Bacillus velezensis with growth promoting effect and application thereof

Abstract

The invention relates to the technical field of application of microorganisms, and particularly discloses bacillus velezensis with bacteriostatic and growth-promoting effects and application thereof. The preservation number of the bacillus velezensis BPC37 disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.34544, and the bacillus velezensis BPC37 has the functions of dissolving phosphorus and potassium, and has the capability of inhibiting a bacterial disease, namely Xanthomonas campestris (Xcc). Tests prove that the strain has a growth promoting effect on lettuce, peach seedlings and sweet potatoes, is green and safe, and has a relatively good development prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial application, and particularly relates to a bacillus velezensis with bacteriostatic and growth-promoting effects and application thereof. BACKGROUND

[0002] The rhizosphere is a highly competitive environment, where microorganisms constantly compete for resources to survive (Sasse, J.; Martinoia, E.; Northen, T. Feed your friends: Do plant exudates shape the root microbiome? Trends Plant Sci. 2018, 23, 25-41.). Some bacteria associated with the rhizosphere, i.e. plant growth-promoting rhizobacteria (PGPR, recognized for their ability to promote plant weight gain and crop yield increase (Beneduzi, A.; Ambrosini, A.; Passaglia, L.M.P. Plant growth-promoting rhizobacteria (PGPR): Their potential as antagonists and biocontrol agents. Genet. Mol. Biol. 2012, 35, 1044-1051.). Bacillus spp. are considered important plant growth-promoting bacteria (PGPR), which directly modulate plant physiology by mimicking the synthesis of plant hormones or producing volatile substances; or by increasing the availability of minerals and nitrogen in the soil to promote plant growth.

[0003] Bacillus spp. are more favored in agricultural systems because they are able to form endospores, which can survive in hot and dry environments and can be made into stable dry powders with a long shelf life (Chowdhury, S.P.; Dietel, K.; Rändler, M.; Schmid, M.; Junge, H.; Borriss, R.; Hartmann, A.; Grosch, R. Effects of Bacillus amyloliquefaciens FZB42 on Lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community. PLoS ONE 2013, 8, 1-10.). The most widely used in agriculture is Bacillus subtilis (B. subtilis ), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B. amyloliquefaciens ), Bacillus licheniformis (Bacillus licheniformis) B. licheniformis ), Bacillus pumilus (Bacillus pumilus) B. pumilus ) and Bacillus velezensis (Bacillus velezensis) B. velezensis In recent years, many B. velezensis Secondary metabolites produced are considered to be able to inhibit pathogenic fungi and bacteria and promote the growth of plants (Rabbee M F, Ali M S, Choi J, et al. 2019. Bacillus velezensis: A Valuable Member of Bioactive Molecules within Plant Microbiomes. Molecules, 24.). The present patent obtains a strain of Bacillus velezensis with disease resistance and growth promotion from the rhizosphere soil of well-grown lettuce. SUMMARY

[0004] The present application obtains a growth-promoting strain from the rhizosphere soil sample of lettuce, and determines that it is Bacillus velezensis (Bacillus velezensis) through strain identification and effect test, which has the functions of phosphorus and potassium solubilization and bacterial inhibition, and thus the present application is proposed. Bacillus velezensis

[0005] The present application provides a Bacillus velezensis (Bacillus velezensis) Bacillus velezensis , which has a preservation number of CGMCC No.34544, and is preserved in the China General Microbiological Culture Collection Center on May 14, 2025, and is classified and named as Bacillus velezensis (Bacillus velezensis) Bacillus velezensis .

[0006] The present application further provides the application of the Bacillus velezensis in preventing and treating bacterial diseases.

[0007] Specifically, the bacteria are Xanthomonas.

[0008] The present application also provides a bactericide prepared from the Bacillus velezensis, which contains active bacteria of the Bacillus velezensis.

[0009] The present application also provides the application of the Bacillus velezensis in promoting plant growth.

[0010] Specifically, the plant is lettuce, peach seedlings or sweet potatoes.

[0011] The present application also provides a growth-promoting bacterium agent prepared from the Bacillus velezensis, which contains active bacteria of the Bacillus velezensis.

[0012] ​​​​​​​​In the specific embodiment, it is in the form of a bacterial solution, preferably, it is 1x10 7 -1x10 9 cfu / mL bacterial suspension.

[0013] The application also provides a method for growing plants, which uses the growth-promoting bacterial agent to irrigate the planted plants to promote the growth of the plants.

[0014] In particular, the plants are lettuce, peach seedlings or sweet potatoes.

[0015] The Bacillus velezensis BPC37 of the application has the functions of phosphorus and potassium release and the ability to inhibit bacterial diseases caused by Xanthomonas campestris pathovar campestris (Xcc). The strain has been proved to have a growth-promoting effect on lettuce, peach seedlings and sweet potatoes, is green and safe, and has a good development prospect. Xanthomonas campestris campestiss BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The flat plate confrontation experiment is used to screen strains with strong antibacterial function.

[0017] Figure 2 The growth-promoting effect of each strain is shown.

[0018] Figure 3 The BPC37 significantly promotes the growth of lettuce.

[0019] Figure 4 The morphological characteristics of the strain BPC37 are shown. A is the colony morphology, B is the gram staining, and C is the spore shape.

[0020] Figure 5 The phosphorus release (A) and potassium release (B) functions of the strain BPC37 are shown.

[0021] Figure 6 The growth characteristics of the strain BPC37 under different concentrations of NaCl (A) and pH (B) conditions are shown.

[0022] Figure 7 The growth-promoting effect of BPC37 on peach seedlings is shown.

[0023] Figure 8 The growth-promoting effect of BPC37 on sweet potatoes is shown. A shows the shapes of the above-ground stems and tubers of sweet potatoes treated in different ways, B shows the total fresh weight results of sweet potatoes, C shows the fresh weight results of sweet potatoes (tubers), and D shows the number of sweet potatoes.

[0024] Biological material preservation information:

[0025] ​Bacillus velezensis BPC37 was preserved in China General Microbiological Culture Collection Center (CGMCC, Beijing, China) on May 14, 2025, with the preservation number of CGMCC No.34544, and the classification name of Bacillus velezensis Bacillus velezensis . DETAILED DESCRIPTION

[0026] The present application will be further described in detail below in combination with the accompanying drawings and specific embodiments.

[0027] Example 1, isolation and screening of growth-promoting strains

[0028] 1. Soil sample and test strains

[0029] The lettuce rhizosphere soil sample was collected from a vegetable planting base in Maizhuang Village, Xingshou Town, Changping District, Beijing. The test pathogenic bacteria was Xanthomonas campestris pv. campestris (Xcc), which was preserved in the Institute of Biotechnology, Beijing Academy of Agriculture and Forestry Sciences. Xanthomonas campestris campestiss

[0030] 2. Isolation and screening of growth-promoting strains

[0031] The soil sample was serially diluted and plated on LB solid medium to obtain single bacterial colonies. Each single colony was transferred to 10 mL of LB culture medium and incubated at 180 r / min and 28°C for 12 h to obtain a bacterial suspension. 100 μL of Xcc bacterial suspension (OD600=0.2) was spread on LB medium, and four 7-mm-diameter holes were punched symmetrically at a distance of 2 cm from the center of the plate. 100 μL of the test bacterial solution was added, and LB culture medium was used as a control. The plates were incubated at 28°C for 3 days, and the inhibition effect was observed to screen strains with strong inhibition effect.

[0032] Growth-promoting test was performed on lettuce seedlings with consistent growth (3-4 leaves). The treatment group: the above strains were adjusted to a bacterial suspension of 2×10 8 cfu / mL after propagation culture and irrigated the lettuce; the negative control: sterile water irrigation, treated every 10 days, and normal watering at other times. Each group had 12 lettuce plants, and the test was repeated three times. After 30 days, the fresh weight and dry weight of the lettuce were measured, and the differences between the treatment group and the control group were compared to screen strains with significant growth-promoting effect. Through the growth-promoting test, one strain with significant growth-promoting effect on lettuce was obtained, named BPC37.

[0033] Example 2, morphological and molecular biological identification of strain BPC37

[0034] ​The strain BPC37 was inoculated on the surface of LB solid medium and cultured at 28°C for 24h. The colony size, elevation, shape, transparency, etc. were observed, and Gram staining and spore staining were performed (Dong Xiuzhu, Cai Miaoying. Common Bacteria System Identification Manual [M]. Beijing: Science Press, 2001, 186-188). The bacterial genome extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing) was used to extract the genomic DNA of strain BPC37. The 16S rDNA sequence was amplified using universal bacterial primers and homologous comparison was performed with known 16S rDNA sequences in GenBank. The average insert fragment length of 500bp library was constructed using the rapid DNA library kit (Illumina platform), and double-end 150bp (PE150) sequencing was performed on the Illumina HiSeq sequencing platform with a sequencing depth of about 100x. After removing the reads with adapters, low quality and repeats in the sequencing data, SPAdes V3.11.0 was used for assembly. The MUMmer software was used for sequence alignment to obtain the average nucleotide identity (ANI) value of the whole genome of strain BPC37 and related strains in the NCBI library.

[0035] Example Three, Phosphorus and Potassium Solubilizing Function of Strain BPC37

[0036] BPC37 was inoculated in LB medium and cultured at 28°C, 180rpm for 12h. The inoculation needle was used to dip the bacterial solution and streak on the identification medium plate. The inorganic phosphorus bacterial culture medium plate (Kuleibo) was used for phosphorus solubilizing function identification, and the potassium solubilizing bacterial solid culture medium plate (Kuleibo, MM5081) was used for potassium solubilizing function identification.

[0037] Example Four, Salt Tolerance Analysis of Strain BPC37

[0038] The PBC37 bacterial suspension cultured to the logarithmic phase was inoculated in LB medium with different initial pH (4, 5, 6, 7, 8, 9, 10) and different NaCl concentrations (0, 1%, 3%, 5%, 7%, 10%, 15%) at a 1% inoculation ratio. The un-inoculated LB medium was used as a control group, and three replicates were set for each experiment. The OD600 was measured every 1h to explore the acid and alkali tolerance of the target strain. The OD600 value of the strain was measured every 12h to explore the salt tolerance of the target strain.

[0039] Example Five, Promoting Growth of Peach Seedlings by Strain BPC37

[0040] Promoting growth test was performed on peach tree seedlings with consistent growth. The treatment group: BPC37 was adjusted to 2x10 8The peach seedlings were irrigated with the bacterial suspension of 2 x 108cfu / mL, and the negative control was irrigated with sterile water. The treatment was performed every 10 days, and the rest of the time was normal watering. Each group of treatment was 12 peach seedlings, and repeated 3 times. After 30 days, the fresh weight and dry weight of the peach seedlings were weighed, and the number and length of lateral roots were counted.

[0041] Example 6, Promoting effect of strain BPC37 on sweet potato

[0042] The growth-promoting test was performed on the uniform sweet potato seedlings. The treatment group: BPC37 was adjusted to 2 x 108cfu / mL after expansion culture, and the negative control was irrigated with sterile water. The treatment was performed every 10 days, and the rest of the time was normal watering. Each group of treatment was 12 sweet potato seedlings, and repeated 3 times. After 60 days, the fresh weight and dry weight of the sweet potato seedlings were weighed. 8 The peach seedlings were irrigated with the bacterial suspension of 2 x 108cfu / mL, and the negative control was irrigated with sterile water. The treatment was performed every 10 days, and the rest of the time was normal watering. Each group of treatment was 12 peach seedlings, and repeated 3 times. After 30 days, the fresh weight and dry weight of the peach seedlings were weighed, and the number and length of lateral roots were counted.

[0043] Experimental research results

[0044] 1. BPC37 has the effects of inhibiting bacteria and promoting growth

[0045] Since Bacillus velezensis has the effects of disease resistance and growth promotion, it was used to screen strains with disease resistance function using pathogenic bacteria Xcc of Xanthomonas campestris pathovar. A total of 5 strains with strong inhibition effect were screened (BPC37, BPC38, BPC39, BPC40, and BPC41), which had similar inhibition effect compared with BPC6 (CGMCC No. 17805) which has been applied for patent. Figure 1

[0046] The 5 strains screened were subjected to lettuce growth-promoting test, and the fresh and dry weight of lettuce was measured after one month. A strain BPC37 was screened which significantly promoted the growth of lettuce. The fresh weight of lettuce in the treatment group of BPC37 increased by 66.28% compared with the control group (CK, LB treatment), and the dry weight increased by 50%, indicating that BPC37 significantly promoted the growth of lettuce (P < 0.05). Figure 2 Figure 3 Compared with BPC6, the fresh weight of lettuce in the treatment group of BPC37 increased by 20%, and the dry weight increased by 10%. Figure 3 The other 4 strains had no significant difference compared with the control (P > 0.05). Figure 3 A strain of Bacillus cereus DW019 was reported to have the effects of disease prevention and growth promotion in the literature (Liu Hongyu. Study on the disease prevention and growth promotion function of a strain of Bacillus cereus DW019, Jiangxi University of Technology. Master's thesis). The growth-promoting effect of a strain of Bacillus cereus DW019 on lettuce seedlings was reported to be 10 8 ​​Treatment with CFU / mL of bacterial solution increased lettuce yield by 62.16%, and the yield increase of this strain (66.28%) was significantly better than that of the other strain (62.16%). Li Fuyan reported that B. massiliogorillae and B. badius increased lettuce yield by 51.08%-59.69% (Li Fuyan (2021). Screening of Bacillus indoleacetic acid-producing bacteria and its growth-promoting effect, South China Agricultural University. Master's thesis.), and the growth-promoting effect of BPC37 was significantly higher than that of these two strains. Apart from this study, there are currently no reports on the effects of Bacillus belyss on promoting lettuce yield.

[0047] 2. Identification of BPC37

[0048] The 16S rDNA sequence of BPC37 is 1446 bp in length. Blast alignment revealed it to be similar to *Bacillus belyssae* (…). Bacillus velezensis The sequence showed the highest homology, reaching 99.86%. Sequence alignment analysis of the assembled BPC37 genome sequence showed that the ANI values ​​between BPC37 and 216 *Bacillus belyssii* strains in the NCBI database ranged from 97.77% to 99.70%, with an average of 98.4%, and were similar to *Bacillus belyssii* FZB42. T The ANI value is 98.67. Therefore, BPC37 is Bacillus belye, and it was submitted to the China General Microbiological Culture Collection Center on May 14, 2025, with accession number CGMCC No. 34544.

[0049] 3. Physiological and biochemical characteristics of BPC37

[0050] When incubated on LB solid medium at 28°C for 24 hours, BPC37 colonies were milky white, nearly round, opaque, with neat edges, a smooth, moist, slightly convex surface, and no odor. Figure 4 (A) Gram staining is positive, and the staining is uniform. Figure 4 (B) The bacterial cells are rod-shaped or short rod-shaped, mostly arranged in chains. Spore staining reveals oval spores (…). Figure 4 (C)

[0051] Phosphorus and potassium solubilization experiments showed that BPC37 could grow normally on both selection media. Figure 5 Figures A and B show the results of its phosphorus-solubilizing and potassium-solubilizing experiments, respectively, indicating that PBC37 has the function of phosphorus-solubilizing and potassium-solubilizing.

[0052] 4. Growth characteristics of strain BPC37 under different concentrations of NaCl and pH conditions

[0053] Rhizosphere growth-promoting bacteria can improve the osmotic balance of plant tissues and reduce the effects of ion toxicity in multiple ways, thereby alleviating the salt and alkali stress experienced by plants and promoting their growth in saline-alkali soils. Figure 6 It can be seen that, except for its inability to grow normally in 15% NaCl, strain BPC37 can grow at the other four NaCl concentrations. Figure 6 A) As the NaCl concentration increases, the growth of strain BPC37 shows a decreasing trend, with more significant growth declines at 7% and 10%. At a 3% NaCl concentration, the growth lag time of BPC37 is 1 day, reaching its maximum value (OD600=3.73) after 2.5 days; at a 7% NaCl concentration, the growth lag time is 2 days, requiring 2.5 days to reach its maximum value (OD600=3.10); and at a 10% NaCl concentration, the growth lag time is 4.5 days, requiring 5 days to reach its maximum value (OD600=2.77). This indicates that BPC37 can tolerate salt concentrations of 10% and below, classifying it as a halophilic bacterium, thus offering potential applications in saline-alkali land.

[0054] Except for its inability to grow normally at pH 4.0 and 10.0, strain BPC37 can grow in the pH range of 5.0 to 10.0. Figure 6 B). The strain exhibits significant growth advantage within the pH range of 6-9, entering a plateau phase after 18 hours; however, growth is inhibited at pH 4 and 10.0. This indicates that strain BPC37 possesses a certain degree of acid and alkali tolerance.

[0055] 5. Growth-promoting effect of strain BPC37 on peach seedlings

[0056] Peach seedlings were treated with BPC37 and BPC6 irrigation methods, respectively. Compared with the control group that was only watered, the fresh weight of the seedlings treated with BPC37 increased by 43.6%, while the fresh weight of the seedlings treated with BPC6 increased by 6.5%. The dry weight of the seedlings treated with BPC37 increased by 52.9%, while the dry weight of the seedlings treated with BPC6 increased by 48.8%. Figure 7 The above data demonstrate that BPC37 significantly promotes peach seedling growth and increases biomass. Observation of the peach seedling roots reveals that the BPC37-treated group had a greater number of lateral roots. Figure 7 ).

[0057] 6. Growth-promoting effect of strain BPC37 on sweet potato

[0058] Sweet potato seedlings were irrigated with BPC37 and BPC6 respectively (morphology as shown). Figure 8 As shown in Figure A), compared with the control group that only received water, the total fresh weight, fresh weight of sweet potatoes, and number of sweet potatoes in the BPC37 treatment were all higher than those in the control group (as shown in Figure A). Figure 8The fresh weight of BPC6 treatment group was not significantly different from the control group (P > 0.05), but the fresh weight of BPC37 treatment group was significantly higher than the control group (P < 0.05), and the yield of sweet potato was increased by 14%, which was significantly higher than the control group; the fresh weight of BPC6 treatment group was not significantly different (P > 0.05). Figure 8 The above data show that BPC37 can significantly improve the yield of sweet potato.

Claims

1. A Bacillus velezensis, characterized in that, The classification name is Bacillus velezensis , and the preservation number is CGMCC No. 34544.

2. The use of Bacillus velezensis according to claim 1 for controlling bacterial diseases.

3. Use according to claim 2, wherein the compound is ###0002### The bacteria are Xanthomonas.

4. The bactericide prepared from Bacillus velezensis according to claim 1, characterized in that, It contains the active bacteria of the Bacillus velezensis.

5. The use of Bacillus velezensis according to claim 1 for promoting plant growth.

6. The use according to claim 5, wherein the compound is ###0002### The plant is lettuce, peach seedlings or sweet potato.

7. The probiotic agent prepared from Bacillus velezensis according to claim 1, characterized in that, It contains the active bacteria of the Bacillus velezensis.

8. The probiotic of claim 7, wherein the probiotic is a strain of Lactobacillus rhamnosus. It is present in the form of a bacterial solution, preferably it is 1 x 10 7 -1 x 10 9 cfu / mL bacterial suspension.

9. A method of planting a plant, characterized by, The plant is lettuce, peach seedlings or sweet potato.

10. The method of claim 9, wherein, The plant is lettuce, peach seedlings or sweet potato.

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