Bacillus velezensis s-34 and application thereof in preventing and treating mango leaf anthracnose

Abstract

This invention belongs to the field of agricultural microbiology technology and relates to a strain of Bacillus velezensis S-34 and its application in controlling mango leaf anthracnose. It was isolated from the rhizosphere soil of the anthracnose-resistant mango variety 'Jinhuang' and deposited at the China Center for Type Culture Collection (CCTCC) on May 29, 2025, with accession number CCTCC No. M 20251225. The Bacillus velezensis S-34 provided by this invention not only controls mango leaf anthracnose but also exhibits good antagonistic effects against various mango leaf pathogenic fungi. Furthermore, Bacillus velezensis S-34, its sterile fermentation broth, and volatile gases can inhibit the germination and epidermal penetration of Anthracnose spores, thereby inhibiting the infection process of Anthracnose spores on plants. This invention provides a new biocontrol agent and preparation method for the efficient control of mango leaf anthracnose and other foliar diseases in agricultural production, which is beneficial to the green and healthy development of the mango industry.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology and relates to a strain of Bacillus velezensis S-34 and its application in the control of anthracnose on mango leaves. Background Technology

[0002] Mangoes are an important tropical fruit with immense economic value, but they suffer significant losses due to diseases during growth, development, and post-harvest transportation. Statistics show that there are as many as 88 types of mango diseases in my country, with anthracnose being the most prevalent. Currently, chemical control methods are widely used in agricultural production to combat mango diseases, primarily employing broad-spectrum fungicides such as carbendazim, benomyl, and thiophanate-methyl. However, the extensive use of chemical fungicides has not only led to drug resistance in mango pathogens but has also caused widespread environmental pollution and food safety issues. Biological control refers to the method of using one organism to inhibit, reduce, or eliminate another harmful organism. Biocontrol bacteria are beneficial microorganisms that can reduce or control the impact of pests and diseases, and their application is a common biological control method for plant diseases. Using biocontrol bacteria for biological control of plant diseases has many advantages: it not only has good application effects but also has high safety for humans and the environment. Long-term use does not lead to pathogen resistance, making it a viable alternative to chemical pesticides.

[0003] For mango diseases such as anthracnose, some studies have screened biocontrol bacteria, using methods such as streak dilution to obtain biocontrol bacteria or fungi with certain biocontrol effects from different sources, including *Streptomyces malaysiensis* HSL-9B strain from mangroves, marine bacteria *Stenotrophomonas rhizophila*, and scallop symbiotic fungus *Trichoderma asperellum* KUFA 0042 strain. However, the isolation methods used in these studies for mango fungal disease biocontrol bacteria are complex and time-consuming, resulting in low screening efficiency and long cycles. Furthermore, the screened biocontrol bacteria often come from growing environments far from mangoes, and their application efficacy, impact on mango growth and development, and biosafety cannot be guaranteed. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the prior art by providing a strain of Bacillus velezensis S-34 and its application in biological control, which can control anthracnose disease on mango leaves.

[0005] To achieve the above objectives, the technical solution adopted in this invention is as follows: a strain of Bacillus velezensis, Bacillus velezensis S-34, was isolated from the rhizosphere soil of the anthracnose-resistant mango variety 'Jinhuang', and was deposited at the China Center for Type Culture Collection on May 29, 2025, with accession number CCTCC M No. 20251225.

[0006] The present invention also provides a microbial preparation of Bacillus belyssus S-34.

[0007] The present invention also provides a sterile fermentation broth prepared from Bacillus belyssus S-34.

[0008] This invention also provides a method for preparing sterile fermentation broth of Bacillus belyssus S-34, the steps of which include:

[0009] (1) Streak the Bacillus belye S-34 as described in claim 1 on NA solid medium, pick a single colony and inoculate it into 10 mL NB liquid medium, and incubate at 30 °C for 24 h;

[0010] (2) Inoculate 1 mL of the bacterial culture obtained above into 100 mL of NB liquid medium and incubate at 30 °C for 48 h;

[0011] (3) Centrifuge the bacterial culture obtained above at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain sterile fermentation broth.

[0012] The present invention also provides the application of Bacillus vesicularis S-34 or / and Bacillus vesicularis S-34 microbial preparation or / and Bacillus vesicularis S-34 as a sterile fermentation broth for the prevention and control of plant pathogens.

[0013] Furthermore, the plant pathogens mentioned are used in the application of *Colletotrichum siamense*, *Colletotrichum fructicola*, *Fusarium incarnatum*, and *Fusarium proliferatum*.

[0014] Furthermore, the plant pathogen is mango leaf anthracnose.

[0015] Furthermore, the volatile gas of Bacillus vesiculosus S-34 described in this invention can also be used to control plant pathogens.

[0016] The beneficial technical effects of this invention are:

[0017] The *Bacillus vesiculosus* S-34 provided by this invention can effectively control mango leaf anthracnose, with a relative inhibition rate of 95.5%. This strain also exhibits good inhibitory effects against various other mango leaf pathogens. Furthermore, *Bacillus vesiculosus* S-34, its sterile fermentation broth, and volatile gases can inhibit the germination and epidermal penetration of *Anthracnose sicca* spores, thereby inhibiting the infection process of *Anthracnose sicca* on plants. Compared with existing chemical control methods, the biocontrol agent provided by this invention has advantages in controlling mango leaf anthracnose, including being environmentally friendly and pollution-free, the pathogen being less likely to develop resistance, a clearly identified strain source, high biosafety, good control effect, and a wide range of action. It has good application prospects and can serve as a novel biological pesticide to replace traditional chemical pesticides. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A schematic diagram of the screening process for biocontrol bacteria against mango anthracnose.

[0020] Figure 2 This study demonstrates the antagonistic effect of *Bacillus belyssus* S-34 on mango leaf pathogens. A and B show colony photos and colony areas of *Anthracis siana* and *Bacillus belyssus* S-34 after 5 days of co-culturing on PDA-LA medium. Scale bar: 2 cm. RI: Relative inhibition rate. C and D show colony photos and colony areas of *Anthracis citrinum* and *Bacillus belyssus* S-34 after 7 days of co-culturing on PDA-LA medium. Scale bar: 2 cm. E and F show colony photos and colony areas of *Fusarium oxysporum* and *Bacillus belyssus* S-34 after 7 days of co-culturing on PDA-LA medium. Scale bar: 2 cm. G and H show colony photos and colony areas of *Fusarium oxysporum* and *Bacillus belyssus* S-34 after 7 days of co-culturing on PDA-LA medium. Scale bar: 2 cm. All data shown in the figures are mean ± SEM. Different lowercase letters in the data indicate significant differences between the data (P < 0.05, Student's t-test).

[0021] Figure 3Images of *Bacillus belyssus* S-34 colonies and its phylogenetic tree. A shows an image of *Bacillus belyssus* S-34 colonies grown on NA medium for 24 h. Scale bar: 2 cm. B shows a multi-gene phylogenetic tree constructed based on 16S rDNA and the ropB gene (Bootstrap values ​​= 1000).

[0022] Figure 4 This figure shows the control effect of Bacillus belyceae S-34 on anthracnose on mango leaves. A shows mango leaves 7 days after simultaneous inoculation with Bacillus belyceae S-34 and Anthracnose sicca. Scale bar: 2 cm. B shows the area of ​​lesions on mango leaves 7 days later. Data shown in the figure are mean ± SEM values. Different lowercase letters in the data indicate significant differences between data (P < 0.05, Student's t-test).

[0023] Figure 5 This study investigated the inhibitory effects of *Bacillus vesiculosus* S-34 and its sterile fermentation broth on the germination and epidermal penetration of *Anthracis chinensis* spores. A shows the inhibitory effect of the sterile fermentation broth of *Bacillus vesiculosus* S-34 on *Anthracis chinensis*. Scale bar: 2 cm. S-34-SECs: sterile fermentation broth of strain S-34. B shows the germination rate of *Anthracis chinensis* spores after 7 h of inoculation into the inner epidermis of onions. C shows the relative inhibition rate of *Bacillus vesiculosus* S-34 and its sterile fermentation broth on the germination of *Anthracis chinensis* spores. D shows the effect of *Bacillus vesiculosus* S-34 and its sterile fermentation broth on the rate of *Anthracis chinensis* penetration into the plant epidermis. All data shown in the figures are mean ± SEM. Different lowercase letters in the data indicate significant differences between data (P < 0.05, Tukey's test in one-way ANOVA or Student's t-test).

[0024] Figure 6 This study investigates the inhibitory effect of volatile gases from *Bacillus belyssus* S-34 on the germination and epidermal penetration of *Anthracis chinensis* spores. A and B show colony images and colony areas of *Anthracis chinensis* spores after 5 days of treatment with *Bacillus belyssus* S-34 volatile gases, respectively. Scale bar: 2 cm. S-34-VOCs: Volatile gases from strain S-34. C shows the germination rate of *Anthracis chinensis* spores on the inner epidermis of onions after 7 hours of treatment with *Bacillus belyssus* S-34 volatile gases. D shows the effect of volatile gases from *Bacillus belyssus* S-34 on the rate of *Anthracis chinensis* penetration into the plant epidermis. All data in the figures are mean ± SEM, and different lowercase letters indicate significant differences between data points (P < 0.05, Student's t-test). Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Experimental reagents: All chemical reagents used in this study were purchased from Sinopharm Chemical Reagent Co., Ltd.; 2×PhantaMax Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; primer synthesis and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0027] Experimental instruments: Autoclave (Systec, Germany); Incubator (Shanghai Yuejin Medical Instrument Co., Ltd.); Ultraviolet spectrophotometer (Shanghai Spectrum Instrument Co., Ltd.); Automated tissue homogenizer (QIAGEN, Germany); Fluorescence microscope (LEICA, Germany).

[0028] Culture media: Potato dextrose agar (PDA) medium: PDA powder (39 g / L), agar (5 g / L). LB (Luria-Bertani) liquid medium: Tryptone (10 g / L), Yeast extract (5 g / L), NaCl (10 g / L). PDA-LA mixed medium: Add the appropriate amounts of PDA powder and agar to the prepared LB liquid medium. NB (Nutrient Broth) liquid medium: Peptone (10 g / L), beef extract powder 3 g / L, NaCl (5 g / L). NA (Nutrient Agar) solid medium: Add agar (15 g / L) to the prepared NB liquid medium. All media were sterilized at 121 °C for 15 min before use.

[0029] Example 1: Screening of biocontrol bacteria for mango anthracnose

[0030] The screening process for biocontrol bacteria against mango anthracnose is as follows: Figure 1 As shown. First, a mixed culture method was used for initial screening of biocontrol bacteria for mango anthracnose. Rhizosphere soil samples were collected from around the main root of the highly anthracnose-resistant variety 'Jin Huang' at a depth of 5-10 cm above the soil surface. An appropriate amount of sterile water was added to the rhizosphere soil samples, which were then vortexed for 1 min, allowed to stand for 5 min, and the supernatant was collected and diluted 100 times for later use. An anthracnose spore suspension was prepared and diluted to 2 × 10⁻⁶. 6conidia / mL. Equal volumes of rhizosphere soil dilution and spore suspension were mixed thoroughly. 100 μL of the mixture was evenly spread onto PDA-LA mixed medium. After the surface moisture dried, the medium was incubated at 28 ℃. After 5 days, bacterial strains producing inhibition zones were picked from the plates and transferred to fresh LB liquid medium for further culture. A portion of the bacterial culture was then cryopreserved. The resulting strains were then screened again using the plate confrontation method. 5 μL of *Anthracis sicca* spore suspension (1 × 10⁻⁶) was added to the center of the PDA-LA mixed medium. 7 (conidia / mL) Subsequently, 5 μL of bacterial suspension was added to each of the four corners 2.5 cm from the center of the plate. After the droplets dried, the plate was incubated at 28 ℃. After 5 days, the colony area was counted, and the relative inhibition rate was calculated. Relative inhibition rate = (Sc - St) / Sc × 100%, where Sc represents the colony area of ​​the control group and St represents the colony area of ​​the experimental group. After secondary screening, strain S-34 showed the strongest inhibitory effect against *Bacillus anthracis*, with a relative inhibition rate of 93.1% (e.g., sc - St) / Sc × 100%). Figure 2 (As shown in A and B).

[0031] Example 2: Application of strain S-34 in antagonizing mango leaf pathogens

[0032] The antagonistic effect of strain S-34 against other mango leaf pathogens was assessed using the plate confrontation method. Five μL of strain S-34 was added to each of the four corners (2.5 cm from the center) of a PDA-LA mixed medium, and the medium was dried. Mycelial cakes of *Anthracnose cylindrica*, *Fusarium oxysporum*, and *Fusarium oxysporum* were then collected using a 5 mm punch and placed in the center of the medium. The medium was incubated at 28 °C for 7 days, and the colony area was counted to calculate the relative inhibition rate. Figure 2 According to CH, strain S-34 has a very strong antagonistic effect on *Anthracis cirrhosa*, with a relative inhibition rate of 93.1%; strain S-34 also has a strong antagonistic effect on *Fusarium moniliforme*, with a relative inhibition rate of 84.9%; strain S-34 also has a strong antagonistic effect on *Fusarium moniliforme*, with a relative inhibition rate of 85.5%.

[0033] Example 3 Identification of Bacillus belyssus S-34

[0034] (1) The 16S rDNA and rpoB gene of strain S-34 were amplified by PCR. The PCR primers for 16S rDNA were:

[0035] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0036] 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

[0037] The primers for the rpoB gene are:

[0038] rpoB-F: 5′-AGGTCAACTAGTTCAGTATGGAC-3′;

[0039] rpoB-R: 5′-AGAACCGTAACCGGCAACTT-3′.

[0040] The amplified products were then sequenced for DNA. The S-34-16S rDNA sequence is shown in SEQ ID No. 1, and the S-34-rpoB sequence is shown in SEQ ID No. 2.

[0041] (2) The sequences obtained from sequencing were BLAST aligned on NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and a phylogenetic tree (Bootstrap value = 1,000) was constructed using MEGA11 based on the Neighbor-joining (NJ) method. Figure 3 The phylogenetic tree in section B shows that strain S-34 is Bacillus velezensis. This strain was deposited on May 29, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC M No. 20251225.

[0042] Example 4: Detection of the biocontrol effect of Bacillus belyssus S-34 on mango leaves

[0043] Mango new shoot leaves were collected and disinfected with a 1% sodium hypochlorite solution, followed by washing the leaves three times with sterile water. A twelve-needle bundle was used to puncture holes in the mango leaves, and then 5 μL of LB medium (negative control) and 5 μL of spore suspension (1 × 10⁻⁶) were inoculated at the puncture sites. 7 A mixture of spores (conidia / mL) and LB medium (positive control), and 5 μL of spore suspension (1 × 10⁻⁶) 7 A mixture of *Bacillus cereus* (conidia / mL) and *Bacillus belyssus* S-34 bacterial suspension (experimental group) was inoculated onto at least 10 leaves each time, with a total of 3 replicates. The lesion area was measured 7 days after inoculation, and the relative inhibition rate was calculated. Relative inhibition rate = (Sc - St) / Sc × 100%, where Sc represents the lesion area of ​​the positive control and St represents the lesion area of ​​the experimental group. Figure 4As shown in A and B, Bacillus berberis S-34 has a good biocontrol effect on anthracnose on mango leaves, with a relative inhibition rate of 95.5%.

[0044] Example 5: Detection of the inhibitory effect of Bacillus belyssus S-34 and its sterile fermentation broth on anthrax spore germination and epidermal penetration.

[0045] (1) Preparation of sterile fermentation broth of Bacillus belyssus S-34. Bacillus belyssus S-34 was streaked on NA solid medium, and a single colony was picked and inoculated into 10 mL of NB liquid medium and cultured at 30 ℃ for 24 h. 1 mL of the bacterial culture was inoculated into 100 mL of NB liquid medium and cultured at 30 ℃ for 48 h. The obtained bacterial culture was centrifuged at 12000 rpm for 10 min, and then the supernatant was filtered through a 0.22 μm bacterial filter to obtain sterile fermentation broth.

[0046] (2) The inhibitory effect of sterile fermentation broth of Bacillus vesiculosus S-34 on Bacillus anthracis was detected using the perforated inhibition zone method. 100 μL of Bacillus anthracis spore suspension (1 × 10⁻⁶) was taken. 7 A plate (conidia / mL) was evenly spread onto PDA medium, and then dried in a clean bench. A hole was punched in the center of the medium using a 15 mm diameter punch, and then 150 μL of sterile fermentation broth of *Bacillus belyssus* S-34 was added. The plate was incubated upright at 28 °C for 24 h, and then observed for the formation of inhibition zones. Figure 5 As shown in Figure A, a distinct inhibition zone was formed in the center of the plate, indicating that the aseptic fermentation broth of Bacillus vesiculosus S-34 can inhibit Bacillus anthracis.

[0047] (3) Using onion inner epidermis as material, the effect of Bacillus belyi S-34 and its sterile fermentation broth on the germination process of Bacillus anthracis spores was detected. Onion inner epidermis (2 cm × 2 cm) was spread evenly on 1.5% water agar. 100 μL of *Bacillus sicca* spore suspension, a mixture of *Bacillus anthracis* spore suspension and strain S-34, or a mixture of spore suspension and sterile fermentation broth of strain S-34 were added to the onion inner epidermis, respectively, and then incubated in a 28 ℃ incubator. After 7 h of incubation, the spores were observed under a microscope, and the spore germination rate was counted. Figure 5 As shown in B and 5C, strain S-34 and its sterile fermentation broth can significantly inhibit the spore germination process of Bacillus anthracis, with relative inhibition rates of 96.0% and 83.3%, respectively.

[0048] (4) Using onion inner epidermis as material, the effect of Bacillus belyi S-34 and its sterile fermentation broth on the epidermal penetration of Bacillus anthracis was detected. Three onion inner epidermis pieces (1 cm × 1 cm) were spread evenly on the same PDA medium. 2 μL of Bacillus anthracis spore suspension, a mixture of Bacillus anthracis spore suspension and strain S-34, or a mixture of Bacillus anthracis spore suspension and sterile fermentation broth of strain S-34 were added to each of the three onion inner epidermis pieces, respectively. The plates were then incubated upright at 28 ℃. After 30 h, the onion inner epidermis was removed, and the plates were cultured for another 36 h to allow the mycelia that had penetrated the epidermis to continue growing. Larger colonies at this point indicate a faster rate of Bacillus anthracis penetration of the plant epidermis, and vice versa. Figure 5 As shown in D, strain S-34 and its secretory compounds significantly inhibited the process of anthracnose penetrating the plant epidermis.

[0049] Example 6: Detection of the inhibitory effect of volatile gases from Bacillus belyss S-34 on anthrax spore germination and epidermal penetration.

[0050] (1) The inhibitory effect of volatile gases from Bacillus belyceta var. berberis S-34 on Bacillus anthracis was detected using the double-plate inverted method. 100 μL of bacterial suspension of strain S-34 was evenly spread onto NA medium and then incubated at 30 ℃ for 24 h to allow the strain S-34 to fully colonize the plate. On another PDA plate, 5 μL of Bacillus anthracis spore suspension (1 × 10⁻⁶) was added to the center of the plate. 7 (conidia / mL), after the droplets dried, invert them onto a blank NA plate or an NA plate confluent with strain S-34 and seal with sealing film. Place the NA plate below and the PDA plate above, and incubate at 28 ℃ for 5 days. Count the colony area and calculate the relative inhibition rate. Figure 6 As shown in A and 6B, the volatile gases of Bacillus belyss S-34 significantly inhibited Bacillus anthracis, with a relative inhibition rate of 93.4%.

[0051] (2) Detection of the effect of volatile gases from *Bacillus belyssioides* S-34 on the germination process of *Anthracis chinensis* spores. Onion inner epidermis (2 cm × 2 cm) was spread evenly on 1.5% water agar medium. A spore suspension of *Anthracis chinensis* was added to the onion inner epidermis. After the spore suspension on the onion inner epidermis was dried, it was inverted onto a blank plate or a NA plate confluent with strain S-34, and then placed in an incubator at 28 ℃. After 7 h of incubation, the spores were observed under a microscope, and the spore germination rate was counted. Figure 6 C indicates that the volatile gases of Bacillus belyss S-34 significantly inhibited the germination process of anthrax spores, with a relative inhibition rate of 54.9%.

[0052] (3) Detection of the effect of volatile gases from *Bacillus belyssus* S-34 on the anthrax epidermal penetration process. One onion inner epidermis (1 cm × 1 cm) was placed on each of two different PDA plates (35 mm in diameter). 2 μL of spore suspension was then inoculated onto each plate. After the droplets dried, they were inverted onto either a blank plate or a NA plate confluent with strain S-34, and then incubated at 28 ℃. After 30 h, the onion inner epidermis was removed, and the NA plate was discarded. The PDA plates were incubated for another 36 h before observing the colony size. Figure 6 As shown in D, the volatile gases of strain S-34 significantly inhibited the process of anthracnose penetrating the plant epidermis.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain of Bacillus velezensis, characterized in that, Bacillus belesiensis ( Bacillus velezensis S-34 was isolated from the rhizosphere soil of the anthracnose-resistant mango variety 'Jinhuang' and deposited at the China Center for Type Culture Collection on May 29, 2025, with accession number CCTCC M No. 20251225.

2. A microbial preparation comprising Bacillus belyssus S-34 as described in claim 1.

3. A sterile fermentation broth prepared from *Bacillus belye* S-34 as described in claim 1, characterized in that, The method for preparing the sterile fermentation broth includes the following steps: (1) Streak the Bacillus belye S-34 as described in claim 1 on NA solid medium, pick a single colony and inoculate it into 10 mL NB liquid medium, and incubate at 30 °C for 24 h; (2) Inoculate 1 mL of the bacterial culture obtained above into 100 mL of NB liquid medium and incubate at 30 °C for 48 h; (3) Centrifuge the bacterial culture obtained above at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain sterile fermentation broth.

4. The application of the sterile fermentation broth prepared from the Bacillus vesicularis S-34 of claim 1 or / and the Bacillus vesicularis S-34 microbial preparation of claim 2 or / and the Bacillus vesicularis S-34 of claim 3 in the prevention and control of plant pathogens.

5. In the application according to claim 4, the plant pathogen is *Anthracis sinensis* (Siamese anthracnose fungus). Colletotrichum siamense ), fruit anthracnose ( Colletotrichum fructicola Fusarium rosenbergii ( ), Fusarium incarnatum ) and Fusarium moniliformes ( Fusarium proliferatum Applications in ).

6. The application of the Bacillus vesicularis S-34 of claim 1 or / and the Bacillus vesicularis S-34 microbial preparation of claim 2 or / and the sterile fermentation broth prepared from Bacillus vesicularis S-34 of claim 3 in the prevention and control of anthracnose on mango leaves.

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