Bacillus velezensis ld4111 and application thereof in preventing and treating phyllosticta cryptotaeniae leaf spot disease

Abstract

The application provides bacillus velezensis Ld4111 and application thereof in prevention and treatment of phyllosticta viticola leaf spot, and belongs to the technical field of biological control. The bacillus velezensis strain Ld4111 is first obtained by separation from grape rhizosphere soil, and the strain has been preserved in China Center for Type Culture Collection (CCTCC) on April 21, 2025, with a preservation number of CCTCC M 2025833. The strain can be used as a biological pesticide or a microbial inoculant to prevent and treat phyllosticta viticola leaf spot. By using a plate confrontation culture method, the mycelium growth inhibition rate of the bacillus velezensis Ld4111 strain on phyllosticta viticola is 81.43%. By using a spraying method, the prevention and treatment effects of 100 billion / gram bacillus velezensis Ld4111 inoculant 100 times dilution liquid on phyllosticta viticola leaf spot are 83.46% and 73.85%, respectively.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to Bacillus belye Ld4111 and its application in controlling leaf spot disease of berries. Background Technology

[0002] genus *Vaccaria* ( Cayratia This plant is distributed throughout most parts of my country, adapting to different types of terrain, with a suitable altitude of 300-2500 meters. It is divided into two types: woody vines and herbaceous vines, the main one being *Vaccaria segetalis* (*Vaccaria spp.*). C. japonica ), Three-leafed Blackberry ( C. trifolia ) and Huazhong blackberry ( C. oligocarpa It has a long history of medicinal use. Among its many uses, *Caulis Caulis Persicae* (also known as *Caulis Caulis Persicae*) is the most common, with the whole plant used medicinally. *Caulis Caulis Persicae* is cold in nature and bitter in taste, and enters the heart, liver, and stomach meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, reducing swelling and relieving pain. The stems and leaves are mainly used to treat carbuncles, sore throat, rheumatoid arthritis, damp-heat jaundice, and snake and insect bites. It is a widely used medicinal plant in my country. In surgical clinical practice, it has good effects in treating scabies, mumps, erysipelas, and herpes zoster. *Caulis Caulis Persicae* can be processed into Chinese herbal milk tea, facial masks, and health products, and can also be developed into high-end health products using flavonoids and terpenes as raw materials. In 2025, the international market demand for raw materials for traditional Chinese medicine surged, and as a specialty medicinal material, the export volume of *Caulis Caulis Persicae* increased by 12% year-on-year. Driven by national policies on standardized planting of Chinese medicinal herbs and integration of the industrial chain, the scale of the Chinese purslane industry is expected to exceed one trillion yuan from 2025 to 2030. Therefore, it is very important to prevent and control diseases and pests of Chinese purslane and increase its yield.

[0003] Research on diseases and pests of *Strombus haematomarginatus* is limited. The only reported disease, both domestically and internationally, is leaf spot, a newly discovered foliar disease affecting *Strombus haematomarginatus* in recent years. Leaf spot is divided into bacterial and fungal types. Regarding bacterial leaf spot, Su Qian et al. isolated the pathogenic strain SF01 from diseased *Strombus haematomarginatus* plants in Jinhua, Zhejiang. Morphological observation and molecular biological identification confirmed it to be *Enterobacter* (…). Enterobacter mori (Su Qian, Fan Shuyu, Chen Zi, et al. Isolation and identification of the pathogen causing bacterial leaf spot disease of *Strombus haematomarginatus* [J]. Acta Phytopathologica Sinica, first published online: 2025-11-26). Regarding fungal leaf spot diseases, the inventors isolated and identified *Anthracnose siamensis*, the pathogen causing fungal leaf spot disease of *Strombus haematomarginatus*, from diseased leaves. Colletotrichum siamense(Qing-Hai Wang, Ming Xu, Yu-Kun Qi, et al. Morphological and molecular identification of Colletotrichum siamense associated with Cayratia japonicaanthracnose in China[J]. Crop Protection 174 (2023) 106385). Fungal leaf spot disease causes a disease rate of over 30%, affecting its application value. Initially, small dark brown spots appear on the leaves, which then expand into circular or nearly circular dark brown lesions with yellow halos around the edges. The central necrotic area is sunken and the edges are dark brown. Currently, there are no reports on the control of leaf spot disease in *Colletotrichum japonica*. To reduce pesticide residues, the use of chemical pesticides is greatly restricted in the control of leaf spot disease in *Colletotrichum japonica*. Therefore, the development of efficient and green control technologies for leaf spot disease is of great significance to the development of the *Colletotrichum japonica* industry.

[0004] Microbial biopesticides have become the primary choice for biopesticide registration due to their rapid reproduction, long-lasting control effects, multiple development and utilization pathways, low likelihood of resistance development, simple fermentation processes, and low production costs. Currently, the most common pathogens of fungal leaf spot diseases include those of the genus *Cercospora* (…). Cercospora Alternaria ( ) Alternaria ), genus *Syngonium* Septoria ), Deschoensis ( Drechslera ) and Helicobacter genus ( Helminthosporium Furthermore, the target crop is not *Brassica juncea*, but the pathogen of the fungal leaf spot disease of *Brassica juncea* in this invention is *Anthracnose sicca*. Colletotrichum siamense) Due to differences in crops, pathogens, and application environments, current biological control methods for leaf spot diseases in other crops are not applicable to leaf spot disease in berries.

[0005] Bacillus belesiensis ( Bacillus velezensis (This is a new species of Bacillus, and its metabolites have broad-spectrum antibacterial activity. It is the most important biocontrol bacterial resource and the main biocontrol bacteria currently registered as biological pesticides. Currently, there are 6 strains registered as pesticides in China, of which 4 are used as fungicides, including...) B. velezensis CGMCC No. 14384 B. velezensis M173 B.velezensis MBI600 and B.velezensis BJ-1 is mainly used to control tobacco powdery mildew, red spot disease, and black shank disease; tea anthracnose; cucumber powdery mildew; tomato bacterial wilt; grape gray mold; and tomato gray mold. One nematicide plant is also included. B.velezensis MBI600 (for control of wheat cyst nematode and cucumber root-knot nematode); 1 acaricideB.velezensis LM-W2 (Citrus Red Spider Mite). Although the registered strains are all *Bacillus belyssioides*, their target pests and diseases differ significantly. Furthermore, *Bacillus belyssioides* is registered for use on annual herbaceous plants such as tobacco, cucumber, tomato, and wheat, as well as perennial woody plants like tea trees and citrus and perennial vines like grapes. Currently, there are no reports on the use of *Bacillus belyssioides* for the control of leaf spot disease in *Strombus haematomyces*. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides Bacillus belye Ld4111 and its application in controlling leaf spot disease of berry. This invention first isolates Bacillus belye strain Ld4111 from grape rhizosphere soil. This strain can be used as a biological pesticide or microbial agent to control leaf spot disease of berry, showing promising application prospects.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: The *Bacillus belye* provided by this invention (… Bacillus velezensis Strain Ld4111 was isolated from the rhizosphere soil of grapevines in Yantai City, Shandong Province, and was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC M 2025833. It exhibits the following microbiological characteristics: colonies are milky white, irregularly shaped, opaque, with irregular and uneven edges, a raised center, and a dull surface. This strain is typically preserved using NA medium, and for long-term storage, 20% glycerol is used.

[0008] The present invention also provides a fermentation broth or biological agent with Bacillus vesiculosus Ld4111 as the active ingredient.

[0009] The preparation method of the above fermentation broth is as follows: 1) Propagation and culture of the strain: Pick a small number of colonies of Bacillus belye Ld4111 from the test tube slant, transfer them to a basal medium plate, and incubate at 28-30℃ for 1.5-2.5 days; 2) Preparation of seed culture of strain: Transfer the propagated Bacillus belye Ld4111 to liquid basal medium and culture in a shaker at 28-30℃ for 12-14 h; 3) Fermentation culture: The seed culture obtained by shaking is added to the fermentation medium to obtain the fermentation broth. The culture temperature is 28-32℃, the initial pH is 7.0-7.5, the inoculum amount is 5-10%, the culture time is 12-36h, and the viable cell count can reach 300-500 billion / g.

[0010] Preparation method of biological agent: Add water-soluble carrier industrial glucose to fermentation broth, stir evenly, and then spray dry to obtain bacterial powder. Then add water-soluble carrier industrial glucose to bacterial powder, stir thoroughly, and finally obtain biological agent with a live bacteria count of 80-120 billion / g.

[0011] Basic culture medium (solid): 3g beef extract, 5g peptone, 5g sodium chloride, 15g agar, 1000mL water, pH 7.3, prepared according to the above proportions.

[0012] Liquid basal culture medium: 3g beef extract, 5g peptone, 5g sodium chloride, 1000mL water, pH 7.3, prepared according to the above proportions.

[0013] Fermentation medium (weight percentage): soybean meal powder 3.0-3.5%, corn starch 2.0-2.5%, glucose 0.4-0.6%, sodium chloride 0.1-0.2%, manganese sulfate 0.01-0.03%, the remainder being water, pH 7.0-7.5. Preferably: soybean meal powder 3.15%, corn starch 2.15%, glucose 0.5%, sodium chloride 0.15%, manganese sulfate 0.02%, the remainder being water, pH 7.0-7.5.

[0014] The present invention also provides the application of Bacillus berberis strain Ld4111, its fermentation broth or biological agent in the prevention and control of leaf spot disease in Chinese purslane.

[0015] This invention also provides a method for controlling leaf spot disease in *Bacillus vesca* Ld4111 fermentation broth or a biological agent. The method includes applying the fermentation broth of *Bacillus vesca* Ld4111 or the biological agent to *Bacillus vesca* plants infected with leaf spot disease. The agent is applied by spraying after dilution.

[0016] The technical effects of this invention are: 1. Currently, there are no reports on the biological control of leaf spot disease in *Strombus haematomarginatus*. This invention utilizes *Bacillus belye* Ld4111 for the control of leaf spot disease in *Strombus haematomarginatus*, which is of great significance for the prevention and control of this disease.

[0017] 2. Using the plate confrontation culture method, the mycelial growth inhibition rate of *Bacillus vesca* Ld4111 strain against *Bacillus vesca* leaf spot pathogen reached 81.43%. Using the spray method, compared with the control, a 100-fold dilution of 10 billion CFU / g *Bacillus vesca* Ld4111 inoculum showed preventive and curative effects of 83.46% and 73.85% on *Bacillus vesca* leaf spot, respectively, with no significant difference compared to the control agent, a 1000-fold dilution of 40% prochloraz emulsion.

[0018] 3. Because Bacillus vesicularis Ld4111 inoculant leaves no pesticide residues and has no toxic side effects, it can reduce the use of chemical pesticides. Taking all factors into consideration, Bacillus vesicularis Ld4111 strain has significant application potential in the biological control of leaf spot disease in Chinese tallow tree strawberries. Attached Figure Description

[0019] Figure 1 This is a single colony morphology diagram of Bacillus belyss Ld4111 strain; Figure 2 The images show the indoor inhibition effect of Bacillus belyss Ld4111, with the left image representing the control group and the right image representing the treatment group. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0021] Example 1: Isolation and Identification of Bacillus belyss Ld4111 strain 1. Separation: The Bacillus berles of this invention was isolated from the rhizosphere soil of grapevines in Yantai City, Shandong Province, using a soil dilution separation method. The isolation method was as follows: (1) Soil sampling: After cleaning the topsoil, take 200g of soil sample from 0-20cm around the root zone, sieve it initially, and take 10g for separation; (2) Weigh 10 g, add 90 mL of sterile water, add to an Erlenmeyer flask, and place on a shaker at 28℃ and 180 r·min. -1 Shake for 30 minutes, remove the conical flask and let it stand for about 30 minutes to dilute to 10. -6 ~10 -9 g·mL -1 Soil suspension; (3) Take concentrations of 10 -6 10 -7 10 -8 10 -9 g·mL -1 100 μL of soil suspension was dropped onto a PDA medium plate, spread with a glass rod, and incubated at a constant temperature of 28–30 °C for 2–3 days. (4) Pick single colonies with different morphologies with an inoculation needle, purify them by streak plate method, and incubate them in an incubator at 28-30℃. At the same time, number the different isolated strains and transfer them to slant culture medium for later use. (5) Using the leaf spot pathogen of *Cymbidium goeringii* as a target, a strain with strong inhibitory activity against the leaf spot pathogen of *Cymbidium goeringii* was screened by plate confrontation method. It was derived from soil sample 4.046T01, numbered 4-1-1, and renumbered Ld4111 during preservation.

[0022] The PDA culture medium formula is as follows: 200g potato (peeled and diced), 20g glucose, 14g agar, and 1000mL distilled water.

[0023] 2. Strain identification (1) Microbiological characteristics: see Figure 1 The Ld4111 colonies are milky white, irregular in shape, opaque, with irregular and uneven edges, a raised center, and a dull surface.

[0024] (2) Molecular biological characteristics: The 16S rRNA gene sequence determination results are shown in SEQ No. 1; gyrA The gene sequencing results are shown in SEQ No. 2.

[0025] The 16S rRNA amplification sequence of strain Ld4111 and one housekeeping gene ( gyrA The sequence was compared with the NCBI database, and the 16S rRNA sequence was found to be... B. velezensis GL9 has a homology of 99.41%. gyrA Gene sequence and B. velezensis The homology with NJAU-Z9 was 100%. Based on the morphological characteristics of strain Ld4111, strain Ld4111 was identified as belonging to *Bacillus belyes* (…). Bacillus velezensis ).

[0026] This strain was deposited on April 21, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC M 2025833.

[0027] Example 2: Indoor efficacy verification of Bacillus vesicles Ld4111 against the leaf spot pathogen of Ulva lactuca. 1. Method The plate confrontation culture method was used to culture the tested *Strombus haematomarginatus* leaf spot pathogen (… Colletotrichum siamense 9mm mycelial discs were transferred to the center of the plate, and Ld4111 strain was symmetrically inoculated 2 cm away from the discs. The plates were then incubated at 28℃ in a biochemical incubator. Each treatment was repeated three times, with a control group consisting of only 9mm mycelial discs inoculated in the center of the plate. When the mycelium in the control group had fully colonized the plate, the colony diameter was measured using the cross-crossing method, and the relative inhibition rate was calculated.

[0028] 2. Results Depend on Figure 2 It can be seen that Bacillus vesiculosus Ld4111 has a strong inhibitory effect on the mycelial growth of the leaf spot pathogen of Ulmus pumila, with a calculated mycelial growth inhibition rate of 81.43%.

[0029] Example 3: Preparation of Bacillus belye Ld4111 inoculum Basic culture medium: 3g beef extract, 5g peptone, 5g sodium chloride, 15g agar, 1000mL water, pH 7.3.

[0030] Liquid basal culture medium: 3g beef extract, 5g peptone, 5g sodium chloride, 1000mL water, pH 7.3.

[0031] Fermentation medium (weight percentage): soybean meal powder 3.15%, corn starch 2.15%, glucose 0.5%, sodium chloride 0.15%, manganese sulfate 0.02%, the remainder is water, pH 7.0-7.5.

[0032] Preparation method of microbial agent: 1) Propagation and culture of the strain: Pick a small number of colonies of Bacillus belye Ld4111 from the test tube slant, transfer them to a basal medium plate, and incubate at 28°C for 2 days; 2) Preparation of seed culture of strain: The propagated Bacillus belye Ld4111 was transferred to liquid basal medium and cultured in a shaker at 30°C for 12-14 h; 3) Fermentation culture: The seed culture from the shaker culture is added to the fermentation medium for culture. The culture temperature is 32℃, the initial pH is 7.0-7.5, the liquid volume is 70%, the inoculum volume is 10%, the rotation speed is 200 r / min, and the culture time is 24h. The viable cell count can reach 300-500 billion / g. 4) Add 10% of water-soluble carrier industrial glucose to the mixing tank, stir evenly, and then spray dry to obtain bacterial powder. Then add water-soluble carrier industrial glucose to the bacterial powder and stir thoroughly for 30 minutes to make the viable bacterial count reach 10 billion / g.

[0033] Example 4: Verification of the effect of Bacillus belyss Ld4111 inoculant This embodiment provides relevant experiments on the application of 10 billion / g Bacillus berberis Ld4111 inoculum against leaf spot disease of Ulva arborescens.

[0034] 1. Method 1.1 Test reagent: Bacillus belyssus Ld4111 bacterial agent, viable count 10 billion / g.

[0035] 1.2 Test crops and target pests: The test crop was *Strombyx mori* (potted seedlings); the target disease to be controlled was leaf spot.

[0036] 1.3 Methods 1.3.1 Determination of preventive effect Using a manual sprayer (E532), Bacillus berberis Ld4111 inoculum was sprayed at a 100-fold dilution onto the surface of *Bacillus berberis* leaves to form a uniform film. Water was used as a control, and 40% prochloraz emulsion was used as a control, diluted 1000 times. 24 hours after treatment, the leaves were lightly pricked with a sterilized inoculation needle to create micro-wounds. 100 μL of a suspension of *Bacillus berberis* spores (10 μL / 1000 μL) was then injected. 6 (Spores / mL) were dripped onto the wound, and two leaves were treated per plant. One inoculation site was placed on each leaf. The inoculated *Strombax ceiba* plants were placed in a greenhouse (25 ℃; L:D = 16h:8h; relative humidity above 45%) for cultivation and disease observation. The disease index and effect were calculated. The experiment was repeated four times.

[0037] 1.3.2 Determination of therapeutic effect The leaves of *Strombax cuneata* used in the experiment were first inoculated with a suspension of *Strombax cuneata* leaf spot pathogen spores (10) according to the above method. 6 Spores / mL), and after 24 h, spray the same concentration of the drug solution (100-fold dilution of Bacillus vesiculosus Ld4111 inoculant) as described above. The control leaves were sprayed with water. 40% prochloraz emulsion was used as the control agent, diluted 1000 times. Two leaves were treated per plant, and one inoculation was performed on each leaf. The experimental *Strombax ceiba* plants were placed in a greenhouse (25 ℃; L:D = 16h:8h; relative humidity above 45%) for cultivation and disease observation. The disease index and effect were calculated. The experiment was repeated 4 times.

[0038] 1.4 Data Statistics The grading criteria for leaf spot disease in *Strombax cuneata* are shown in Table 1.

[0039] Table 1 Grading Criteria for Leaf Spot Disease in Ulmus pumila

[0040] Incidence rate (%) = (Number of diseased leaves / Total number of surveyed leaves) × 100; Disease index = 100 × ∑[number of diseased leaves at each level × relative grade value] / [total number of leaves surveyed × highest grade value]; Prevention and control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0041] 2. Results Table 2 shows that a 100-fold dilution of 10 billion / g *Bacillus vesalis* Ld4111 inoculum has a good preventive effect against leaf spot disease of *Strombax ceiba pentaphyllum*, reaching 83.46%. The preventive effect of a 1000-fold dilution of the control agent, 40% prochloraz emulsion, was 85.71%. In terms of treatment, the 100-fold dilution of *Bacillus vesalis* Ld4111 inoculum has a therapeutic effect of 73.85%, while the 1000-fold dilution of the control agent has a therapeutic effect of 79.23%, slightly lower than the preventive effect.

[0042] Table 2. Effects of 10 billion CFU / g Bacillus berberis Ld4111 inoculum on leaf spot disease of Ulmus pumila.

[0043] 3. Summary A 100-fold dilution of 10 billion / g Bacillus berberis Ld4111 inoculum has good preventive and curative effects against leaf spot disease of Ulmus pumila, with both reaching over 70%, which is comparable to the control agent. It can partially replace chemical pesticides and reduce the amount of chemical pesticides used.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis Ld4111, the preservation number of the strain is CCTCC M 2025833.

2. A fermentation broth or biological agent with Bacillus berreatus Ld4111 as the active ingredient as described in claim 1.

3. The method for preparing fermentation broth as described in claim 2, characterized in that, 1) Propagation and culture of the strain: Pick a small number of colonies of Bacillus belye Ld4111 from the test tube slant, transfer them to a basal medium plate, and incubate at 28-30℃ for 1.5-2.5 days; 2) Preparation of seed culture of strain: Transfer the propagated Bacillus belye Ld4111 to liquid basal medium and culture in a shaker at 28-30℃ for 12-14 h; 3) Fermentation culture: The seed culture obtained by shaking is added to the fermentation medium to obtain the fermentation broth. The culture temperature is 28-32℃, the initial pH is 7.0-7.5, the inoculum amount is 5-10%, and the culture time is 12-36h.

4. The method for preparing the fermentation broth as described in claim 3, characterized in that, The fermentation medium, by weight percentage, consists of: 3.0-3.5% soybean meal, 2.0-2.5% corn starch, 0.4-0.6% glucose, 0.1-0.2% sodium chloride, 0.01-0.03% manganese sulfate, with the remainder being water, and a pH of 7.0-7.

5.

5. The method for preparing fermentation broth as described in claim 3 or 4, characterized in that, The number of viable bacteria in the fermentation broth is 300-500 billion / g.

6. The method for preparing the biological agent as described in claim 5, characterized in that, Add glucose to the fermentation broth, stir well, and then spray dry to obtain bacterial powder. Then add glucose to the bacterial powder and stir thoroughly to obtain a biological agent, so that the number of live bacteria reaches 80-120 billion / g.

7. The use of the Bacillus berberis strain Ld4111 as described in claim 1, or the fermentation broth or biological agent as described in claim 2, in the prevention and control of leaf spot disease in *Strombocybe pungens*.

8. A method for controlling leaf spot disease of *Bacillus berberis* Ld4111 fermentation broth or biological agent as described in claim 2, characterized in that, The method includes applying a fermentation broth of Bacillus vesicularis Ld4111 or a biological agent to berries with leaf spot disease.

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