Bacillus velezensis rh2 and application thereof

Abstract

This invention belongs to the field of agricultural microbiology and green biological control technology, specifically relating to a strain of Bacillus belye RH2 and its application. The Bacillus belye RH2 strain is deposited at the China Center for Type Culture Collection and is classified as follows: Bacillus from Velez RH2, with accession number CCTCC No: M 2026638, accession date April 10, 2026, and accession address Wuhan University, Wuhan, China. This invention, *Bacillus belyssiensis* RH2, can serve as a microbial resource for the green control of rice sheath blight and root-knot nematodes. The *Bacillus belyssiensis* RH2 provided by this invention can also serve as a new strain resource for developing multifunctional microbial biocontrol agents.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and green biological control technology, specifically involving a strain of Bacillus belye RH2 and its application. Background Technology

[0002] Rice sheath blight is one of the important fungal diseases in rice production, mainly caused by Rhizoctonia solani (Rhizoctonia solani). Rhizoctonia solani Rice sheath blight, caused by infection, can damage rice leaf sheaths, leaves, and stems, leading to lodging, reduced seed setting rate, and yield loss. Currently, the control of rice sheath blight in production still mainly relies on chemical agents such as jinggangmycin and thifluzamide. Long-term use may lead to increased pathogen resistance, unstable field control efficacy, pesticide residues, and environmental pressure. Root-knot nematodes are important plant parasitic nematodes that can infect the root systems of various crops and affect normal plant growth. Their complex living environment and wide host range make control difficult, and existing control measures face the need for reduced pesticide use and green alternatives. Therefore, developing microbial resources with both fungal disease control activity and plant parasitic nematode control activity is beneficial for expanding the scope of action and agricultural application scenarios of biocontrol agents.

[0003] Several strains of Bacillus and their preparations for the control of rice sheath blight have been disclosed in existing technologies. However, some strains are mainly targeted at a single pathogen, or lack verification of efficacy at the rice plant level. Other microbial resources have shown antibacterial or nematicidal activity, but there is insufficient disclosure of their dual activity against both rice sheath blight and root-knot nematodes. Therefore, screening for a strain of Bacillus belyss that can significantly inhibit Rhizoctonia solani and reduce the spread of rice sheath blight lesions, while also exhibiting strong toxic activity against root-knot nematodes, is of great significance for enriching the microbial resources for green pest control in rice, reducing reliance on chemical pesticides, and developing multifunctional biocontrol agents. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus belyssus RH2 and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A strain of Bacillus belye RH2, deposited at the China Center for Type Culture Collection (CCTCC), is classified and named as follows: Bacillus velezensis RH2, accession number CCTCC No: M 2026638, accession date April 10, 2026, accession address Wuhan University, Wuhan, China.

[0006] The application of the aforementioned Bacillus berleis RH2 in the control of rice sheath blight and / or root-knot nematodes.

[0007] The rice sheath blight is caused by Rhizoctonia solani (… Rhizoctonia solani )cause.

[0008] The root-knot nematode is preferably a second-instar larva of the root-knot nematode.

[0009] The application of Bacillus vesiculosus RH2 in the preparation of microbial agents for the control of rice sheath blight and / or microbial preparations for the control of root-knot nematodes.

[0010] A microbial preparation for controlling rice sheath blight, wherein the active ingredient of the microbial preparation is one or more of the following: Bacillus brevis RH2 cells, spores, fermentation broth, fermentation supernatant, or sterile fermentation filtrate.

[0011] Preferably, the fermentation supernatant ratio of the microbial preparation is 40% (fermentation supernatant: sterile water = 40:60).

[0012] Bacillus belye RH2 was inoculated into LB liquid medium and cultured at 25–37°C and 160–220 r / min for 24–72 h to obtain the fermentation broth of Bacillus belye RH2. Centrifuge and / or filter the Bacillus brevis RH2 fermentation broth to obtain fermentation supernatant or sterile fermentation filtrate.

[0013] The microbial preparation for controlling rice sheath blight is used to inhibit the growth of Rhizoctonia solani and reduce the extent of rice sheath blight lesion expansion.

[0014] A microbial preparation for controlling root-knot nematodes, wherein the active ingredient of the microbial preparation is one or more of the following: Bacillus belyss RH2 cells, spores, fermentation broth, fermentation supernatant, or sterile fermentation filtrate.

[0015] Bacillus belye RH2 was inoculated into LB liquid medium and cultured at 25–37°C and 160–220 r / min for 24–72 h to obtain the fermentation broth of Bacillus belye RH2. Centrifuge and / or filter the Bacillus brevis RH2 fermentation broth to obtain fermentation supernatant or sterile fermentation filtrate.

[0016] The microbial preparation for controlling root-knot nematodes is used to kill second-instar larvae of root-knot nematodes.

[0017] Furthermore, the microbial preparations for controlling rice sheath blight or for controlling root-knot nematodes also include one or more of the following agriculturally acceptable carriers, adjuvants, protectants, wetting agents, dispersants, stabilizers, nutrients, or organic fertilizer substrates.

[0018] The effective viable bacteria content of Bacillus vesiculosus RH2 in the microbial preparation for controlling rice sheath blight or root-knot nematodes is preferably 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

[0019] The formulations of the microbial agents for controlling rice sheath blight or for controlling root-knot nematodes are aqueous solutions, suspensions, wettable powders, water-dispersible granules, pellets, or seed treatment agents.

[0020] A method for controlling rice sheath blight involves applying a microbial agent for controlling rice sheath blight to rice plants or their growing environment.

[0021] Application methods include foliar spraying, stem base spraying, root irrigation, root dipping, seed soaking, seed mixing, soil application, or application with irrigation water.

[0022] Preferably, foliar spraying or stem base spraying is applied at the early stage of rice sheath blight.

[0023] The rice plant consists of rice seeds, seedlings, plant leaves, and stem base; the growing environment is the rhizosphere soil or paddy field water layer.

[0024] Microbial agents for controlling rice sheath blight should be applied during the tillering, jointing, booting, or early stages of rice sheath blight.

[0025] Microbial agents for controlling rice sheath blight can be applied alone or in conjunction with conventional water and fertilizer management practices. One method for controlling root-knot nematodes involves applying a microbial agent for root-knot nematode control to the plant roots or rhizosphere soil.

[0026] The present invention has the following beneficial effects: (1) This invention verified the biocontrol activity of Bacillus vesiculosus RH2 through plate confrontation test, fermentation supernatant antibacterial test, rice pot control efficacy test, and indoor toxicity test of root-knot nematodes. The plate confrontation test showed that Bacillus vesiculosus RH2 inhibited Rhizoctonia solani by 83.0%; when the proportion of fermentation supernatant was 40%, the inhibition rate against Rhizoctonia solani reached 82.0%. The rice pot test showed that both RH2 bacterial suspension and RH2 fermentation broth could significantly inhibit the expansion of rice sheath blight lesions, with control effects of 73.5% and 77.8%, respectively. (2) The Bacillus vesiculosus RH2 fermentation broth, fermentation supernatant and sterile fermentation filtrate of the present invention have strong toxicity to second-instar larvae of root-knot nematodes, and the mortality rate of nematodes after 6 hours of treatment is over 97%. (3) The Bacillus RH2 strain of the present invention is easy to cultivate and can be used in various forms such as bacterial cells, spores, fermentation broth, fermentation supernatant or sterile fermentation filtrate for formulation development, providing new microbial resources for the synergistic green control of rice sheath blight and root-knot nematodes. Attached Figure Description

[0027] Figure 1 This is a plate confrontation inhibition effect diagram of Bacillus belyss RH2 against plant pathogens, where A: Rhizoctonia solani; B: Botrytis cinerea; C: Anthracnose; D: Alternaria alternata; E: Fusarium graminearum; F: Apple leaf spot fungus; G: Maize leaf spot fungus; H: Verticillium wilt fungus.

[0028] Figure 2 This is a colony morphology diagram of Bacillus belyss RH2.

[0029] Figure 3 Gram-stained microscopic morphology of Bacillus belyssus RH2.

[0030] Figure 4 Phylogenetic tree of Bacillus belyss RH2.

[0031] Figure 5 The diagram shows the control efficacy of Bacillus berberis RH2 against rice sheath blight, where A: sterile water; B: LB medium; C: Bacillus berberis RH2 fermentation broth; and D: Bacillus berberis RH2 bacterial suspension.

[0032] A strain of Bacillus belye RH2, deposited at the China Center for Type Culture Collection (CCTCC), is classified and named as follows: Bacillus velezensis RH2, accession number CCTCC No: M 2026638, accession date April 10, 2026, accession address Wuhan University, Wuhan, China. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to specific embodiments. Examples 1-4 illustrate the identification, antibacterial activity, and control effect of Bacillus belye RH2 on rice sheath blight; Example 5 illustrates the indoor toxicity of its fermentation product against root-knot nematodes; and Example 6 illustrates the preparation method of the inoculant. The scope of protection of the present invention is not limited to the following embodiments.

[0034] Example 1 Identification of Bacillus belyssus RH2 (1) Isolation and screening of Bacillus belye RH2 Strain strain RH2 was isolated from soil samples collected in Shenyang, Liaoning Province. 1 g of soil sample was weighed and added to 9 mL of sterile physiological saline or sterile water, then thoroughly shaken to mix, preparing a 10...-1 Soil suspension; subsequently diluted 10-fold stepwise to 10. -6 100 μL of the corresponding dilution was spread onto LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, distilled water to 1000 mL, pH adjusted to 7.0) plates and incubated at 30 °C for 48 h. Single colonies with obvious morphological differences were picked and purified to obtain pure culture strain RH2.

[0035] The antagonistic activity of isolated strains against *Rhizoctonia solani* was determined using the plate confrontation method. *Rhizoctonia solani* mycelial cakes were inoculated in the center of PDA plates (200g peeled and diced potato juice, 20g glucose, 17g agar, and distilled water to a final volume of 1000mL). The test strain RH2 was inoculated approximately 2.5cm from the center of the plate. The plates were incubated at 28℃ for 5 days, and the inhibition of pathogen colony growth was observed. Results are shown below. Figure 1 .

[0036] The results showed that strain RH2 had a significant inhibitory effect on Rhizoctonia solani, exhibiting strong antagonistic activity.

[0037] (2) Mycological characteristics of Bacillus belyss RH2 Strain RH2 was inoculated onto LB agar plates and incubated at 30°C for 24 hours. Figure 2 As shown, the colonies are milky white to pale yellow, opaque overall, with a dry and rough surface, and are wrinkled or flocculently raised; the edges of the colonies are irregular, wavy or leaf-like, extending outwards, with a distinctly raised central area and a thicker texture, exhibiting the typical morphological characteristics of Bacillus colonies: rough, wrinkled, and with irregular edges.

[0038] Observe under a microscope, such as Figure 3 As shown, strain RH2 has rod-shaped cells, is Gram-positive, and can form spores.

[0039] (3) Molecular biological identification Genomic DNA was extracted from strain RH2, amplified by PCR using universal primers for bacterial 16S rDNA, and sequenced. The obtained sequences were compared with known sequences in the GenBank database. The results showed that strain RH2 is similar to *Bacillus belye* (…). Bacillus velezensis They have high homology, see Figure 4 Based on the analysis of colony morphology, microscopic morphology, and 16S rDNA sequence, strain RH2 was identified as Bacillus belesiensis.

[0040]

[0041] Example 2 Antibacterial spectrum determination of Bacillus vesiculosus RH2 The inhibitory activity of strain RH2 against various plant pathogenic fungi was determined using the plate confrontation method. Fungal discs of each pathogen were inoculated in the center of PDA plates, and strain RH2 was inoculated at a certain distance from the center of the plate. The plates were incubated at 28℃ for 5–7 days. Plates inoculated only with pathogenic fungi served as controls. When the pathogenic fungal colonies in the control group nearly covered the entire culture dish, the colony diameters of the pathogenic fungi in both the treatment and control groups were measured, and the inhibition rate was calculated.

[0042] The results are shown in Table 1. *Bacillus belye* RH2 exhibited inhibitory effects against all tested plant pathogenic fungi. The strongest inhibitory effect was observed against *Rhizoctonia solani*, with an inhibition rate of 83.0%; the inhibition rate against *Botrytis cinerea* was 80.9%; and the inhibition rates against other tested pathogenic fungi ranged from 66.9% to 76.2%. These results indicate that *Bacillus belye* RH2 has a broad spectrum of inhibition and possesses potential for biocontrol against various plant pathogenic fungi.

[0043] Table 1. Antimicrobial spectrum of Bacillus belye RH2 against common plant pathogenic fungi. Note: The data in the table are the results of three repeated measurements, expressed as mean ± standard deviation.

[0044] Example 3 Inhibitory effect of Bacillus vesiculosus RH2 fermentation broth on Rhizoctonia solani Strain strain RH2 was inoculated into LB liquid medium and cultured at 30℃ and 220 rpm for 48 h with shaking to obtain fermentation broth. The fermentation broth was centrifuged at 5000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain sterile fermentation supernatant.

[0045] The inhibitory effect of RH2 fermentation supernatant on Rhizoctonia solani was determined using the mycelial growth rate method. A certain volume of fermentation supernatant was added to PDA medium cooled to approximately 50°C, mixed thoroughly, and plated. Medium with an equal volume of sterile water was used as a control. After the plates solidified, Rhizoctonia solani mycelial cakes with a diameter of 5 mm were inoculated into the center of the plates and incubated at 28°C for 3 days. The colony diameter was measured, and the inhibition rate was calculated. The results are shown in Table 2.

[0046] The inhibition rate is calculated using the following formula: Inhibition rate (%) = [(Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelium cake diameter)] × 100%.

[0047] As shown in Table 2, using sterile water treatment as a control, the inhibitory effect of RH2 fermentation supernatant on pathogens was evaluated by colony growth inhibition rate. The results showed that as the proportion of RH2 fermentation supernatant increased, the colony diameter of the pathogens gradually decreased, and the inhibition rate gradually increased. When the proportion of RH2 fermentation supernatant was 5%, 10%, 20%, 30%, and 40%, the inhibition rates were 27.4%, 49.4%, 53.6%, 66.2%, and 82.0%, respectively, indicating that RH2 fermentation supernatant had a significant concentration-dependent inhibitory effect on this pathogen.

[0048] Table 2. Inhibitory effect of Bacillus belye RH2 fermentation supernatant on Rhizoctonia solani. Note: The data in the table are the results of three repeated measurements, expressed as mean ± standard deviation.

[0049] Example 4 The control effect of Bacillus vesicularis RH2 on rice sheath blight under pot conditions Rice seedlings of uniform growth were cultured in a greenhouse under greenhouse conditions, using a suspension of Bacillus berberis RH2 (1×10⁻⁶). 8 CFU / mL) and fermentation broth were evenly sprayed onto the leaves and leaf sheaths of rice plants, with sterile water and LB medium used as controls. After 24 hours of treatment, pre-activated Rhizoctonia solani (CFU / mL) was used... Rhizoctonia solani The mycelium was inoculated onto the leaf sheath of rice plants. High humidity was maintained after inoculation to promote disease development. The incidence of rice sheath blight was investigated 7–10 days after inoculation, and the length of lesions was recorded to calculate the control effect. Control effect (%) = [(Control lesion length - Treatment lesion length) / Control lesion length] × 100%.

[0050] The results are shown in Table 3 and Figure 5 LB medium treatment had virtually no control effect on rice sheath blight; both RH2 bacterial suspension and RH2 fermentation broth significantly inhibited lesion expansion. The lesion length treated with RH2 bacterial suspension was 12.9 mm, with a control effect of 73.5%; the lesion length treated with RH2 fermentation broth was 10.8 mm, with a control effect of 77.8%. These results indicate that the RH2 strain and its fermentation products have good control effects on rice sheath blight.

[0051] Table 3. Control efficacy of Bacillus belye RH2 against rice sheath blight Note: The data in the table are the results of three repeated measurements, expressed as mean ± standard deviation.

[0052] Example 5 Assay of the toxic activity of Bacillus vesiculosus RH2 fermentation products against second-instar larvae of root-knot nematodes (1) Preparation of bacterial suspension, fermentation supernatant or sterile fermentation filtrate Bacillus brevis RH2 was inoculated into LB liquid medium and cultured with shaking at 30℃ and 180 rpm for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 5000 rpm for 5 min to collect the supernatant. The supernatant was then filtered through a 0.22 μm microporous membrane to remove bacteria and obtain sterile fermentation filtrate for use.

[0053] (2) Acquisition and treatment of root-knot nematodes Root-knot nematode egg sacs were collected from the roots of diseased plants and placed in an incubation apparatus (200-mesh sieve + 9cm glass petri dish) containing sterile water (with 1 / 1000 gentamicin added). After incubation at 25°C in the dark for 5 days, second-instar larvae (J2) were collected. The collected nematodes were washed three times with sterile water to remove impurities, and then surface-sterilized with 1% sodium hypochlorite solution for 1 minute. The sterilization solution was then removed by centrifugation, and the nematodes were repeatedly washed with sterile water at least five times until no chlorine odor remained. Finally, the nematodes were resuspended in sterile water, adjusted to a concentration of approximately 50 nematodes / 100μL, and examined under a microscope to confirm good viability before being used in experiments.

[0054] (3) Indoor toxicity assay A 24-well plate was used, with 100 μL of the test sample (RH2 fermentation broth, fermentation supernatant, or sterile fermentation filtrate) and 100 μL of nematode suspension (approximately 50 second-instar root-knot nematode larvae) added to each well, bringing the final volume of the system to 200 μL. A blank control (sterile water) and a culture medium control (sterile LB medium diluted by the same factor) were included, with at least three replicates for each treatment. After sample addition, the plates were incubated in the dark. At 6 h and 24 h post-treatment, the total number of nematodes and the number of dead nematodes were counted under a stereomicroscope, and the mortality rate and corrected mortality rate were calculated. If the corrected mortality rate in the treatment group was significantly higher than that in the control group, the test sample was considered to have toxic activity. The criterion for experimental validity was: the mortality rate in the control group should be less than 10%; if it exceeded 20%, the data for that batch was invalid and the experiment needed to be repeated.

[0055] The experimental results are shown in Table 4. After 6 hours of treatment, the nematode mortality rates in the RH2 fermentation broth, fermentation supernatant, and sterile fermentation filtrate treatment groups were 98.33%, 98.67%, and 98.00%, respectively, with corrected mortality rates all exceeding 97%. Furthermore, the mortality rates in the blank control and culture medium control were both below 10%. These results indicate that the fermentation products of *Bacillus belye* RH2 possess rapid and strong indoor toxicity against second-instar larvae of root-knot nematodes.

[0056] Table 4. Toxicity of strain RH2 against nematodes Note: Data are expressed as mean ± standard error. Different lowercase letters indicate significant differences at the 0.05 level.

[0057] Example 6 Preparation of Bacillus belyss RH2 microbial preparation A single colony of strain RH2 was inoculated into LB liquid medium and cultured with shaking at 30℃ and 220 r / min for 8 h to obtain seed culture; the seed culture was then inoculated into fermentation medium and cultured with shaking at 30℃ and 220 r / min for 48 h to obtain fermentation broth.

[0058] Bacillus vesiculosus RH2 can be used alone or in combination with agriculturally permitted nutrient adjuvants, organic fertilizer substrates, humic acid, amino acids, seaweed extracts, or other beneficial microorganisms. Before combination, indoor compatibility tests should be conducted to determine compatibility stability, and efficacy tests for rice sheath blight control and root-knot nematode control should be carried out separately according to the target application scenario.

[0059] The formulations of Bacillus vesiculosus RH2 include, but are not limited to, aqueous solutions, suspensions, wettable powders, water-dispersible granules, granules, seed treatment agents, bio-organic fertilizers, and compound microbial fertilizers. When used for the control of rice sheath blight, it can be applied during seedling raising, transplanting, field growth, or the early stages of disease occurrence. When used for the control of root-knot nematodes, it can be applied to the plant roots or rhizosphere soil through methods such as root irrigation, root dipping, or soil treatment. The fermentation broth can also be processed into different formulations through centrifugation, filtration, concentration, adsorption, spray drying, or freeze drying, and agriculturally acceptable adjuvants such as dispersants, wetting agents, protectants, or carriers can be added as needed.

[0060] In summary, the *Bacillus vesiculosus* RH2 strain provided by this invention possesses two outstanding biocontrol activities: firstly, this strain exhibits significant antagonistic activity against *Rhizoctonia solani*, and its bacterial suspension and fermentation broth can significantly reduce the extent of rice sheath blight lesion expansion under pot conditions; secondly, the fermentation broth, fermentation supernatant, and sterile fermentation filtrate of this strain demonstrate rapid and strong indoor toxicity against second-instar larvae of root-knot nematodes. Therefore, *Bacillus vesiculosus* RH2 can serve as a microbial resource for the green control of both rice sheath blight and root-knot nematodes. The *Bacillus vesiculosus* RH2 strain provided by this invention can also serve as a new strain resource for developing multifunctional microbial biocontrol agents.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Modifications, substitutions, or equivalent transformations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit and substance of the present invention should all fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus velezensis RH2, characterized by: The Bacillus velezensis RH2 strain is deposited at the China Center for Type Culture Collection (CCTCC), classified as Bacillus velezensis RH2, with accession number CCTCC No: M 2026638, deposit date of April 10, 2026, and deposit address at Wuhan University, Wuhan, China.

2. The application of Bacillus berleis RH2 as described in claim 1 in the control of rice sheath blight and / or root-knot nematodes.

3. The application according to claim 2, characterized in that: The rice sheath blight is caused by Rhizoctonia solani. Rhizoctonia solani cause.

4. The application according to claim 2, characterized in that: The root-knot nematode is a second-instar larva of the root-knot nematode.

5. The application according to claim 2, characterized in that: The application of Bacillus vesiculosus RH2 in the preparation of microbial agents for the control of rice sheath blight and / or microbial preparations for the control of root-knot nematodes.

6. A microbial preparation for controlling rice sheath blight, characterized in that: The active ingredient of the microbial preparation is one or more of the following: Bacillus brevis RH2 cells, spores, fermentation broth, fermentation supernatant, or sterile fermentation filtrate as described in claim 1.

7. A microbial preparation for controlling root-knot nematodes, characterized in that: The active ingredient of the microbial preparation is one or more of the following: Bacillus brevis RH2 cells, spores, fermentation broth, fermentation supernatant, or sterile fermentation filtrate as described in claim 1.

8. A method for controlling rice sheath blight, characterized in that: Apply the Bacillus oryzae RH2 of claim 1 or the microbial preparation for controlling rice sheath blight of claim 6 to rice plants or their growth environment.

9. The method according to claim 8, characterized in that: Application methods include seed soaking, seed mixing, root dipping, root irrigation, foliar spraying, stem base spraying, soil application, or application with irrigation water.

10. A method for controlling root-knot nematodes, characterized in that: Apply the Bacillus berleis RH2 of claim 1 or the microbial preparation for controlling root-knot nematodes of claim 7 to the roots of plants or rhizosphere soil.

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