A Bacillus species and its applications

Abstract

This invention provides a Bacillus strain and its applications, belonging to the field of microbial germplasm resource development and utilization technology. The Bacillus strain of this invention includes *Bacillus belyssae* BEV2 and / or *Bacillus amyloliquefaciens* BAM7. The *Bacillus belyssae* BEV2 has the accession number CGMCC NO. 26741, and the *Bacillus amyloliquefaciens* BAM7 has the accession number CGMCC NO. 26742. The Bacillus strain of this invention has a significant inhibitory effect on *Potato scab* pathogens and promotes potato growth. It also has significant inhibitory effects on *Anthracis coccidioides*, *Helicobacter pylori*, *Alternaria alternata*, *Rhizoctonia solani*, and *Fusarium oxysporum*, and also has a phosphorus-solubilizing effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial germplasm resource development and utilization technology, specifically relating to a Bacillus species and its applications. Background Technology

[0002] In recent years, with the continuous increase in potato planting area, the occurrence and severity of potato scab have become increasingly serious, leading to increasing losses to the potato industry year by year. Therefore, the prevention and control of this disease has become a key research focus. Considering environmental pollution, crop quality, and human health, the use of biological agents to replace chemical agents has become a key control measure to improve the agricultural ecological environment. Currently, although many biocontrol agents antagonizing potato scab have been reported domestically and internationally, there are still relatively few commercially available agents. Furthermore, research on potato scab has primarily focused on single biocontrol strains. The diverse species and complex distribution of potato scab pathogens result in the occurrence of multiple pathogenic Streptomyces infections, making control extremely difficult. Currently, single biocontrol strains are insufficient to effectively address the complex infection of multiple pathogenic Streptomyces in the complex field environment. Therefore, exploring the combined control of multiple strains against potato scab has become a key research focus at this stage. In addition, existing biocontrol bacteria are all effective against the same type of pathogens, but are ineffective or effective against a very small number of other pathogens. Exploring biocontrol bacteria with effective broad-spectrum antibacterial effects is also a technical problem that urgently needs to be solved at this stage. Summary of the Invention

[0003] This invention provides a Bacillus and its application. The Bacillus has a significant inhibitory effect on potato scab fungus, anthracnose fungus, large spotted umbelliferous worm, Alternaria alternata, Rhizoctonia solani, and Fusarium oxysporum, and also has the effects of phosphorus solubilization and promoting potato growth.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0005] This invention provides a Bacillus species, including Bacillus belyceae BEV2 and / or Bacillus amyloliquefaciens BAM7, wherein the Bacillus belyceae BEV2 has the accession number CGMCC NO.26741 and the Bacillus amyloliquefaciens BAM7 has the accession number CGMCC NO.26742.

[0006] The present invention provides a microbial agent comprising the aforementioned Bacillus.

[0007] Preferably, the microbial agent includes a powder formulation, a liquid formulation, or a solid formulation.

[0008] This invention provides the application of the Bacillus or the bacterial agent in the preparation of products for the prevention and control of potato scab.

[0009] This invention provides the application of the Bacillus or the bacterial agent in the preparation of products for preventing and controlling diseases caused by Bacillus anthracis, Helicobacter pylori, Alternaria alternata, Rhizoctonia solani, and Fusarium oxysporum.

[0010] This invention provides the application of the Bacillus or the bacterial agent in the preparation of products that promote potato growth.

[0011] This invention provides the application of the Bacillus or the bacterial agent in the preparation of phosphorus-solubilizing products.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The Bacillus berberis BEV2 and / or Bacillus amyloliquefaciens BAM7 screened in this invention can significantly inhibit the growth of various Streptomyces, effectively prevent and control potato scab caused by various Streptomyces, and have no soil pollution, thus reducing agricultural pollution.

[0014] The Bacillus vesiculosus BEV2 and / or Bacillus amyloliquefaciens BAM7 screened in this invention have broad antibacterial spectra and inhibit various pathogens, such as Bacillus anthracis, Helicobacter pylori, Alternaria alternata, Rhizoctonia solani, and Fusarium oxysporum, and have good prospects for development and application.

[0015] The Bacillus berreatus BEV2 and / or Bacillus amyloliquefaciens BAM7 screened in this invention have phosphorus-solubilizing effects, which can effectively increase potato plant height, stem circumference, root length, fresh weight and dry weight of aboveground and underground parts, as well as proline content in potato leaves and root activity, thereby effectively promoting potato growth. Attached Figure Description

[0016] Figure 1 Results of 13 Bacillus strains inhibiting the growth of *H. 4*, the causal agent of potato scab.

[0017] Figure 2 Phylogenetic trees of five biocontrol bacteria were constructed based on the gyrB sequence.

[0018] Figure 3 Determination of the antibacterial activity of four strains of Bacillus and their combinations against three pathogenic bacteria.

[0019] Figure 4 Determination of the antibacterial activity of four Bacillus strains against three pathogenic Streptomyces strains and their combined treatments.

[0020] Figure 5 The antibacterial activity of two Bacillus strains and their combination against three pathogenic Streptomyces strains and their combination.

[0021] Figure 6Growth of Bacillus spp. BEV2, BAM7 and BEV2+BAM7 on phosphorus-solubilizing, nitrogen-fixing and potassium-solubilizing media.

[0022] Figure 7 The antibacterial spectrum of Bacillus BAM7 and BEV2 against plant pathogens.

[0023] Figure 8 Standard curve of proline content.

[0024] Figure 9 Standard curve of root vitality.

[0025] Figure 10 Effects of Bacillus cereus BEV2, BAM7 and their combination treatments on potato plant height.

[0026] Figure 11 Effects of Bacillus spp. BEV2, BAM7 and their combinations on stem circumference of potato plants.

[0027] Figure 12 Effects of Bacillus subtilis BEV2, BAM7 and their combinations on root length of potato plants.

[0028] Figure 13 Effects of Bacillus subtilis BEV2, BAM7 and their combinations on the fresh weight of aboveground and underground parts of potato plants.

[0029] Figure 14 Effects of Bacillus subtilis BEV2, BAM7 and their combinations on the aboveground and underground dry weight of potato plants.

[0030] Figure 15 Determination of the effects of Bacillus subtilis BEV2, BAM7 and their combinations on potato root vigor.

[0031] Figure 16 Determination of proline content in potato leaves by Bacillus spp. BEV2, BAM7 and their combinations.

[0032] Biological Preservation Instructions

[0033] Bacillus velezensis (BEV2) was deposited on March 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO. 26741. It is classified and named Bacillus velezensis.

[0034] Bacillus amyloliquefaciens BAM7 was deposited on March 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.26742. It is classified as Bacillus amyloliquefaciens. Detailed Implementation

[0035] This invention provides a Bacillus species, including Bacillus velezensis BEV2 and / or Bacillus amyloliquefaciens BAM7. Both Bacillus velezensis BEV2 and Bacillus amyloliquefaciens BAM7 were deposited on March 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC). The accession number for Bacillus velezensis BEV2 is CGMCC NO. 26741, and the Latin name is Bacillus velezensis. The accession number for Bacillus amyloliquefaciens BAM7 is CGMCC NO. 26742, and the Latin name is Bacillus amyloliquefaciens.

[0036] This invention provides a microbial agent comprising the aforementioned Bacillus. The microbial agent of this invention further includes other excipients that do not affect the activity of the Bacillus. The microbial agent of this invention includes powder formulations, liquid formulations, or solid formulations.

[0037] This invention provides the application of the aforementioned Bacillus or the aforementioned bacterial agent in the preparation of products for controlling potato scab. The Bacillus of this invention includes *Bacillus bellis* BEV2 and / or *Bacillus amyloliquefaciens* BAM7, preferably a combination of *Bacillus bellis* BEV2 and *Bacillus amyloliquefaciens* BAM7. The potato scab pathogen of this invention includes *Streptomyces scabies* HP4, *S. scabies* SCA3, *S. turgidiscabies* SUR1, and *S. stelliscabiei* STE2, as well as combinations of the above strains.

[0038] This invention provides the application of the aforementioned Bacillus or the aforementioned bacterial agent in the preparation of products for controlling diseases caused by *C. cocci*, *Hylocereus macrocarpa*, *Alternaria alternata*, *Rhizoctonia solani*, and *Fusarium oxysporum*. The *Bacillus amyloliquefaciens* BAM7 and *Bacillus belyssus* BEV2 of this invention exhibit strong inhibitory effects against *C. coccodes*, with inhibition bands exceeding 11 mm. *Bacillus amyloliquefaciens* BAM7 shows inhibition bands ≥11 mm against *E. turcicum*, the causal agent of maize leaf spot, and *A. solani*, the early blight causal agent of potato. *Bacillus belyssus* BEV2 shows inhibition bands of 8.55 mm against both pathogens.

[0039] This invention provides the application of the aforementioned Bacillus or the aforementioned inoculant in the preparation of products that promote potato growth. The Bacillus of this invention includes *Bacillus bellis* BEV2 and / or *Bacillus amyloliquefaciens* BAM7, preferably a combination of *Bacillus bellis* BEV2 and *Bacillus amyloliquefaciens* BAM7. The Bacillus of this invention can effectively increase potato plant height, stem circumference, root length, fresh and dry weight of aboveground and underground parts, as well as the proline content of potato leaves and root activity, achieving an effective effect in promoting potato growth.

[0040] This invention provides the application of the aforementioned Bacillus or the aforementioned bacterial agent in the preparation of phosphorus-solubilizing products. The two Bacillus strains BAM7 and BEV2, and their combination, described in this invention, all exhibited distinct transparent halos on phosphorus-solubilizing media, demonstrating their ability to promote effective phosphorus absorption by plants.

[0041] Unless otherwise specified, all components used in this invention are commercially available products well known to those skilled in the art.

[0042] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] Example 1: Screening and identification of Bacillus antagonistic to potato scab causal agent

[0044] 1. Preparation of a suspension of S. scabies HP4, the causal agent of potato scabies.

[0045] Given that *S. scabies* is the dominant species of potato scab pathogen in the main potato-producing areas of Hebei Province, this invention selects *S. scabies* HP4 (isolated from Chengde, Hebei Province) as the target bacterium for preliminary screening of antagonistic bacilli against potato scab pathogen. 6 mL of sterile water was pipetted into an OMA plate contaminated with *S. scabies*. The bacterial cells were gently scraped off with a spreader, and the mycelium was filtered out using a sterile syringe filled with absorbent cotton. The filtrate (mostly spores) was temporarily stored at -20°C. The filtrate (mostly spores) was serially diluted to 10⁻⁶. -6 Spread 50 μL onto OMA medium. After 5 days, count single colonies to calculate the spore suspension concentration, and adjust the spore suspension concentration to 10. 6 CFU / mL.

[0046] 2. Preparation of Bacillus bacterial culture

[0047] Single colonies of Bacillus strains BEV2 and BAM7 were picked and transferred to 50 mL Erlenmeyer flasks containing 30 mL of liquid LB medium. The flasks were then incubated at 37°C and 200 rpm in a shaker until the OD value of the bacterial culture was reached. 600 Only when the value is 1 can it be used for subsequent experiments. For combined treatments, the bacterial suspensions of individual strains are mixed at a 1:1 ratio.

[0048] 3. Screening of Bacillus species antagonistic to S. scabies, the causal agent of potato scabies.

[0049] Between 2015 and 2020, 766 Bacillus strains were isolated from potato rhizosphere soil samples. Among them, 124 strains showed varying degrees of antibacterial activity against *Bacillus subtilis*, the causal agent of potato scab. Thirteen strains with relatively good antibacterial effects (BEV2, BAM7, GF3, BPU6, BMO8, Q3911, HZ7, FM2-4, Q395, HZ15, Q394, F17, and F7) were selected for further screening. The antibacterial antagonism test on agar plates was as follows: 100 μL of a 10% concentration prepared in step 1 was used... 6 A CFU / mL suspension of *S. scabies* HP4 bacteria was evenly spread onto an OMA agar plate; a 5 mm diameter sterile filter paper was placed in the center of the petri dish, and 5 μL of a 10 CFU / mL solution was added to each plate. 8 The above-mentioned Bacillus spp. bacterial suspension was prepared at CFU / mL; an equal volume of sterile water was set up as a control group, and each treatment group was repeated four times; finally, OMA culture dishes were placed in an incubator at 28℃ and inverted for 7 days, and the diameter of the inhibition zone (minus the diameter of the filter paper) was measured. The results are shown in Table 1 and 2. Figure 1 .

[0050] Table 1.1 Antibacterial activity of 113 Bacillus strains against HP4, the causal agent of potato scab.

[0051]

[0052]

[0053] As shown in Table 1, among the 13 Bacillus strains, BEV2 showed the best inhibitory effect against HP4, the scab causal agent of potato scab, with an inhibition zone diameter of 12.3 ± 0.6 mm. In addition, BAM7, GF3, BPU6 and BMO8 showed relatively significant inhibitory effects, with inhibition diameters all exceeding 9.00 mm.

[0054] 4. Molecular identification of Bacillus species

[0055] Five Bacillus strains (BEV2, BAM7, GF3, BPU6, and BMO8) exhibiting strong inhibitory effects against *S. scabies* HP4 were selected for molecular identification to clarify their taxonomic position. Total DNA was extracted from the Bacillus strains using a bacterial DNA extraction kit. PCR amplification of this locus was performed using universal gyrB primers (forward primer gyrB-F: 5′-TGRCGGHRGYGGHTATAAAGT-3′, reverse primer gyrB-R: 5′-TCCDCCSTCAGARTCWCCCTC-3′). The amplification program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 1 min, extension at 72℃ for 2 min, 35 cycles; final extension at 72℃ for 10 min, and storage at 4℃. The PCR products were separated and purified by electrophoresis on a 1% agarose gel and then sequenced (Shanghai Sangon Biotech). A phylogenetic tree based on the gyrB locus was constructed using the NJ method. Results are shown below. Figure 2 .

[0056] through Figure 2 Identification revealed that BEV2 was *Bacillus velezensi*; BAM7 was *Bacillus amyloliquefaciens*; GF3 was *Bacillus velezensi*; BPU6 was *Bacillus pumilus*; and BMO8 was *Bacillus mojavensis*. Since BEV2 and GF3 are the same species of spore-forming bacillus, we selected four strains—BEV2, BAM7, BPU6, and BMO8—for subsequent experiments.

[0057] 5. Antimicrobial Activity of Four Bacillus Strains and Combinations Against Three Other Potato Scab Pathogens Since Bacillus Strains BEV2 and GF3 were identified as belonging to the same genus *Bacillus amyloliquefaciens*, BEV2, which showed better antimicrobial activity, was retained for subsequent interspecies combination optimization screening. In addition to the pathogen *S. scabies* HP4, *S. scabies* SCA3, isolated from Zhangbei, was selected as a target bacterium to test the stability of the antagonistic effect of Bacillus Strains against isolates from different regions. Simultaneously, two other relatively common scab pathogens besides the dominant *S. scabies*, *S. turgidiscabies* SUR1 and *S. stelliscabiei* STE2, were selected as target bacteria. The antimicrobial effects of multiple (2–4) Bacillus Strains mixtures against the three scab pathogens were then measured. The bacterial counts in the mixed strain treatment groups were mixed in equal proportions according to the number of strains, and the bacterial concentration was 10⁻⁶. 8 CFU / mL. Six mixed bacterial strain treatment groups were tested (pairwise combinations: BEV2+BAM7, BPU6+BAM7, BMO8+BAM7; three-strain combinations: BEV2+BAM7+BPU6, BEV2+BAM7+BMO8; four-strain combinations: BEV2+BAM7+BPU6+BMO8). The method for determining the antibacterial effect of the Bacillus mixed treatment groups on the pathogen was the same as in steps 2 and 3. The results are shown in Table 2 and... Figure 3 .

[0058] Table 2. Determination of the antibacterial activity of four Bacillus strains and their combinations against three pathogenic bacteria.

[0059]

[0060] The results in Table 2 show that among the 11 treatments, BAM7 exhibited the most significant antibacterial effect against SUR1, with an inhibition zone diameter of 34.50 mm. In addition, BAM7 also showed good antibacterial effects against STE2 and SCA3. Specifically, BAM7+BPU6+BMO8 and BAM7+BMO8 showed better antibacterial effects against STE2. Treatments with good antibacterial effects against SCA3 included BMA7+BPU6, BMA7+BMO8, BMA7, and BPU6+BEV2. All of the above treatments showed antibacterial effects ≥29.00 mm against the three potato scab pathogens.

[0061] Depend on Figure 3The results showed that among the four Bacillus strains, BAM7 exhibited strong inhibitory effects against three of the scab pathogens. BPU6 showed a strong inhibitory effect against SCA3 (inhibition zone diameter 29.00±0.82ab), followed by STE2, but had no significant inhibitory effect against SUR1. BEV2 showed some inhibitory effect against all three scab pathogens, but the effect was relatively weak. BMO8 showed no significant inhibitory effect against SUR1, and had inhibitory effects against STE2 and SCA3, but the effects were not significant. Furthermore, mixing it with other Bacillus strains did not enhance the effect. Considering all factors, this strain was eliminated.

[0062] 6. Determine the antibacterial activity of three Bacillus strains against different combinations of scab causative agents.

[0063] To determine the stability of the antagonistic effect of Bacillus against mixed pathogens when multiple pathogens are present simultaneously, this experiment set up four mixed treatment groups: SUR1+STE2, SUR1+SCA3, STE2+SCA3, and SUR1+STE2+SCA3 pathogens. The inhibitory effect of applying one of Bacillus BEV2, BAM7, or BPU6 against multiple mixed pathogens was then tested. The pathogen amounts in the mixed treatment groups were mixed in equal proportions, and the bacterial concentration of each treatment group was 10⁻⁶. 6 CFU / mL. The determination method is described in steps 2 and 3. The results are shown in Table 3. Figure 4 .

[0064] Table 3. Determination of the antibacterial activity of four Bacillus strains against three pathogenic Streptomyces strains and their combination strains.

[0065]

[0066] From Table 3 and Figure 4 The results showed that BPU6 had a good inhibitory effect on STE2+SCA3, with an average inhibition zone diameter of 34.00±0.82 mm, but no significant inhibitory effect on other combination treatments. BEV2 had a certain inhibitory effect on all four combination treatments, with the most significant inhibitory effect on SUR1+STE2 and SUR1+SCA3, with inhibition zone diameters ≥32.00 mm. BAM7 had the most significant inhibitory effect on all four treatments, with inhibition zone diameters all above 30.00 mm, and the most significant inhibitory effect on STE2+SCA3, with an inhibition zone diameter of 35.25±2.36 mm. In summary, although BPU6 has a certain inhibitory effect on some potato scab pathogens, its inhibitory effect is unstable when multiple pathogens are present simultaneously. Considering the complex ecological environment in the field, this strain was eliminated. BEV2 and BAM7 both had certain inhibitory effects on the three scab pathogens and their combination strains; therefore, these two Bacillus strains were selected for subsequent experiments.

[0067] Example 2: Determination of the antibacterial activity of Bacillus spp. BEV2 and / or Bacillus spp. BAM7 against three strains of potato scab pathogen and their combinations.

[0068] The antibacterial effects of the BEV2+BAM7 group, BAM7 group, and BEV2 group against the mixed treatment groups of four pathogens: SUR1+STE2, SUR1+SCA3, STE2+SCA3, and SUR1+STE2+SCA3 were determined. For the mixed treatment, single-spore suspensions were mixed at a ratio of 1:1 or 1:1:1. The determination method was the same as step 3 in Example 1. The results are shown in Table 4. Figure 5 .

[0069] Table 4. Determination of the antibacterial activity of BEV2 and / or BAM7 against three pathogenic Streptomyces strains and their combined treatments.

[0070]

[0071] From Table 4 and Figure 5 The experimental results showed that BEV2+BAM7 had the best antibacterial effect against STE2+SCA3, with an inhibition zone diameter of 33.25±3.95 mm. The antibacterial effect of this strain against the four mixed treatment groups of potato scab pathogens was not weakened compared to BEV2 and BAM7 alone; at the same time, the effect of BEV2+BAM7 was improved compared to BAM7 alone.

[0072] Example 3: Determination of the phosphorus-solubilizing, nitrogen-fixing, and potassium-solubilizing abilities of BEV2, BAM7, and their combination strains

[0073] Sterile filter paper discs with a diameter of 5 mm were placed in the center of phosphate-solubilizing, nitrogen-fixing, and potassium-solubilizing agar plates, respectively. 5 μL of bacterial suspensions of BEV2, BAM7, and BEV2+BAM7 (refer to step 2 of Example 1) were then added to the filter paper discs. The plates were incubated upside down at 37°C for 7 days, and the appearance of a transparent halo around the Bacillus strains was observed. This experiment consisted of three treatments, with three replicates for each treatment, for a total of three replicates. Results are shown below. Figure 6 .

[0074] Depend on Figure 6 The results showed that both Bacillus strains BAM7 and BEV2, and their combination, exhibited good phosphorus solubilization effects, with obvious transparent halos appearing on the phosphorus solubilization medium. However, neither strain nor their combination showed significant potassium solubilization or nitrogen fixation abilities, and no obvious transparent halos appeared on either potassium solubilization or nitrogen fixation medium.

[0075] Example 4: Determination of the antibacterial spectrum of Bacillus subtilis BAM7 and BEV2

[0076] To investigate the antagonistic effects of BEV2 and BAM7 Bacillus strains against other plant pathogens, the inhibitory activities of the two strains against various plant pathogenic fungi and bacteria were determined.

[0077] (1) Plant pathogenic fungi: *C. coccodes* F3-3 (A), *Fusarium oxysporum* Z5-2 (B), *R. solani* R18 (C), *Exserohilum turcicum* (D), and *Alternaria solani* HWC-168 (E), the causal agent of potato black spot, were selected as target fungi. The target fungal discs were placed in the center of a PDA agar plate. Sterile filter paper discs with a diameter of 5 mm were placed at equal intervals in all four directions (top, bottom, left, and right) of the discs. 5 μL of a 10⁻⁶ solution was added to each filter paper disc. 8 Bacillus spp. culture at CFU / mL. Sterile water was used as a control. The culture was incubated upside down in the dark at 25°C for 7 days, and the size of the inhibition zone was measured. Statistical analysis was performed using the same method as step 3 of Example 1. Results are shown in Table 5. Figure 7 .

[0078] (2) Plant pathogenic bacteria: Pectobacterium brasiliense B412(F), the causal agent of potato soft rot, was selected as the target bacterium. Single colonies of the pathogenic bacteria were picked and cultured in liquid LB medium at 28°C and 180 rpm with shaking until OD. 600 The concentration was 0.8. Pipette 1 mL of the prepared bacterial culture into a 1.5 mL centrifuge tube and centrifuge at 10000 rpm for 10 min. Collect the supernatant and transfer it to 100 mL of solid LB medium. Finally, use a 10% concentration... 8 CFU / mL Bacillus seed culture was streaked on LB agar plates and incubated upside down at 28°C for 72 h to observe antibacterial activity. Results are shown in Table 5. Figure 7 .

[0079] Table 5 and Figure 7 The results showed that BAM7 and BEV2 had certain inhibitory effects on some pathogens of potatoes and other field crops. They had a strong inhibitory effect on potato anthracnose fungus (C. coccodes), with inhibition bands exceeding 11 mm. BAM7 had inhibition bands ≥11 mm against maize leaf spot fungus (E. turcicum) and potato early blight fungus (A. solani), while BEV2 had inhibition bands of 8.55 mm against both pathogens. In addition, the two biocontrol strains did not show significant inhibitory effects on potato soft rot fungus B412 (P. brasiliense).

[0080] Table 5. Determination of the inhibitory activity of Bacillus subtilis BAM7 and BEV2 against different pathogens.

[0081]

[0082] Example 5: Growth-promoting effects of BEV2, BAM7, and BEV2+BAM7 on potato plants

[0083] 1. Experimental protocol for treating potted plants with Bacillus subtilis

[0084] (1) Potato planting

[0085] Rinse the sprouted potato tubers three times with sterile water, then cut the sprouted potatoes into tubers with single buds. Place the treated tubers in pots (20×18cm) filled with soil, using a 2:1 ratio of potting soil to vermiculite. Place them in a smart greenhouse at 25℃, with an average of 16 hours of sunlight per day, and water them every three days.

[0086] (2) This experiment included four treatments. Seven days after planting potatoes, 30 ml of inoculum solutions of three strains of BEV2, BAM7, and BEV2+BAM7 (prepared using the method described in step 2 of Example 1) were taken, diluted with water, and then poured into the soil along the roots. Sterile water was used as a control. Twelve pots were planted for each treatment, for a total of 48 pots in each experiment. After 30 days, the growth-promoting ability of the three Bacillus strains was measured from two dimensions: physical (plant height, stem circumference, root length, fresh and dry weight of aboveground and underground parts) and chemical (proline content, root activity). Four batches of potatoes were planted. The results are shown in […]. Figures 8-15 .

[0087] Plant height: Measure the height of the entire potato plant from the base of the stem to the top of the plant using a ruler; Stem circumference: Measure the circumference of the same part of the potato plant stem using a tape measure; Root length: Measure the length from the junction of the base of the stem and the root to the lowest point of the root using a ruler; Above-ground fresh weight: Weigh all parts of the potato plant except the roots using an electronic balance; Underground fresh weight: Weigh the roots of the potato plant using an electronic balance; Above-ground dry weight: Weigh the parts of the potato plant except the roots after drying under natural conditions for 4 days using an electronic balance; Underground dry weight: Weigh the roots of the potato plant after drying under natural conditions for 4 days using an electronic balance.

[0088] Determination of proline content in potato leaves: (1) Preparation of standard curve: Prepare a proline standard solution with a concentration of 100 μg / mL, and add each chemical reagent to a glass test tube according to the requirements in Table 6. Then boil in boiling water for 40 min, cool on ice for 10 min to room temperature, add 5 mL of toluene and shake to extract, let stand in the dark for 2 h, the solution separates into layers, take the upper pink solution into a cuvette, and measure the absorbance at 520 nm. Plot the standard curve with absorbance as the ordinate and proline standard solution content as the abscissa, see Figure 8 .

[0089] Table 6. Reagent dosage for standard curve preparation.

[0090]

[0091] (2) Take 1.0g of fresh leaves, cut them into small pieces, and transfer them to a glass test tube with tweezers. Add 4mL of sulfosalicylic acid, boil in boiling water for 20min, and then filter. Collect the filtrate. Add 0.5mL of the filtrate, 1.5mL of water, 2mL of ninhydrin, and 2mL of glacial acetic acid to the test tubes respectively, shake well, and boil in boiling water for 40min. Then cool on ice for 10min to room temperature, add 5mL of toluene to each tube, shake thoroughly to extract, and let stand in the dark for 2h. The solution will separate into layers. Take the upper pink solution into a cuvette and measure its absorbance at a wavelength of 520nm. Calculate the proline content according to the standard curve.

[0092] Potato root vigor determination: (1) Standard curve preparation: Add 0.01g sodium dithionite, 200μL of 4% TTC solution and 9.8mL of ethyl acetate to a clean beaker to produce a red TTF solution. Take 0.25mL, 0.50mL, 1.00mL, 1.50mL and 2.00mL of TTF solution to five test tubes respectively, and add ethyl acetate to 10mL. Ethyl acetate is used as a blank control. Take the test solution into a cuvette and measure the absorbance at a wavelength of 485nm. Plot the standard curve with absorbance as the ordinate and TTF content as the abscissa. See Figure 9 .

[0093] (2) Take 0.5g of fresh root sample, cut it into small pieces and place it in a test tube. Add 5mL of 0.4% TTC solution and 5mL of pH 7.0 phosphate buffer to each tube, and incubate in the dark at 37℃ for 2 hours. Immediately afterwards, add 2mL of 1mol / L sulfuric acid solution to stop the reaction. Transfer the root tissue to sterile filter paper, blot dry, and place it in a sterile mortar. Add 4mL of ethyl acetate to the mortar containing the root tissue and grind for 5-10 minutes. Obtain the red extract, transfer it to a test tube using a pipette, and wash the residue with ethyl acetate 2-3 times, continuing to grind during washing. Transfer all washing solutions to the test tube, and finally add ethyl acetate to the test tube to a final volume of 10mL. Transfer the red solution from the test tube to a cuvette and measure its absorbance at a wavelength of 485nm. Calculate the TTF content using a standard curve.

[0094] Depend on Figure 10The results showed that the potato plant height in all three treatment groups was significantly higher than that in the control group (P<0.05). Specifically, the average plant height in the BEV2 treatment group reached 53.30±2.91 cm, 11.32% higher than the control group; the BAM7 treatment group showed no significant difference from the BEV2 group, with an average plant height of 53.80±3.43 cm; the BEV2+BAM7 treatment group showed the strongest growth-promoting effect on potato plant height, significantly higher than the single-strain treatment group (P<0.05), with an average height of 57.00±3.00 cm.

[0095] Depend on Figure 11 The results showed that the stem circumference of potato plants in the BEV2, BAM7, and BEV2+BAM7 treatment groups was significantly higher than that in the water control (P<0.05), indicating a significant promoting effect on stem circumference. The mean stem circumference of potato plants in the BEV2 treatment group was 3.45±0.35 cm, which was significantly increased by 15.56% compared with the control group; the mean stem circumference of potato plants in the BAM7 treatment group was 3.25±0.25 cm; and the stem circumference of potato plants in the BEV2+BAM7 treatment group was significantly higher than that in the single-plant treatment group, with a mean stem circumference of 3.58±0.28 cm, indicating a good promoting effect on stem circumference.

[0096] Depend on Figure 12 The results showed that the root lengths of potato plants treated with BEV2, BAM7, and BEV2+BAM7 were all longer than those of the water control, and all three treatments promoted root growth in potatoes. The mean root length of potato plants treated with BEV2 was 32.40±2.76 cm; the root length of the BAM7 treatment group was longer than that of BEV2, with a mean of 33.00±3.33 cm, which was 14.09% higher than that of the control group; the mean root length of the BEV2+BAM7 treatment group reached 35.20±3.08 cm, which was significantly higher than that of the single-plant treatment group (P<0.05).

[0097] Depend on Figure 13The results showed that the aboveground and underground fresh weights of the treatment groups were significantly higher than those of the control group (P<0.05), indicating that the treatment groups had a significant growth-promoting effect on both the aboveground and underground parts of potato plants. There was no significant difference in the aboveground and underground fresh weights of potato plants between the BEV2 and BAM7 treatment groups, but both were significantly higher than the control group. Specifically, the mean aboveground and underground fresh weights of BEV2 were 63.30±4.05 cm and 9.32±0.98 cm, respectively, while those of BAM7 were 65.00±3.13 cm and 9.69±0.84 cm, respectively. The aboveground and underground fresh weights of the BEV2+BAM7 treatment group were significantly higher than those of the single-strain treatment group. The mean aboveground fresh weight of this treatment group was 71.28±5.40 cm, 35.82% higher than the control; the mean underground fresh weight reached 12.44±1.47 cm, 35.69% higher than the control. In conclusion, the BEV2+BAM7 treatment group showed the most significant effect in increasing the fresh weight of both the aboveground and underground parts of potato plants, and had a strong growth-promoting effect on potato plants.

[0098] Depend on Figure 14 The results showed that the aboveground and underground dry weights of Bacillus subtilis BEV2, BAM7, and their combinations were significantly higher than those of the water control (P<0.05). Therefore, all three treatments significantly promoted the dry weight of potato plants. The aboveground and underground dry weights of the BEV2+BAM7 treatment group were significantly higher than those of the single-strain treatment group. The mean aboveground dry weight of this treatment was 34.19±1.66 cm, an increase of 58.91% compared to the control; the mean underground dry weight was 1.46±0.16 cm, an increase of 46.58% compared to the control. There was no significant difference in the aboveground and underground dry weights of potato plants between the BEV2 and BAM7 treatment groups, but both were significantly higher than those of the water control. The average aboveground dry weight of BEV2 was 26.85±1.82 cm, and the average underground dry weight was 1.26±0.16 cm. For BAM7, the average aboveground and underground dry weights were 27.17±1.83 cm and 1.20±0.14 cm, respectively. In conclusion, the BEV2+BAM7 treatment group showed the most significant increase in potato plant dry weight and had a strong promoting effect on plant growth.

[0099] Depend on Figure 15The results showed that the root activity of plants treated with Bacillus cereus BEV2, BAM7, and their combinations was significantly higher than that of the water control group (P<0.05). Furthermore, the root activity of plants treated with BEV2+BAM7 was significantly higher than that of the two single-strain treatment groups, with a mean TTF content of 20.12±3.12 μg / mL, an increase of 51.69% compared to the control group. In addition, the root activity of plants treated with BEV2 and BAM7 was also significantly higher than the control, with mean TTF contents of 17.00±3.12 μg / mL and 15.96±3.60 μg / mL, representing increases of 42.82% and 39.10%, respectively. This indicates that all three biocontrol bacteria can effectively improve the root activity of potato plants.

[0100] Depend on Figure 16 The results showed that BEV2, BAM7, and their combinations significantly increased the proline content in potato leaves compared with the control group (P<0.05). Specifically, the proline content in potato leaves treated with BEV2 and BAM7 was significantly higher than that in the control group. The proline content in the BEV2 treatment reached 4.31±0.26 μg / mL, an increase of 60.56% compared with the control group; the proline content in the BAM7 treatment reached 3.70±0.15 μg / mL, an increase of 54.05% compared with the control group; and the proline content in the BEV2+BAM7 treatment was as high as 6.14±0.69 μg / mL, significantly higher than that in the BEV2 and BAM7 treatments alone (P<0.05).

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Bacillus species, characterized in that, The Bacillus species is Bacillus belesiensis (B. belesiensis) Bacillus velezensis BEV2 or Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens BAM7; The Bacillus belyceae BEV2 has the accession number CGMCC NO.26741, and the Bacillus amyloliquefaciens BAM7 has the accession number CGMCC NO.26742.

2. A microbial agent, characterized in that, Includes Bacillus belyssus BEV2 as described in claim 1 and / or Bacillus amyloliquefaciens BAM7.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent may be a liquid or solid formulation.

4. The use of the Bacillus as described in claim 1 or the inoculum as described in claim 2 in the preparation of products for the prevention and control of potato scab.

5. The use of the Bacillus as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of products for preventing and controlling diseases caused by Bacillus anthracis, Helicobacter pylori, Alternaria alternata, Rhizoctonia solani, and Fusarium oxysporum.

6. The use of the Bacillus as described in claim 1 or the inoculant as described in claim 2 in the preparation of products that promote potato growth.

7. The use of the Bacillus as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of phosphorus-solubilizing products.

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