Bacillus sp. c1-74 and use thereof

Abstract

This invention belongs to the field of microbial technology, specifically disclosing a Bacillus C1-74 strain and its applications. More particularly, it relates to a Bacillus C1-74 strain that promotes growth and significantly inhibits garlic root rot. This Bacillus C1-74 strain was deposited on April 3, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, with accession number CCTCC M 2025700. This invention discloses a Bacillus C1-74 strain and its applications. This Bacillus C1-74 strain significantly inhibits garlic root rot, effectively alleviating the harmful symptoms of root rot such as browning and decay of garlic roots. Simultaneously, this strain has a significant growth-promoting effect on seedlings of vegetables such as garlic, scallions, cucumbers, and tomatoes.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Bacillus C1-74 and its applications. Background Technology

[0002] garlic( Allium sativum Garlic (Allium chinense), an important crop with both medicinal and edible uses, has bulbs rich in organic sulfur compounds, polysaccharides, and polyphenols, among other bioactive components. It has wide applications in food seasoning, medicine, and health care, making it a vital crop in my country's agricultural production, offering both economic and ecological benefits. However, with the promotion of intensive and large-scale garlic cultivation and the increase in continuous cropping years, soil-borne diseases have become increasingly prominent, with root rot caused by various pathogenic fungi, such as Fusarium oxysporum, being the most severe.

[0003] This disease is characterized by its insidious onset and diverse transmission routes. After the pathogen infects the garlic roots, it causes browning and rotting of the roots, leading to yellowing and wilting of the above-ground parts of the plant, severely impairing the plant's water and nutrient absorption capabilities. Statistics show that garlic root rot can generally cause 20%–30% yield loss, with severely affected fields experiencing losses exceeding 50%. Furthermore, the quality of garlic bulbs produced after infection declines, significantly reducing their commercial value. It has become a key bottleneck restricting the sustainable development of the garlic industry in my country's major garlic-producing areas.

[0004] Currently, production mainly relies on chemical fungicides to control garlic root rot. However, long-term use not only leads to increased pathogen resistance, soil microecological degradation, and environmental pollution, but also contradicts international agricultural product safety standards and the direction of green agriculture development. Although measures such as disease-resistant variety breeding and agricultural ecological regulation have been applied, progress in traditional breeding methods has been slow due to garlic's unique propagation method, narrow genetic background, and scarcity of resistant germplasm resources. Against this backdrop, biological control, especially green control technologies based on beneficial rhizosphere microorganisms, has gradually become a research focus due to its environmental compatibility and sustainable disease control potential.

[0005] Against this backdrop, biological control, especially green control technologies based on beneficial rhizosphere microorganisms, has gradually become a research focus due to its environmental compatibility and sustainable disease control potential. Bacillus ( Bacillus spBacteria are widely distributed in soil and plant rhizosphere, exhibiting strong resistance, rapid reproduction, and a broad spectrum of inhibition. They can inhibit plant pathogens through various mechanisms, including competition, production of antimicrobial substances, and induction of systemic resistance. They also promote plant growth and improve fertilizer utilization efficiency, making them an important resource for the development of biocontrol agents. In recent years, they have shown good results in the control of soil-borne diseases in various crops. However, research on the systemic biological control of garlic root rot using Bacillus is still in its early stages. A systematic review and in-depth evaluation of their rhizosphere colonization capacity, antimicrobial mechanisms, agent construction, and field application potential are still lacking. Summary of the Invention

[0006] This invention aims to provide a Bacillus C1-74 strain and its application. This Bacillus C1-74 strain can significantly reduce the disease index of garlic root rot and effectively alleviate the harmful symptoms of root rot such as browning and rotting of garlic roots. At the same time, this strain has a significant growth-promoting effect on seedlings of vegetables such as garlic, scallions, cucumbers, or tomatoes.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A type of Bacillus ( Bacillus sp. Bacillus C1-74 was deposited on April 3, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, with accession number CCTCC M2025700.

[0008] Preferably, the ITS sequence of the Bacillus C1-74 is shown in SEQ ID NO:1.

[0009] The present invention also provides the application of the Bacillus C1-74 in the prevention and control of garlic root rot.

[0010] The present invention also provides the application of the Bacillus C1-74 in the preparation of biological agents for the prevention and control of garlic root rot.

[0011] The present invention also provides a biological agent for preventing and controlling garlic root rot, comprising the aforementioned Bacillus C1-74.

[0012] The present invention also provides the application of the Bacillus C1-74 in promoting plant growth.

[0013] Preferably, the plant includes one of garlic, scallions, cucumber, or tomato.

[0014] The present invention also provides the application of the Bacillus C1-74 in the preparation of biological agents that promote plant growth.

[0015] The present invention also provides a biological agent for promoting plant growth, comprising the Bacillus C1-74.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a Bacillus C1-74 strain and its application. This Bacillus C1-74 strain exhibits highly efficient antagonistic activity against the main pathogens causing garlic root rot, with an inhibition rate of over 70% against Fusarium oxysporum. Applying this strain after pathogen infection can reduce the garlic root rot disease index from 80.27% to 21.09%, effectively alleviating root rot symptoms such as browning, rotting, and yellowing of the above-ground parts of garlic, significantly reducing the damage of the disease to garlic growth, and enabling green control of garlic root rot using beneficial rhizosphere microorganisms.

[0017] As a microbial resource, the Bacillus C1-74 strain has good environmental compatibility. Its application will not disrupt the soil rhizosphere microecological balance or cause pathogens to develop drug resistance. Compared with chemical pesticides, it is more in line with the needs of green agricultural development and has a wide range of applications. It provides an efficient and sustainable technical solution for the green prevention and control of garlic root rot and for increasing the yield and quality of multiple crops, and has important production and application value.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Bacillus C1-74; Figure 2 It is a phylogenetic tree of Bacillus C1-74; Figure 3 This is the result of Bacillus C1-74 inhibiting the growth of garlic root rot pathogens; Figure 4 The results are from the detection of the growth-promoting properties of Bacillus C1-74; Figure 5 The results show that Bacillus C1-74 inoculation promotes the growth of garlic seedlings; Figure 6 This is the result of promoting the growth of scallion seedlings after inoculation with Bacillus C1-74; Figure 7 This is the result of promoting the growth of cucumber seedlings after inoculation with Bacillus C1-74; Figure 8 This is the result of promoting the growth of tomato seedlings after inoculation with Bacillus C1-74; Figure 9 The effect of Bacillus C1-74 inoculation on the prevention and control of root rot in garlic seedlings. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] In this embodiment, the tested vegetable species were improved garlic, scallion, cucumber inbred line Q16, and tomato variety "Provence".

[0023] The inbred lines for garlic and cucumber were provided by the Vegetable Quality Regulation and New Germplasm Innovation Team of the College of Horticulture, Northwest A&F University. The seeds for scallions and tomatoes were provided by Yangling Yufeng Seed Industry Co., Ltd., and were either core germplasm of the research group or local main varieties.

[0024] The culture medium used in the embodiments of this invention is as follows: PDA medium: Boil 200g of peeled potatoes for about 20 minutes until they change color. Filter the boiled solution through double gauze. Boil the filtrate, add 20g of agar, and after the agar melts, add 20g of glucose. Heat for a few more minutes until completely melted. While hot, bring the volume to 1L and dispense into Erlenmeyer flasks or test tubes. Sterilize at 121℃ for 21 minutes before use.

[0025] PDB liquid culture medium: Weigh 200g of peeled potatoes, boil for 20min, filter to remove solids, and collect the filtrate; then weigh 20g of sucrose, heat and dissolve it in the filtrate, and after the solution cools, make up to 1L with a graduated cylinder, autoclave at 121℃ for 20min, and then cool for later use.

[0026] LB solid medium: 5.0g yeast extract, 10g peptone, 10g sodium chloride, 18g agar, pH 7.0±0.1, 1L distilled water, autoclaved at 121℃ for 15min.

[0027] LB liquid medium: 5.0g yeast extract, 10g peptone, 10g sodium chloride, pH 7.0±0.1, 1L distilled water, autoclaved at 121℃ for 15min.

[0028] Assumption medium: 0.2g potassium dihydrogen phosphate, 10.0g mannitol, 0.2g sodium chloride, 0.1g calcium sulfate, 5.0g calcium carbonate, 15.0g agar, 0.2g magnesium sulfate, pH 7.0±0.1, 1L distilled water, autoclaved at 121℃ for 15min.

[0029] Protein detection culture medium: 10g skim milk powder, 20g agar, heated and dissolved in 1L distilled water, autoclaved at 105℃ for 20min to avoid the formation of flocculent protein.

[0030] Organophosphate bacteria culture medium: glucose 10.0g, lecithin 0.2g, ammonium sulfate 0.5g, yeast extract 0.5g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, calcium carbonate 1.0g, sodium chloride 0.3g, agar 15.0g, pH 7.0-7.5. Add 1L of distilled water and autoclave at 121℃ for 15min.

[0031] Inorganic phosphorus bacteria culture medium: glucose 10.0g, ammonium sulfate 0.5g, yeast extract 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, calcium phosphate 5.0g, agar 15.0g, pH 7.0-7.5, distilled water 1000mL, autoclave at 121℃ for 15min.

[0032] Cellulase detection medium: 10.0g peptone, 10.0g yeast extract, 10.0g sodium carboxycellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, 18g agar, 1L distilled water, autoclaved at 121℃ for 15min.

[0033] Silicate bacteria culture medium: sucrose 5.0g, magnesium sulfate 0.5g, calcium sulfate 0.1g, disodium hydrogen phosphate 2.0g, ferric chloride 0.005g, glass powder 1.0g, agar 15.0g, pH 7.0±0.2, distilled water 1000mL, autoclave at 121℃ for 15min.

[0034] CAS detection medium: Chromium azurite S (CAS) 0.06g, hexadecyltrimethylammonium bromide (HDTMA) 0.07g, ferric chloride hexahydrate 0.002g, sodium dihydrogen phosphate dihydrate 0.3g, disodium hydrogen phosphate dodecahydrate 0.3g, ammonium chloride 0.1g, potassium dihydrogen phosphate 0.04g, sodium chloride 0.06g, agar 9g, pH 6.8±0.1, distilled water 1000mL, autoclaved at 116℃ for 30min.

[0035] Chitinase detection culture medium: 1% colloidal chitin, 1g ammonium dihydrogen phosphate, 0.2g potassium chloride, 0.2g magnesium sulfate, 20g agar, and sterile water to a final volume of 1000mL, pH 7, autoclaved at 121℃ for 21min.

[0036] Preparation of colloidal chitin: Dissolve 20g of chitin in 350mL of concentrated hydrochloric acid, place at 4℃ for 24 h, filter with glass wool, add 2L of ice-cold anhydrous ethanol to the filtrate and incubate at -20℃ overnight, centrifuge at 10000r / min for 20min, wash the precipitate continuously with running tap water until the pH is neutral, and store in a sealed container at -20℃.

[0037] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0038] Example 1 Isolation and identification of strains Healthy garlic plants were collected on May 10, 2025, from the garlic experimental field at the Caoxinzhuang Experimental Demonstration Station of Northwest A&F University, at which time root rot was clearly observed in the field. Before sampling, a sterile stainless steel tray (30cm×40cm) was placed under a sterile stainless steel sieve with a 2mm aperture as a receiving container. Sterile plastic bags, self-sealing bags, scrapers, brushes, and gloves were also provided. Five to six healthy garlic plants were selected from the area surrounding the root rot site. Using a shovel, the roots were dug at a 45° angle downwards, 20cm from the base of the plant stem, ensuring that the roots within a 30cm depth were completely removed.

[0039] Rhizosphere soil was collected from garlic plants using the root-shaking method, with approximately 100g of rhizosphere soil collected from each plant. The collection method involved gently removing the dug-up plant along with its roots and soil. The garlic shoot was then gently lifted to 15cm above the ground, and the roots were lightly shaken or tapped to allow approximately 80% of the non-rhizosphere soil to fall to the ground naturally. The soil adhering to the root surface (1-5mm) was retained as the rhizosphere soil sample. Using a sterile brush or similar tool, this sample was carefully separated into a plastic bag, removing any remaining roots, stones, animal remains, or other impurities. The collected rhizosphere soil was then sieved through a 2mm sterile stainless steel sieve into a tray, mixed thoroughly, and placed into a resealable bag. The bag was labeled with the collection location name, sampling time, and other information. After collection, the sampling bags were placed in an icebox and transported back to the laboratory as soon as possible for storage at -80℃.

[0040] Isolation and preservation of rhizosphere bacteria Bacteria were isolated using the plate dilution method. First, 10g of each soil sample was weighed and added to a 250mL Erlenmeyer flask containing 90mL of sterile water. The flask was then placed on a shaker at 28℃ and 200rpm for 20min. After shaking, the flask was allowed to stand until soil particles precipitated. 1mL of the supernatant was then added to a test tube containing 9mL of sterile water for serial dilution, resulting in a 10-10 dilution. -1 ~10 -5 The diluted solution. Next, take the solution with a dilution of 10... -3 10 -4 and 10 -5200 μL of soil suspension was plated on LB solid medium, with each gradient repeated three times. After sealing with a sealing film, the plates were incubated at 28°C for 2 days. Once single colonies had grown, single colonies of different colors, morphological characteristics, and sizes were picked with a sterile inoculation loop and streaked for reculturing. The purified bacterial cultures were then stored at -20°C with 50% glycerol for later use.

[0041] Morphological and molecular identification Take the purified strain, streak it on LB solid medium, seal it with sealing film, and incubate it in a biochemical incubator at 28℃ for 2 days. Then take out the culture dish and observe the basic morphological characteristics such as colony morphology, size, color, texture viscosity, gloss, transparency, and edge smoothness. Take pictures and record them.

[0042] The colony morphology of strain C1-74 is as follows Figure 1 As shown. By Figure 1 It can be seen that the colonies of strain C1-74 on LB medium are round, milky white and transparent, with irregular and uneven edges, slightly moist and viscous cells, easy to pick up, and a clear depression can be seen in the center of the colony.

[0043] Single colonies of the purified strain were picked and dissolved in 10 μL of sterile water, and bacterial DNA was extracted using the boiling lysis method. Specifically, the lysis was performed at 100°C for 10 min to obtain bacterial DNA. PCR amplification of the selected strain was performed using primers 27F and 1492R. The nucleotide sequence of primer 27F is shown in SEQ ID NO:2, and the nucleotide sequence of primer 1492R is shown in SEQ ID NO:3.

[0044] SEQ ID NO: 2: 5'-AGAGTTTGATCCTGGCTCAG-3'.

[0045] SEQ ID NO: 3: 5'-GGTTACCTTGTTACGACTT-3'.

[0046] The PCR reaction system consisted of 50 μL of the following components: 4 μL of template (genomic DNA), 2 μL each of the front and rear primers (27F and 1492R), 17 μL of ddH2O, and 25 μL of MIX (2×Taq plus Master Mix).

[0047] PCR amplification conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min; 35 cycles, final extension at 72℃ for 10 min. PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The obtained bacterial sequences were compared using BLAST in the NCBI database, and a phylogenetic tree was constructed using MAGA-X software.

[0048] The ITS sequence of strain C1-74 is shown in SEQ ID NO:1.

[0049] SEQ ID NO:1:

[0050] Sequencing results were used to locate sequences from different genera within the same species in the NCBI database for multiple alignment. A phylogenetic tree was then constructed using MEGA-X software for comparison. The results are as follows: Figure 2 As shown.

[0051] Based on homology comparison analysis, the gene sequence of strain C1-74 is within the same clade as OP364585.1:9-1451Bacillus samyloliquefaciens, OQ423166.1:13-1451 Bacillus subtilis, and KM659226.1:9-1450Bacillus. Therefore, C1-74 may be a Bacillus subtilis strain. Bacillus subtilis ) or Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Based solely on 16S rDNA molecular identification assays, C1-74 can be identified as belonging to the genus *Bacillus*. It was named... Bacillus sp. C1-74, hereinafter referred to as strain C1-74.

[0052] Bacillus C1-74 was deposited on April 3, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC M 2025700.

[0053] The efficacy of the above-mentioned Bacillus C1-74 was verified.

[0054] 1. Antibacterial activity test Fermentation broth preparation: Use a sterile inoculation loop to pick a single colony of the strain and inoculate it into LB liquid medium. Incubate on a shaker at 28℃ and 200r / min for 16h. The resulting culture medium is the fermentation broth, which should be stored for later use.

[0055] Initial screening stage: *Garlic root rot pathogen* was selected as the target bacterium for antagonistic bacteria screening. Using a 5mm diameter punch, holes were made along the edge of the pathogen colony to obtain 5mm diameter mycelial discs, which were then placed in the center of PDA medium. Colonies were then inoculated from purified C1-74 single colonies using an inoculation loop at approximately 2.5cm from the top, bottom, left, and right of the mycelial disc, with four inoculations per PDA medium and three replicates. A blank control (CK) was used, inoculated only with the target bacterium. After sealing, the culture was incubated at 28℃ for 7 days, and the inhibition rate was measured using the cross-hatching method.

[0056] Secondary screening stage: The following method was used to further verify the antagonistic effect of the initially screened bacterial strains (preliminary procedures were the same as the initial screening): Four holes were punched approximately 2.5 cm above, below, to the left, and to the right of the bacterial cake using a 5 mm diameter punch. 30 μL of C1-74 fermentation broth was inoculated into each hole. A blank control (CK) was used, inoculated only with the target bacteria. The cultures were incubated at 28℃ for 7 days. Each treatment was repeated three times. The inhibition radius was recorded, and the inhibition rate was calculated. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Coronavirus colony diameter of control pathogen - Coronavirus colony diameter of treatment pathogen) / (Coronavirus colony diameter of control pathogen - Initial diameter of pathogen mycelial cake) × 100%.

[0057] The results are as follows Figure 3 As shown in Table 1.

[0058] Table 1. Inhibition diameter and inhibition rate of Bacillus C1-74 against garlic root rot pathogen.

[0059] As shown in Table 1, strain C1-74 exhibited a 76.5% inhibition rate on mycelial growth of Fusarium oxysporum in the initial screening and a 77.7% inhibition rate in the rescreening. Both the initial and rescreening inhibition rates were above 70%, demonstrating strong antibacterial ability.

[0060] Depend on Figure 3 It can be seen that Bacillus C2-35 can significantly inhibit the mycelial growth of Fusarium oxysporum, and the inhibitory effect remains stable in both the primary and secondary screening stages, and is significantly higher than that of the control group (CK), indicating that this strain has strong potential and consistency in inhibiting Fusarium oxysporum.

[0061] 2. Assay of extracellular enzyme inhibition activity and growth-promoting function of Bacillus C1-74 The specific experimental protocol for determining the protease, chitinase, and cellulase activities of Bacillus C1-74 is as follows: Fermentation broth preparation: Use a sterile inoculation loop to pick a single colony of the strain and inoculate it into LB liquid medium. Incubate on a shaker at 28℃ and 200r / min for 16h. The resulting culture medium is the fermentation broth, which should be stored for later use.

[0062] Assay for protease and chitinase activities: The strain was streaked on LB solid medium for 24 hours, and then single colonies were picked up with a sterile inoculation needle and inoculated onto protease and chitinase detection medium plates, respectively. The plates were incubated at 28°C for 3–7 days, and the presence or absence of a clear zone was observed. The diameter of the colony (d) and the diameter of the clear zone (D) were measured. The presence or absence of enzyme activity was determined by the presence or absence of a clear zone on the medium, and the enzyme activity was positively correlated with the HC value (D / d).

[0063] Cellulase activity assay: The strain was streaked in LB solid medium for 24 h, and then colonies were picked up using a live inoculation needle and inoculated onto cellulase activity assay plates. The plates were incubated at 28°C for 3 days. The entire culture dish was then completely immersed in 1 mg / mL Congo red solution for staining. The staining solution was left for 30 min, then discarded, and the plates were washed with 1 mol / L NaCl solution. The observation and measurement methods were the same as above.

[0064] Protease production capacity: Bacillus C1-74 was inoculated onto a protein detection medium plate, and the presence or absence of a clear zone was observed after 3 days.

[0065] Cellulase production capacity: Bacillus C1-74 was inoculated onto cellulase detection medium plates. After 2-3 days, 1 mg / mL Congo red solution was added for staining for 30 min. The staining solution was discarded, and the plates were washed with 1 mol / L sodium chloride for 30 min. The presence or absence of a clear zone was observed.

[0066] Chitinase production capacity: Bacillus C1-74 was inoculated onto chitinase detection medium plates and cultured for 7 days. Observe whether a clear zone appears.

[0067] The growth-promoting characteristics of Bacillus C1-74 were determined, including its nitrogen fixation capacity, ability to dissolve inorganic and organic phosphorus, potassium solubilization, siderophore production, and IAA production capacity. The specific experimental protocol is as follows: Nitrogen fixation capacity: Bacillus C1-74 was inoculated into Assumption medium and cultured for 7 days to observe whether bacterial cells grew.

[0068] Phosphate-solubilizing ability: Bacillus C1-74 was inoculated onto solid organic and inorganic phosphate-solubilizing medium plates and cultured for 7 days. The presence or absence of transparent phosphate-solubilizing zones was then observed.

[0069] Siderogenic carrier: Bacillus C1-74 was inoculated onto chroma blue CAS plates, and the presence of a yellow-green halo was observed after 7 days.

[0070] IAA detection: Take 1 mL of the cultured C1-74 bacterial suspension and inoculate it into a liquid medium containing L-tryptophan. Incubate at 28℃ and 200 r / min for 24 h. Take 100 μL of the bacterial suspension and drop it onto a white ceramic plate. Add the same volume of Salkowski colorimetric reagent and mix well. React in the dark at room temperature for 30 min. If the color turns red, it indicates that the strain has the ability to produce IAA; otherwise, it does not.

[0071] Potassium solubilization capacity: Bacillus C1-74 was inoculated into silicate medium and cultured for 7 days to observe whether it produced an oily liquid.

[0072] The test results are as follows Figure 4 And Table 2.

[0073] Table 2. Results of detection of extracellular enzyme inhibition activity and growth-promoting properties of Bacillus C1-74

[0074] Note: "+" sign indicates that the ability exists, and "-" sign indicates that the ability does not exist.

[0075] Depend on Figure 4 As shown in Table 2, the extracellular enzyme inhibition activity of strain C1-74 is mainly manifested in the production of protease and cellulose, but not in the production of chitinase, with a protease activity value of 3.56 and a cellulase activity value of 2.64; the growth-promoting characteristics are manifested in the functions of nitrogen fixation and potassium solubilization, but not in the functions of dissolving inorganic phosphorus, dissolving organic phosphorus, producing IAA and producing siderophores.

[0076] 3. Verify the growth-promoting effect on garlic, scallion, cucumber, and tomato seedlings. The tested varieties were "Improved Garlic" garlic, scallions, "Q16" inbred cucumber, and "Provence" tomato.

[0077] Fermentation broth preparation: A single colony of the strain was picked using a sterile inoculation loop and inoculated into LB liquid medium. The medium was then incubated on a shaker at 28°C and 200 rpm for 16 h. The resulting culture broth was the fermentation broth. After centrifugation at 10,000 rpm for 5 min, the supernatant was collected and diluted with ddH2O to adjust the OD600 value to 0.8 (10⁻⁶). 8 (cfu / mL) and set aside for later use.

[0078] The pot experiment will be conducted in April 2025 in the artificial climate chamber of Northwest A&F University.

[0079] The cultivation conditions for cucumber and tomato seedlings were as follows: light intensity of 360 µmol·photons·m -2 ·s -1 The temperature is 25±1℃ / 18±1℃ (day / night), the photoperiod is 16h / 8h (day / night), and the relative humidity is 60-80%.

[0080] The cultivation conditions for garlic and scallion seedlings were: light intensity of 200 µmol·photons·m -2 ·s -1 The temperature is 22±1℃ / 17±1℃ (day / night), the photoperiod is 16h / 8h (day / night), and the relative humidity is 50-70%.

[0081] Experiment on promoting the growth of garlic seedlings: Garlic cloves were soaked and germinated until they rooted and sprouted. When the sprouts reached 2cm in length, they were sown in flowerpots containing high-pressure heat-sterilized substrate. The flowerpot diameter × bottom diameter × height was 19.5cm × 15.5cm × 14cm. Four garlic seedlings of uniform growth were planted in each pot, and the top of the garlic cloves was covered with soil to a thickness of 3-5cm. When the garlic seedlings reached 10cm in height, four holes about 2cm deep were made at the base of the seedling stem. The roots were then drenched with the prepared Bacillus C1-74 fermentation broth, 10mL per seedling. The control (CK) was inoculated with an equal amount of water. Each treatment was inoculated with 50 seedlings and repeated 3 times. Seven days later, the bacterial solution was applied again. Seven days after the second treatment, 30 uniformly growing plants were randomly selected from each treatment, with 3 plants constituting one replicate. Seedling height, leaf length, leaf width, stem diameter, number of leaves, dry weight, and fresh weight were measured. The measurement methods were based on Li Xixiang's "Specifications for the Description of Garlic Germplasm Resources," and the robust seedling index was calculated using Li Hesheng's method, as shown in the following formula: Strong seedling index = (stem diameter / plant height + root dry weight / aboveground dry weight) × total plant dry weight.

[0082] The test results are as follows Figure 5 As shown in Table 3.

[0083] Table 3. Growth-promoting effect of Bacillus C1-74 on garlic seedlings

[0084] Note: Different lowercase letters in the same column indicate significant differences. P <0.05) From Table 3 and Figure 5 It can be seen that inoculation with strain C1-14 significantly promoted the growth of garlic seedlings in multiple indicators. Compared with the control group (CK), the plant height and leaf length of the C1-74 treatment group increased slightly, but the difference was not significant. P >0.05); while leaf width, stem diameter, number of leaves, fresh weight of plants, dry weight of plants, and seedling vigor index all showed significant improvements ( P <0.05). Among these changes, leaf width increased from 6.28 mm to 8.61 mm, stem diameter increased from 3.52 mm to 4.70 mm, and plant fresh weight and dry weight increased significantly by 61.60% and 63.29%, respectively, indicating that the C1-74 treatment significantly promoted garlic biomass accumulation. Furthermore, the seedling vigor index also increased from 0.28 to 0.40, indicating that the seedlings were generally more robust.

[0085] In summary, strain C1-74 can effectively promote the morphogenesis and material accumulation of garlic seedlings, and has significant potential for rhizosphere growth promotion. It can be applied as a potential plant rhizosphere growth promoter (PGPR) in garlic cultivation.

[0086] Growth promotion experiment of scallion seedlings: Scallion seeds were sown in 72-cell trays. When the seedlings reached 10cm in height, uniform scallion seedlings were selected and transplanted into flowerpots containing autoclaved and heat-sterilized substrate. The tillering nodes (root-stem junction) should be kept 0.5-1cm below the soil surface to avoid burying the heart leaves. Four uniformly grown scallion seedlings were planted in each pot. The pot diameter × bottom diameter × height was 19.5cm × 15.5cm × 14cm. After 2 days of acclimatization, four holes approximately 2cm deep were made at the base of the seedling stem. The roots were drenched with the prepared Bacillus C1-74 fermentation broth. The control (CK) was inoculated with only an equal amount of water. Each treatment was inoculated with 50 seedlings and repeated three times. Seven days later, the bacterial culture was inoculated again. Seven days after the second inoculation, 30 uniformly growing plants were randomly selected from each treatment, with 3 plants constituting one replicate. Seedling height, leaf length, stem diameter, plant dry weight, and plant fresh weight were measured according to Li Xixiang's "Specifications for the Description of Garlic Germplasm Resources," and the seedling vigor index was calculated using the same formula. Results are as follows: Figure 6 As shown in Table 4.

[0087] Table 4. Growth-promoting effect of Bacillus C1-74 on scallion seedlings

[0088] Note: Different lowercase letters in the same column indicate significant differences. P <0.05).

[0089] From Table 4 and Figure 6 It can be seen that, compared with the control group (CK), the scallion seedlings treated with strain C1-74 showed significant improvements in multiple growth indicators. Although plant height and leaf length both increased, the differences did not reach a statistically significant level. P >0.05). However, in terms of stem diameter, fresh plant weight, dry plant weight, and seedling vigor index, the C1-74 treatment group was significantly higher than the control group ( P <0.05). Among them, the fresh weight of the plant increased significantly from 4.31g to 6.43g, an increase of 49.19%; the dry weight of the plant increased from 0.41g to 0.77g, an increase of 87.80%. The seedling vigor index also increased from 0.06 to 0.16, more than doubling, indicating that the C1-74 treatment significantly enhanced the vigor of scallion seedlings.

[0090] The results above indicate that strain C1-74 has a significant promoting effect on the growth of scallion seedlings, especially in increasing biomass accumulation, thickening stems, and improving the seedling vigor index. This further proves that the strain has the potential to be used as a plant rhizosphere growth promoter (PGPR) and can be used for the green cultivation and production of scallions.

[0091] Experiments on promoting the growth of cucumber and tomato seedlings: After disinfection, soaking, and germination, cucumber and tomato seeds were sown in flowerpots containing autoclaved substrate. Cucumber seeds were sown at a depth of 1.5-2 cm, with the radicle facing down to avoid emergence with the seed coat still attached. Tomato seeds were sown at a depth of 0.5-1 cm. The flowerpot diameter × bottom diameter × height was 19.5 cm × 15.5 cm × 14 cm. When cucumber seedlings had two leaves and one bud, and tomato seedlings had three leaves and one bud, uniformly growing seedlings were selected. Four holes, approximately 2 cm deep, were made about 2 cm from the base of the seedling stem. The roots were then drenched with the prepared Bacillus C1-74 fermentation broth. Each treatment was inoculated with 50 seedlings and repeated three times. CK was inoculated with an equal volume of water, followed by a second inoculation with bacterial solution 7 days later. Seven days after the second inoculation, 30 uniformly growing plants were randomly selected from each treatment. Plant height, stem diameter, leaf area, number of leaves, dry weight, and fresh weight of cucumber and tomato seedlings were measured. Measurement methods followed the guidelines in Li Xixiang et al.'s "Description Specifications and Data Standards for Cucumber Germplasm Resources" and Wang Xiaojing et al.'s "Study on Genetic Diversity of Quality Traits in Tomato Germplasm Resources." The seedling vigor index was also calculated using the same formula. The growth-promoting effect on cucumber seedlings is shown in Table 5. Figure 7 As shown in Table 6; the growth-promoting effect on tomato seedlings is shown in Table 6 and... Figure 8 As shown.

[0092] Table 5. Growth-promoting effect of Bacillus C1-74 on cucumber seedlings.

[0093] Note: Different lowercase letters in the same column indicate significant differences. P <0.05).

[0094] From Table 5 and Figure 7 As shown, strain C1-74 exhibited a comprehensive growth-promoting effect on cucumber seedlings. Compared with the control group (CK), seedlings inoculated with C1-74 showed significant differences in all measured indicators. P <0.05). Plant height increased significantly by 43.5%, leaf area and stem diameter increased by 25.8% and 27.0% respectively, and the number of leaves also increased significantly. More notably, the C1-74 treatment promoted a significant accumulation of plant biomass, with fresh weight and dry weight increasing by 53.9% and 56.5% respectively, indicating that this strain not only promotes vegetative growth but also significantly enhances dry matter synthesis capacity. At the same time, the seedling vigor index increased by 33.3% compared with the control, showing that the overall vitality and robustness of seedlings were effectively improved.

[0095] The results indicate that strain C1-74 can effectively promote cucumber seedling growth through multiple pathways, significantly improve agronomic traits and biomass accumulation, and has the potential to be developed as a highly efficient microbial inoculant in cucumber cultivation.

[0096] Table 6. Growth-promoting effect of Bacillus C1-74 on tomato seedlings

[0097] Note: Different lowercase letters in the same column indicate significant differences. P <0.05).

[0098] From Table 6 and Figure 8 It can be seen that, compared with the control group (CK), the vaccination treatment showed significant advantages in all measured indicators. P <0.05). Plant height and leaf length increased significantly by 50.1% and 48.0%, respectively, while stem diameter increased significantly by 18.3%. Regarding biomass accumulation, the C1-74 treatment significantly increased both fresh and dry weight, particularly dry weight, by 136.5%, indicating that this strain significantly promoted the accumulation of photosynthetic products and material transformation. Simultaneously, the seedling vigor index increased by 122.7%, reflecting a significant enhancement in the overall seedling vitality and stress resistance.

[0099] The results showed that strain C1-74 could effectively improve the agronomic traits and physiological state of tomato seedlings through multiple pathways, such as promoting plant height growth, increasing leaf area, thickening stems, and increasing dry matter ratio, and has good potential to be developed into a tomato-specific microbial inoculant.

[0100] 4. The effect of inoculation with Bacillus C1-74 on the control of garlic root rot Preparation of root rot pathogen spore suspension: Fusarium oxysporum (…) preserved in the laboratory… Fusarium oxysporum Using a punch, take a 5mm diameter mycelial cake and inoculate it onto PDB liquid medium. Incubate on a shaker at 28℃ and 200 rpm for 3 days. Filter the mycelium through three layers of gauze to obtain the supernatant, i.e., the sporangium suspension. Take 10μL of the filtered sporangium suspension and drop it onto a hemocytometer. Count the spore concentration under a microscope and dilute it to 1×10⁻⁶ with sterile water. 7 CFU / mL is prepared for later use.

[0101] Fermentation broth preparation: A single colony of the strain was picked using a sterile inoculation loop and inoculated into LB liquid medium. The medium was then incubated on a shaker at 28°C and 200 rpm for 16 hours. The resulting culture broth was the fermentation broth. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was collected and diluted with ddH2O to adjust the OD600 value to 0.8 (1×10⁻⁶). 8 (cfu / mL) and set aside for later use.

[0102] The cultivation and management methods for garlic seedlings are the same as those for the above-mentioned garlic seedling growth promotion experiment.

[0103] Two inoculation methods were set up for the experiment. Treatment T1: Inoculated with 5 mL of pathogen sporangium suspension; Treatment T2: Inoculated with 5 mL of pathogen sporangium suspension 1 day prior to inoculation with 5 mL of C1-74 fermentation broth.

[0104] The pathogen was inoculated using the root-injury method. Before inoculation, the substrate was kept moist by watering appropriately. A hole was made about 2 cm away from the seedling rootstock using a glass rod, carefully exposing part of the seedling rootstock. A small wound was made at the root hairs with a sterile blade, and 5 mL of sporangium suspension was poured into the hole. For strain C1-74 fermentation broth, four holes about 2 cm deep were made about 2 cm from the base of the seedling stem, and the bacterial solution was poured evenly into the holes. The control (CK) was inoculated with an equal amount of water. Each treatment was inoculated with 21 seedlings, and the inoculation was repeated 3 times. After 30 days, the seedling height, leaf length, leaf width, stem diameter, number of leaves, dry weight, and fresh weight were measured. The garlic plants were also observed for yellowing and wilting of leaves and rotting of the stem base. Intact plants were dug up to check whether the roots were brown and soft rotten, and the disease status was recorded. The disease grading standards are shown in Table 7 below (Zhang Bo, 2008).

[0105] Table 7 Disease Grading Standards

[0106] Calculate the disease index and incidence rate using the following formula: Disease index = ∑ (number of diseased plants × number of representative levels) / (total number of plants × highest level) × 100; Incidence rate (%) = Number of infected plants / Total number of plants × 100; Prevention efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100.

[0107] The control efficacy of Bacillus C1-74 against garlic root rot is shown in Table 8. The growth and development of garlic seedlings under different treatments are shown in Tables 9 and 9. Figure 9 As shown.

[0108] Table 8. Control efficacy of Bacillus C1-74 against garlic root rot

[0109] Table 8 shows that there were significant differences in disease incidence among garlic seedlings under different treatments. Compared with the treatment of Fusarium oxysporum inoculation alone (T2), the treatment group treated with C1-74 (T3) significantly reduced the incidence of garlic root rot. The disease index of the T2 treatment was as high as 80.27%, while the disease index of the T3 treatment dropped to 21.09%, a reduction of 73.73%. Meanwhile, the relative control efficacy of the C1-74 treatment group (T3) was 73.73%, indicating that this strain has a significant therapeutic effect on garlic root rot. These results demonstrate that strain C1-74 can not only effectively inhibit pathogen infection but also significantly alleviate disease development, showing good biocontrol potential and providing a basis for the application of this strain in the green control of garlic.

[0110] Table 9. Growth and development of garlic seedlings under different treatments

[0111] Depend on Figure 9 As shown in Table 9, different treatments had a significant impact on the growth and development of garlic seedlings. Compared with the water control group (CK), the treatment with pathogen inoculation alone (T1) significantly inhibited all growth indicators, with plant height, fresh weight, and dry weight decreasing by 49.4%, 83.8%, and 87.5%, respectively, indicating that Fusarium oxysporum infection severely hindered the normal growth of garlic seedlings. However, after treatment with C1-74 bacterial suspension (T2), all growth indicators of garlic seedlings recovered significantly compared to the T1 treatment. Plant height, leaf width, stem diameter, and dry weight increased by 72.0%, 31.9%, 64.9%, and 589.5%, respectively, and most indicators showed no significant difference from the control group. The results indicate that strain C1-74 can not only effectively alleviate the inhibitory effect of pathogen on the growth of garlic seedlings but also promote plant morphogenesis and nutrient accumulation, demonstrating good potential for biocontrol applications.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A Bacillus C1-74, characterized in that, The Bacillus C1-74 was deposited on April 3, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, with accession number CCTCC M2025700.

2. The Bacillus C1-74 according to claim 1, characterized in that, The ITS sequence of the Bacillus C1-74 is shown in SEQ ID NO:

1.

3. The application of Bacillus C1-74 as described in claim 1 in the prevention and control of garlic root rot.

4. The application of Bacillus C1-74 as described in claim 1 in the preparation of biological agents for the prevention and control of garlic root rot.

5. A biological agent for controlling garlic root rot, characterized in that, Includes the Bacillus C1-74 as described in claim 1.

6. The application of Bacillus C1-74 as described in claim 1 in promoting plant growth.

7. The application according to claim 6, characterized in that, The plant includes one of garlic, scallions, cucumber, or tomato.

8. The use of Bacillus C1-74 as described in claim 1 in the preparation of biological agents that promote plant growth.

9. A biological agent for promoting plant growth, characterized in that, Includes the Bacillus C1-74 as described in claim 1.

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