free culture medium, combined nitrogen-fixing culture medium, Assabeth medium for nitrogen-fixing bacteria, inorganic phosphorus bacteria culture medium, organic phosphorus bacteria culture medium, silicate bacteria culture medium, and CAS detection culture medium were purchased from Qingdao Haibo Biotechnology Co., Ltd., with product numbers HB8752, HB8541, HB8540, HB8549-2, HB8673, HB8548-2, and HB9132, respectively.

[0066] The methods for Gram staining, morphological observation and biochemical identification in Example 3 were based on the references "Manual of Systematic Identification of Common Bacteria [M]. Science Press, Dong Xiuzhu and Cai Miaoying. 2001." and "Bergey's Manual of Bacterial Identification. 8th Edition [M]. Science Press, Buchanan and Gibbons. 1984.".

[0067] Solanaceae Raulella ( Ralstonia pseudosolanacearum The XCqk strain, with NCBI accession number PRJNA1329957 and ID 1329957, is the pathogen of bacterial wilt of mulberry (Ralstonia solanacearum).

[0068] Example 1: Isolation and purification of endophytic bacteria from mulberry rhizomes Thirteen samples of stems and leaves from mulberry trees (a hybrid of Kangqing 283 and Kangqing 10) were cleaned with tap water, dried, and then cut into 3cm sections. Under sterile conditions, the stems were disinfected with 75% ethanol for 3 minutes, and the leaves were disinfected with alcohol for 30 seconds and then rinsed four times with sterile water. The stems were then disinfected with 0.1% mercuric chloride solution for 5 minutes, and the leaves were disinfected with 0.1% mercuric chloride solution for 1 minute. Finally, the samples were rinsed five times with sterile water.

[0069] After blotting the surface moisture of the mulberry stem sample with sterile filter paper, cut off both ends. Split the remaining section in half lengthwise to remove the xylem. Under sterile conditions, cut the xylem into 0.1cm pieces. 3 The leaves were cut into small pieces, while the mulberry leaves were simply chopped into 0.1cm pieces. 3The fragments were placed in 10 mL of sterile water and shaken in a shaker at 28°C and 140 r / min for 15–30 min to obtain a bacterial suspension containing endophytic bacteria. The bacterial suspension was then serially diluted 8 times according to the 10-fold serial dilution method to obtain a dilution factor of 10. -1 ~10 -8 The bacterial culture dilution. Take 100 μL of each of the 10... -1 ~10 -8 The bacterial suspension was diluted and evenly spread on LB and NA agar plates using a spreader, with each dilution repeated three times. The plates were incubated upside down in a 28°C incubator for 2 days. Single colonies of different morphologies were picked and streaked onto LB agar plates to purify and obtain multiple pure strains, which were then stored in glycerol tubes at -80°C.

[0070] Example 2: Detection and Screening of Growth-Promoting and Antagonistic Characteristics of Mulberry Endophytic Fungi I. Experimental Methods The endophytic bacteria isolated from mulberry leaves in Example 1 were tested for their growth-promoting and disease-resistant abilities. Then, functional strains were evaluated and screened by comprehensively considering their nitrogen fixation, phosphorus solubilization, potassium solubilization, iron carrier production, and biocontrol abilities.

[0071] (1) Analysis of nitrogen fixation characteristics The isolated and purified bacteria were streaked, spot-inoculated, and punched onto nitrogen-free medium, combined nitrogen-fixing medium, and Assabeth medium for nitrogen-fixing bacteria. After sealing, the plates were placed in a constant temperature incubator at 28°C for culture. The growth of bacteria on each nitrogen-fixing medium was observed. Strains that could grow on nitrogen-free medium were identified as nitrogen-fixing bacteria with nitrogen-fixing function.

[0072] (2) Analysis of phosphorus solubility characteristics The isolated and purified bacteria were cultured on phosphate-solubilizing bacterial culture media (inorganic phosphorus bacteria culture media and organic phosphorus bacteria culture media) by streak plating and spot inoculation, respectively. After sealing, the plates were placed in a constant temperature incubator at 28℃ and the growth of bacteria on each phosphate-solubilizing bacterial culture medium was observed.

[0073] In addition, the phosphorus-solubilizing ability of the isolated mulberry endophytic bacteria was detected using the phosphorus-solubilizing zone method. Single colonies of the isolated and purified bacteria were picked and cultured in nutrient broth (NB) medium at 28℃ and 180 r / min for 3 days to obtain the bacterial fermentation broth. Using a perforator, small holes with a diameter of 5 mm were evenly punched on an inorganic phosphorus bacteria medium plate. Then, 50 μL of the prepared bacterial fermentation broth was added to each hole. After the sample was added, the plate was placed in a 28℃ incubator for cultivation. The formation of a transparent phosphorus-solubilizing zone around the inoculated bacterial solution was observed. Strains that formed a transparent phosphorus-solubilizing zone were identified as phosphorus-solubilizing bacteria.

[0074] (3) Analysis of potassium solubilization characteristics The isolated and purified bacteria were cultured on silicate bacteria medium with glass powder as the potassium source by streak plating, spot inoculation, and perforation with bacterial solution. The plates were sealed and placed in a constant temperature incubator at 28°C. The growth of the strains was observed. Strains that could grow on silicate bacteria medium plates were determined to have potassium solubilizing ability.

[0075] (4) Analysis of the characteristics of iron-producing carriers The isolated and purified bacteria were cultured by punching holes and adding bacterial solution on CAS detection medium plates and by spot injection culture. After sealing, the plates were placed in a constant temperature incubator at 28℃. The presence of orange-yellow halos around the strains was observed. Bacteria that formed orange-yellow halos were identified as siderogenic bacteria with siderogenic function.

[0076] (5) Disease resistance analysis The isolated and purified bacteria were subjected to a plate confrontation experiment on TTC agar plates. 100 μL of Ralstonia solanacearum (XCqk) fermentation broth (OD) was taken. 600 =0.1) After adding to the plate, spread it evenly, then punch holes and add the bacterial solution obtained from isolation and purification. Place it in a constant temperature incubator for 72 h and observe and record.

[0077] II. Experimental Results This invention screened a growth-promoting bacterium with nitrogen fixation, phosphorus solubilization, potassium solubilization, and siderophore production functions by detecting nitrogen fixation, phosphorus solubilization, potassium solubilization, and siderophore production. This growth-promoting bacterium strain was named Pan-64.

[0078] (1) The results of nitrogen fixation detection of Pan-64 strain are as follows: Figure 1 As shown, the results indicate that strain Pan-64 can thrive in combined nitrogen-fixing medium (…). Figure 1 Figures a, b, and c), nitrogen-free culture medium ( Figure 1 Figures d, e, and f) and Assumption medium used for nitrogen-fixing bacteria ( Figure 1 It can grow on all three (g, h, and i) medium; and it forms a transparent zone after spot inoculation and perforation in nitrogen-free medium and Assumption medium, indicating that the Pan-64 strain has nitrogen-fixing activity.

[0079] (2) The results of the phosphorus solubilization test of Pan-64 strain are as follows: Figure 2 As shown, the results indicate that strain Pan-64 can thrive on inorganic phosphorus bacteria culture medium (…). Figure 2 This strain can grow on (Figures a and b) and form transparent phosphate-solubilizing zones in inorganic phosphorus bacteria culture media; moreover, it can also grow on organic phosphorus bacteria culture media (Figures a and b). Figure 2 The bacteria grew on the surface shown in Figures c and d, and formed a transparent phosphate-solubilizing zone in the organic phosphorus bacteria culture medium, indicating that the Pan-64 strain has phosphate-solubilizing activity.

[0080] (3) The results of the potassium solubilization test of Pan-64 strain are as follows: Figure 3 As shown, the results indicate that strain Pan-64 can thrive in silicate bacteria culture medium ( Figure 3 The Pan-64 strain showed good growth in all three figures (a, b, and c), indicating that it has potassium-solubilizing ability.

[0081] (4) Results of siderophore production detection of Pan-64 strain are as follows Figure 4 As shown, the results indicate that strain Pan-64, when inoculated with bacterial culture and perforated on CAS detection medium plates (with holes punched),... Figure 4 (Figure a) and spot vaccination ( Figure 4 (Figure b) After cultivation, orange-yellow halos appeared around the strain, indicating that the Pan-64 strain has the ability to produce siderophores.

[0082] (5) The results of the detection of the antagonistic effect of Pan-64 strain against Ralstonia solanacearum are as follows: Figure 5 As shown, the results indicate that strain Pan-64 can produce a significant inhibition zone on TTC medium, suggesting that strain Pan-64 has the ability to antagonize Ralstonia solanacearum.

[0083] In summary, the growth-promoting characteristics of strain Pan-64 are shown in Table 1. The results indicate that strain Pan-64 possesses the abilities of biological nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, and disease resistance.

[0084] Table 1. Growth-promoting and disease-resistant characteristics of Pan-64 strain

[0085] Note: +++ indicates that the effect is very good, ++ indicates that the effect is good, + indicates that the effect is average, and - indicates that there is no effect.

[0086] Example 3 Identification of Pan-64 strain I. Experimental Methods (1) The Pan-64 strain of Example 2 was inoculated into NA medium and cultured at 28°C. The colony morphology and growth status were observed and recorded. Gram staining, morphological observation and determination of relevant physicochemical parameters of biochemical tests were carried out in accordance with the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification".

[0087] (2) Molecular biological identification of Pan-64 strain: 16S rDNA gene sequencing was performed on Pan-64 strain, and the obtained 16S rDNA gene was sequenced by NCBI-BLAST.

[0088] 16S rDNA primer sequence: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0089] PCR amplification system: 1 μL of 27F primer, 1 μL of 1492R primer, 2 μL of DNA template, 12.5 μL of 2×TaqPCR Master Mix II, and ultrapure water to 25 μL.

[0090] PCR amplification conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 8 min.

[0091] The PCR amplification products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0092] II. Experimental Results (1) Colony morphology characteristics of Pan-64 strain on NA medium plates as follows Figure 6 As shown, the results indicate that strain Pan-64 grows well on NA medium plates, with yellow, opaque colonies that are smooth and have regular edges.

[0093] (2) Gram staining results of Pan-64 strain are as follows Figure 7 As shown in the figure, the results indicate that the Pan-64 strain is a Gram-negative bacterium with short rod-shaped cells.

[0094] (3) The biochemical analysis results of strain Pan-64 are shown in Table 2. The results show that strain Pan-64 can utilize glucose, sucrose, xylose, rhamnose, salicin, eugenol and fructose, but cannot utilize lactose, maltose, galactose, sorbitol, eugenol, inositol, calendula alcohol and starch; the glucose acid production test results are positive, while the results of β-galactoside ONPG, Simon's citrate, gluconate, nitrate gas production, nitrite gas production and glucose gas production tests are all negative.

[0095] Table 2 Biochemical analysis results of Pan-64 strain

[0096] Note: "+" represents a positive reaction, and "-" represents a negative reaction. (5) Molecular biological identification of Pan-64 strain The 16S rDNA gene sequence of strain Pan-64 is shown in SEQ ID NO.1. SEQ ID NO.1:

[0097] The 16S rDNA sequencing alignment results of strain Pan-64 are shown in Table 3. The results indicate that the 16S rDNA of strain Pan-64 is similar to that of multiple strains of pantothenic bacteria (Pu-64). Pantoea agglomerans The similarity (identities) of the two objects reached over 99%.

[0098] Table 3. NCBI-BLAST alignment results of 16S rDNA gene in Pan-64 strain.

[0099] Based on the combined results of colony morphology, biochemical analysis, and molecular biological identification, strain Pan-64 was identified as a type of pantothecin (Pan-64). Pantoea agglomerans The strain was named: Pantotheca cumulus ( ). Pantoea agglomerans Pan-64 was deposited on May 14, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 68283), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0100] Example 4: Pot experiment on the control of bacterial wilt of mulberry tree by Pan-64 bacterial suspension. I. Experimental Methods This embodiment aims to test the control effect of Pan-64 clumps on mulberry bacterial wilt.

[0101] Pan-64 bacterial culture: Pan-64 was inoculated into NB medium and cultured at 28℃ and 180 r / min for 48 h to obtain Pan-64 bacterial culture.

[0102] Ralstonia solanacearum XCqk bacterial suspension: Ralstonia solanacearum XCqk was inoculated into CPG liquid medium and cultured in a shaker at 28℃ and 180 r / min for 72 h to obtain Ralstonia solanacearum XCqk bacterial suspension.

[0103] Twenty-four mulberry seedlings of similar growth (120 days old) were randomly divided into four groups (A, B, C, and D, with six seedlings in each group). After root injury treatment, the treatments for each group were as follows: Group A: Apply 20 mL of sterile water; Group B: Apply 20 mL of Pan-64 bacterial suspension to form clumps; Group C: 20 mL of Pan-64 bacterial suspension was applied, followed by 20 mL of Ralstonia vera XCqk bacterial suspension (concentration 10) 7 days later. 8 (CFU / mL) Group D: 20 mL of *Rhizoctonia solani* XCqk bacterial suspension (concentration 10) was applied. 8(CFU / mL).

[0104] After inoculation, the four groups of potted plants were placed at 28±1℃, with 16 h of light and 8 h of darkness, and 70% humidity for 15 days. The growth and disease incidence of the mulberry seedlings were then investigated.

[0105] II. Experimental Results Results of potted plant control experiment as follows Figure 8 , Figure 9 As shown in Tables 4 and 5, the results indicate that the mulberry trees in both groups A and B grew normally, and compared with group A, the growth of group B was better; this shows that the Pan-64 of the present invention does not cause mulberry wilt disease and can promote mulberry growth.

[0106] All six mulberry trees in group D showed varying degrees of wilting, with four of them exhibiting severe wilting. In contrast, only two mulberry trees in group C showed mild symptoms, indicating that the Pan-64 strain isolated in this invention has a good control effect on bacterial wilt of mulberry caused by *Raylorhizium anisopliae*.

[0107] Table 4. Potted plant growth performance data of strain Pan-64 in controlling bacterial wilt of mulberry.

[0108] Table 5. Potted plant growth performance data of strain Pan-64 in controlling bacterial wilt of mulberry.

[0109] Example 5 Safety test of Pan-64 strain of Pantotheca acuminata I. Experimental Methods The safety of Pan-64 strain to silkworms and the environment was evaluated through hemolytic tests and silkworm feeding with mulberry leaves.

[0110] Pan-64 bacterial suspension: Pan-64 bacterial suspension was inoculated onto NB medium and cultured at 28℃ and 180 r / min for 48 h. After culture, the bacterial pellet was collected by centrifugation and resuspended in sterile water. The bacterial suspension concentration was adjusted to OD. 600 =0.1, and Pan-64 bacterial suspension was obtained.

[0111] (1) Hemolytic test: Pan-64 strain was streaked on a blood agar plate and incubated in a 37 ℃ constant temperature incubator for 24 h. The hemolytic activity around the colony was observed and recorded.

[0112] (2) Silkworm feeding experiment: Healthy silkworms were selected, and on the first day of their 4th instar and the first day of their 5th instar, fresh healthy mulberry leaves were soaked in Pan-64 bacterial suspension (OD200). 600=0.1) In the treatment group, silkworms were fed dried mulberry leaves, while silkworms were fed mulberry leaves treated with sterile water as the control group. The silkworms were fed mulberry leaves for 7 days after they spun their cocoons. The survival rate, cocoon weight, cocoon spinning rate, pupa weight, and cocoon layer weight of the silkworms were investigated and recorded as economic indicators.

[0113] II. Experimental Results (1) Blood plate test Blood platelet results as follows Figure 10 As shown, no hemolytic zone was observed around the single colony, indicating that the Pan-64 strain does not have hemolytic activity, which is in line with the "General Technical Guidelines for Biosafety of Microbial Fertilizers (NY / T1109-2017)" issued by the Ministry of Agriculture and Rural Affairs.

[0114] (2) Experiment on feeding silkworms The results of the safety feeding experiment on silkworms with Pan-64 strain are as follows: Figure 11 As shown, after adding bacteria on the first day of the 4th and 5th instars, the silkworms in the treatment group were able to survive and spin cocoons normally, and there was no difference in cocoon weight and cocooning rate compared with the control group. This indicates that Pan-64 is safe for silkworms and will not cause silkworm death or reduced production.

[0115] Example 6: Antibiotic susceptibility testing and resistance gene analysis of Pan-64 strain of Pantotheca acuminata. I. Experimental Methods The disk diffusion method was used to affix disks containing the antibiotic susceptibility profiles of 27 common antibiotics to NA plates inoculated with Pan-64, and the antibiotic susceptibility profile of Pan-64 was detected. Simultaneously, the whole-genome sequencing data of this strain was used to analyze whether transferable resistance genes existed in its genome, in order to assess the biosafety of this strain in agricultural environments.

[0116] II. Experimental Results The results of the antimicrobial susceptibility test for Pan-64 strain are as follows: Figure 12 As shown, the results indicate that Pan-64 is sensitive to aminoglycosides and quinolones, but resistant to macrolides, lincosamides, and penicillins. Combined with whole-genome analysis of this strain, no transferable resistance genes were found, indicating that the resistance genes of this strain exist in its chromosome and are safe for use in the environment.

[0117] Example 7: In vitro co-culture antibacterial test of Pan-64 strain of Pantotheca acuminata I. Experimental Methods YFP (yellow fluorescent protein) and mCherry (red fluorescent protein) expression plasmids were introduced into strain Pan-64 and strain XCqk (Ralstonia solanaceae), respectively, to construct fluorescently labeled strains. Equal volumes of the two fluorescently labeled bacterial solutions were mixed and co-cultured in NB liquid medium for 24 h. Subsequently, fluorescence signals were observed using laser confocal microscopy, co-localization analysis was performed, and the growth and spatial distribution of the two bacteria in the co-culture system were observed and recorded.

[0118] II. Experimental Results The results of the in vitro co-culture experiment between Pan-64 strain and the pathogen of *Rhizoctonia solani* are as follows: Figure 13 As shown, the results indicate that both fluorescent plasmids (YFP and mCherry) can be expressed normally, and the growth of Ralstonia solanacearum XCqk was significantly inhibited in the treatment group. This shows that the Pan-64 strain can significantly inhibit the growth of Ralstonia solanacearum in vitro, which is consistent with the plate antagonism results of Example 2 and the pot experiment results of Example 4.

[0119] Example 8 Colonization experiment of Pan-64 strain of Pantotheca acuminata in mulberry trees I. Experimental Methods Pan-64 bacterial suspension: Pan-64 bacterial suspension was inoculated onto NB medium and cultured at 28℃ and 180 r / min for 48 h. After culture, the bacterial pellet was collected by centrifugation and resuspended in sterile water. The bacterial suspension concentration was adjusted to OD. 600 =0.1, and Pan-64 bacterial suspension was obtained.

[0120] Pan-64 bacterial suspension labeled with YFP (yellow fluorescent protein) was inoculated into sterile mulberry seedlings. After culturing for 7 days at a constant temperature of 28±1℃, 16 h light / 8 h dark, and 70% humidity, root, stem, and leaf tissues of the sterile seedlings were collected and photographed using a laser confocal microscope. The fluorescence signal was detected to assess whether the Pan-64 strain could invade the host through plant roots, establish systematic colonization, and exert a long-lasting effect.

[0121] II. Experimental Results Results of the colonization experiment in mulberry trees as follows Figure 14 As shown, after 7 days of cultivation, obvious fluorescent signals of strain Pan-64 were observed in the root, stem, and leaf tissues of mulberry seedlings. This indicates that strain Pan-64 can be absorbed by plant roots and widely distributed within the mulberry tree, exerting its growth-promoting and disease-resistant capabilities.

[0122] Comparative Example 1 I. Experimental Methods In Example 1, various Pantoea bacteria were isolated, including Pan-64 strain from Example 2, as well as Pan-5 strain (Pantoea sp.) and Pan-6 strain (…). Pantoea ananatis ), Pan-8 strain ( Pantoea ananatis ), Pan-12 strain ( Pantoea agglomerans ), Pan-14 strain ( Pantoea vagans ), Pan-15 strain ( Pantoea ananatis ), Pan-17 strain ( Pantoea sp). The phylogenetic trees of each strain based on 16S rDNA are as follows: Figures 15-21 As shown.

[0123] A plate confrontation experiment was conducted on TTC medium plates. 100 μL of Ralstonia solanacearum (XCqk) fermentation broth (OD) was taken. 600 =0.1) After being added to the plate, it was spread evenly. Then, the bacterial suspensions of several Pan-Bacteria strains (Pan-5, Pan-6, Pan-8, Pan-12, Pan-14, Pan-15, and Pan-17 strains) obtained in Example 1 were added to the plate and then placed in a constant temperature incubator for 72 h before observation and recording.

[0124] II. Experimental Results Experimental results are as follows Figure 22 and Figure 23 As shown, the results indicate that only strain Pan-64 was able to produce a clear inhibition zone on TTC medium, while the other Pantotheca bacteria were unable to produce an inhibition zone.

[0125] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A cluster of pantothecin ( Pantoea agglomerans Pan-64, characterized in that, The Pan-64 strain of *Panthera solani* was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 14, 2026, with accession number GDMCC No: 68283.

2. A microbial preparation, characterized in that, Contains the Pan-64 clump-forming bacteria as described in claim 1 or its fermentation broth.

3. The use of the Pan-64 clump-forming bacteria of claim 1 or the microbial preparation of claim 2 in inhibiting Ralstonia solanacearum, or in the preparation of a product inhibiting Ralstonia solanacearum.

4. The use of the Pan-64 microbial agent of claim 1 or the microbial agent of claim 2 in the prevention and control of bacterial wilt of mulberry, or in the preparation of products for the prevention and control of bacterial wilt of mulberry.

5. The use of the Pan-64 microbial agent of claim 1 or the microbial preparation of claim 2 in promoting plant growth, or in the preparation of products that promote plant growth.

6. The use of the Pan-64 microbial preparation of claim 1 or the microbial preparation of claim 2 in nitrogen fixation, or in the preparation of products with nitrogen fixation capabilities.

7. The use of the Pan-64 clump-forming bacteria of claim 1 or the microbial preparation of claim 2 in phosphorus solubilization, or in the preparation of products with phosphorus solubilization capabilities.

8. The use of the Pan-64 microbial preparation of claim 1 or the microbial preparation of claim 2 in potassium solubilization, or in the preparation of products with potassium solubilization capabilities.

9. The use of the Pan-64 agglomerates of claim 1 or the microbial preparation of claim 2 in the production of siderophores, or in the preparation of products containing siderophores, or in the preparation of products containing siderophores.

10. A method for preventing and controlling bacterial wilt of mulberry trees, characterized in that, Mulberry trees are treated with the Pan-64 microbial agent described in claim 1 or the microbial agent described in claim 2.

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