Bacillus velezensis B16 and application thereof in prevention and treatment of postharvest fungal diseases of kiwi fruits

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus velezensis B16 and application of the bacillus velezensis B16 to prevention and treatment of postharvest fungal diseases of kiwi fruits. The preservation number of the bacillus velezensis B16 is CCTCC (China Center For Type Culture Collection) NO: M 20251598. The bacillus velezensis B16 shows a remarkable effect in the aspect of preventing and treating the diseases of the kiwi fruits. Compared with a traditional chemical bactericide, the bacillus velezensis B16 has an obvious advantage of environmental protection. In the process of preventing and treating the kiwi fruit diseases, the environment is not polluted, and adverse effects on the food safety of fruits are avoided. Due to the characteristics of safety and eco-friendliness, the bacillus velezensis B16 has an important application value in modern green agriculture, and is a biological control resource suitable for sustainable agricultural production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus belye B16 and its application in the prevention and control of postharvest fungal diseases in kiwifruit. Background Technology

[0002] kiwi( Actinidia chinensis Kiwifruit, also known as Chinese gooseberry, is a perennial deciduous vine belonging to the genus *Actinidia* in the family Actinidiaceae, and is rich in nutrients. Kiwifruit is a typical climacteric fruit, highly susceptible to spoilage during post-harvest storage and transportation, primarily due to fungal diseases. These diseases cause significant economic losses during transportation, storage, and sales. Currently, post-harvest disease control in kiwifruit mainly relies on pre-harvest chemical control. However, the extensive use of pre-harvest chemical agents can lead to fungal resistance, reducing the effectiveness of control and negatively impacting human health and the environment.

[0003] Bacillus species possess diverse secondary metabolic mechanisms and can produce a variety of structurally different antagonistic substances, enabling them to effectively inhibit various crop diseases and thus providing direct or indirect promotion of crop growth. However, current technologies do not propose Bacillus species for controlling postharvest fungal diseases in kiwifruit. Summary of the Invention

[0004] In view of the above-mentioned shortcomings in the prior art, the purpose of this invention is to provide a strain of Bacillus belyssus B16 and its application in the prevention and control of postharvest fungal diseases in kiwifruit.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: Firstly, it provides Bacillus vesiculosus (BVV) Bacillus velezensis )B16, its accession number is CCTCCNO: M 20251598.

[0006] Secondly, it provides microbial agents, including Bacillus belesiensis (B. belesiensis). Bacillus velezensis )B16. Thirdly, it provides Bacillus vesiculosus (BVV) Bacillus velezensis B16; or including Bacillus belesiensis ( Bacillus velezensis Application of B16 microbial inoculant in the control of postharvest gray mold in kiwifruit.

[0007] Fourthly, it provides Bacillus vesiculosus (BVV) Bacillus velezensis B16; or including Bacillus belesiensis ( Bacillus velezensis Application of B16 microbial inoculant in the prevention and control of postharvest brown spot disease in kiwifruit.

[0008] Fifthly, it provides Bacillus vesiculosus (BVV)Bacillus velezensis B16; or including Bacillus belesiensis ( Bacillus velezensis Application of B16 microbial inoculant in extending the shelf life of kiwifruit.

[0009] The beneficial effects of this invention are as follows: 1. Improve prevention and control effectiveness: Bacillus velezensis B16 showed significant effectiveness in controlling kiwifruit diseases. In plate confrontation experiments, Bacillus velezensis B16 is effective against gray mold ( Botrytis cinerea ) and brown spot disease ( Alternaria alternata The mycelial growth inhibition rates of the fungi reached 66.5% and 65.7%, respectively, indicating that it has strong antibacterial ability. Further inoculation experiments on kiwifruit showed that the use of... Bacillus velezensis B16 treatment followed by vaccination Botrytis cinerea and Alternaria alternata The diameter of the lesions decreased to 4.8 mm and 5.4 mm, respectively; the incidence of disease decreased to 38.5% and 49.6%, respectively. These results indicate that... Bacillus velezensis B16 can effectively inhibit the growth of pathogens and significantly reduce the severity of diseases in kiwifruit, making it an ideal strain for biological control.

[0010] 2. Reduce environmental pollution and ensure safety: Compared with traditional chemical disinfectants, Bacillus velezensis B16 has significant environmental advantages. In the process of controlling kiwifruit diseases, it does not pollute the environment nor adversely affect the food safety of the fruit. This safety and eco-friendly characteristic makes it... Bacillus velezensis B16 has important application value in modern green agriculture and is a biological control resource suitable for sustainable agricultural production.

[0011] 3. Extend shelf life: In addition to disease prevention and control, Bacillus velezensis B16 also showed the potential to extend the shelf life of kiwifruit. Experimental data showed that... Bacillus velezensis B16 can inhibit the growth of gray mold spots and brown spots, and can suppress the incidence rate, reducing the incidence rate to 38.51% and 49.62%, respectively. Figure 3 , 4 5). After Bacillus velezensis Kiwifruit treated with B16 showed effective control over the expansion of fruit lesions, maintained high fruit firmness, and significantly reduced rot rate. This characteristic indicates that... Bacillus velezensis B16 not only reduces the occurrence of postharvest diseases, but also slows down the ripening and quality decline of fruits, thereby extending the shelf life of kiwifruit during storage and transportation and increasing the market value of the fruit. Attached Figure Description

[0012] Figure 1 for Bacillus velezensis A schematic diagram of plate contrast analysis between B16 and gray mold and brown spot pathogens; Figure 2 for Bacillus velezensis B16 is a phylogenetic tree constructed based on 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf). Figure 3 For vaccination Bacillus velezensis B16 prevention Botrytis cinerea A diagram illustrating the gray mold disease that causes kiwifruit. Figure 4 For vaccination Bacillus velezensis B16 prevention Alternaria alternata A diagram illustrating the brown spot disease in kiwifruit. Figure 5 Inoculation of kiwifruit Botrytis cinerea and Alternaria alternata Statistical graphs of fruit lesion diameter and incidence rate, blank control group (Bc_ck / Aa_ck), B16 treatment group (Bc_B / Aa_B). Figure 6 For vaccination Bacillus velezensis B16 and Botrytis cinerea / Alternaria alternata Changes in the relative expression levels of disease resistance genes in kiwifruit during storage; where A represents the disease resistance gene. GLU Relative expression level; A represents the disease resistance gene. CHI The relative expression level. Detailed Implementation

[0013] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0014] Example 1: Screening of Bacillus subtilis: Soil samples were taken from healthy kiwifruit orchards. 10 g of the mixed soil sample was placed in an Erlenmeyer flask containing 90 mL of sterile PBS buffer and incubated in a constant-temperature shaker at 28°C and 150 r·min. -1 Shake for 30 min under sterile conditions, then dilute with sterile PBS to a concentration of 10. -5 The soil sample was diluted 100 times, and then 100 μL of the prepared diluted solution was spread evenly on an LB plate and incubated in a constant temperature incubator at 37°C. The resulting bacterial colonies were purified multiple times.

[0015] Select purified bacteria and Botrytis cinerea and Alternaria alternata The strains were subjected to confrontation culture on sterile PDA plates to screen for the strain with the highest antibacterial rate. Bacillus velezensis B16, with antibacterial rates reaching 66.5% and 65.7% respectively. Figure 1 ).

[0016] Example 2: Bacillus velezensis Molecular biological identification of B16: Genomic DNA was extracted from the strain, purified, and fragmented using a Covaris sonicator to construct a library and ligate specific adapters. After screening and electrophoresis, the target fragment was obtained and denatured into single-stranded DNA using alkaline denaturation. DNA clusters were then formed on the microarray surface using bridge PCR and linearized as sequencing templates. Finally, sequencing was performed using the Illumina platform, employing cyclic synthesis of fluorescently labeled dNTPs, laser scanning, and signal readout to obtain high-throughput sequence data (sequencing results are shown in SEQ ID No. 1). After sequencing the product, an ML phylogenetic tree was constructed by combining the sequences of 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf). Figure 2 Based on the phylogenetic tree analysis constructed using housekeeping genes, strain B16 is related to several known strains. Bacillus After comparing with reference strains of the genus and related genera, it was determined that they clustered in [the relevant genera]. Bacillus velezensis (GCF_001461825.1) belongs to the same branch and is the closest in evolutionary distance, showing no mixing with other strains, indicating a high degree of phylogenetic similarity. In contrast, B16 and... Bacillus amyloliquefaciens , Bacillus siamensis , Bacillus pumilusThe evolutionary distance between strains is significantly greater, further supporting its classification. In conclusion, strain B16 can be definitively identified as... Bacillus velezensis .

[0017] Example 3: The flat plate confrontation method is used for evaluation. Bacillus velezensis B16's ability to inhibit pathogenic fungi. Stored in a 4°C refrigerator. Bacillus velezensis B16 was inoculated into LB medium and cultured for 48 h. Then, using the confrontation method, it was inoculated at two symmetrical positions 2.5 cm from the center of PDA medium. A 0.6 cm diameter fungal disc was inoculated at the center of the PDA medium. Each biological treatment was repeated in triplicate, with the control group not inoculated. Bacillus velezensis B16 was incubated in a 28℃ incubator for 7 days. The colony diameter was measured and calculated using the following formula. Bacillus velezensis B16's antagonistic ability against different pathogenic fungi.

[0018] 100 Configuration approximately 1 × 10 10 spores / mL Botrytis cinerea and Alternaria alternata Spore suspension. Using a sterile punch, make a circular wound with a diameter of 5 mm and a depth of 5 mm at the equator of each kiwifruit, then collect 20 μL of spore suspension. Bacillus velezensis Inoculate the wound with B16 bacterial suspension. After air drying, inoculate 20 μL into the wound. Botrytis cinerea or Alternaria alternata The spore suspension was used. Another group of wounds were treated with 20 μL of LB medium as a control, and incubated at 28°C for 5 days. Each treatment had 6 biological replicates, and the experiment was repeated 3 times. The diameter of the lesions was measured daily using a crossover method. The results showed that... Bacillus velezensis B16 can inhibit the growth of gray mold spots and brown spots, and can suppress the incidence rate, reducing the incidence rate to 38.51% and 49.62%, respectively. Figure 3 , 4 5).

[0019] Example 4: Configuration approximately 1 × 10 10 spores / mL Botrytis cinerea and Alternaria alternata Spore suspension. Using a sterile punch, make a circular wound with a diameter of 5 mm and a depth of 5 mm at the equator of each kiwifruit, then collect 20 μL of spore suspension. Bacillus velezensis Inoculate the wound with B16 bacterial suspension. After air drying, inoculate 20 μL of the suspension onto the wound. Botrytis cinerea or Alternaria alternataSpore suspensions were prepared (experimental group). Another group had 20 μL of LB medium added to the wound as a control (control group), and cultured at 28℃ for 5 days. Afterwards, kiwifruit from both the control and experimental groups were flash-frozen in liquid nitrogen, ground into powder, and RNA was extracted and measured using real-time quantitative PCR. GLU and CHI The relative expression levels of genes. The results showed that, compared to the control kiwifruit, the expression levels of genes expressed using [the following method] were significantly higher. Bacillus velezensis Disease-resistant genes in B16-treated kiwifruit CHI and GLU Increased. (Explanation) Bacillus velezensis B16 treatment enhanced the disease resistance of kiwifruit itself. Figure 6 ).

[0020] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0021] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Bacillus belye ( Bacillus velezensis B16, characterized in that, Its accession number is CCTCC NO: M 20251598.

2. A microbial inoculant, characterized in that, Including Bacillus belesiensis as described in claim 1 ( Bacillus velezensis )B16.

3. The Bacillus belesiensis as described in claim 1 ( Bacillus velezensis The application of the microbial agent described in claim 2 or B16 in the prevention and control of postharvest gray mold in kiwifruit.

4. The Bacillus belesiensis as described in claim 1 ( Bacillus velezensis The application of the microbial agent described in claim 2 or B16 in the prevention and control of postharvest brown spot disease in kiwifruit.

5. The Bacillus belesiensis as described in claim 1 ( Bacillus velezensis The application of the microbial agent described in claim 2 or B16 in extending the shelf life of kiwifruit.

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