Bacillus amyloliquefaciens and its application in preparing product for preventing and treating black spot of pear

Abstract

This invention relates to *Bacillus amyloliquefaciens* and its application in the preparation of products for controlling pear black spot disease, belonging to the field of biological control technology. This invention provides *Bacillus amyloliquefaciens*. Bacillus amyloliquefaciens The ZG2 strain, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 11868, demonstrates the biocompatibility and strong antifungal activity of this *Bacillus amyloliquefaciens* ZG2 strain. It significantly reduces the incidence of pear black spot disease, thus providing safety evidence and a theoretical basis for the preparation of novel, safe, and efficient postharvest biopreservatives for pears.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to Bacillus amyloliquefaciens and its application in the preparation of products for controlling pear black spot disease. Background Technology

[0002] Jade Dew Pear ( Pyrus 'Yu Lu Xiang' is a high-quality pear variety originating from Shanxi Province, China, with the Korla fragrant pear being a prime example. Pyrus sinkiangensis ) as the parent plant, Snowflake Pear ( Pyrus Developed by hybridizing 'Xue Hua' with the male parent, this variety boasts economic advantages such as thin skin, abundant juice, and a unique aroma, leading to a year-on-year increase in planting area and yield. However, the pear fruit is susceptible to mechanical damage during harvesting and transportation, creating conditions for pathogenic fungal infection. Postharvest diseases have become a major bottleneck restricting the healthy development of the Yulu Xiang pear industry, among which Alternaria alternata (Alternaria) is a significant contributor. Alternaria alternata Black spot disease caused by spores is one of the most serious fungal diseases of pear fruit, which can cause pear fruit to rot rapidly and cause huge economic losses.

[0003] Currently, the control of post-harvest diseases in pears still relies mainly on chemical fungicides. While these methods have some effectiveness, excessive use can lead to a series of problems, including fungicide residues, environmental pollution, and pathogen resistance. Therefore, there is an urgent need to develop safe, efficient, and environmentally friendly preservation technologies to reduce or replace the use of chemical fungicides in the post-harvest supply chain. Summary of the Invention

[0004] The purpose of this invention is to provide Bacillus amyloliquefaciens and its application in the preparation of products for the prevention and control of pear black spot disease, so as to solve the problems of fungicide residue, environmental pollution and pathogen resistance caused by the overuse of chemical fungicides in pear fruit in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Bacillus amyloliquefaciens, wherein the Bacillus amyloliquefaciens is a Bacillus amyloliquefaciens. Bacillus amyloliquefaciens The ZG2 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 11, 2015, and the accession number is CGMCC No. 11868.

[0006] The present invention also provides the application of the aforementioned Bacillus amyloliquefaciens in the preparation of products for the prevention and control of pear black spot disease.

[0007] The present invention also provides a biological preservative for preventing and controlling pear black spot disease, the biological preservative comprising the aforementioned Bacillus amyloliquefaciens.

[0008] Preferably, the initial viable concentration of Bacillus amyloliquefaciens in the biopreservative is ≥1×10⁻⁶. 8 CFU / mL.

[0009] The present invention also provides the application of the aforementioned Bacillus amyloliquefaciens or the aforementioned biological preservative in the prevention and control of pear black spot disease.

[0010] The present invention also provides a method for preventing and controlling pear black spot disease, wherein the aforementioned Bacillus amyloliquefaciens or the aforementioned biological preservative is inoculated onto the diseased spots on the pear fruit.

[0011] Preferably, the pear black spot disease is caused by Alternaria. Alternaria alternata cause.

[0012] Preferably, the inoculation amount of the biological preservative is 30-70 μL / site.

[0013] The present invention has the following technical effects and advantages: The Bacillus amyloliquefaciens ZG2 strain of this invention is practically non-toxic, exhibiting extremely high biocompatibility. It shows significant inhibitory effects against Alternaria alternata and can significantly reduce the incidence and severity of black spot disease in Yulu Xiang pear. It possesses extremely high biocompatibility and strong antifungal activity, thus providing safety evidence and a theoretical basis for the preparation of novel, safe, and efficient postharvest biological preservatives for pears. Attached Figure Description

[0014] Figure 1 To analyze the morphological characteristics of Bacillus amyloliquefaciens strain ZG2, A represents the colony morphology of ZG2 after 24 hours of culture, B represents the single colony morphology of ZG2, C represents the shrunken morphology of a single colony of ZG2 after extended culture, and D represents the Gram staining results. Figure 2 To determine the in vitro antifungal activity of Bacillus amyloliquefaciens strain ZG2, Figure A shows the antifungal effect of different ZG2 treatment solutions on Alternaria alternifolia. In the figure, a is the control plate (CK), b is the plate with ZG2 fermentation broth, c is the plate with ZG2 bacterial suspension, d is the plate with ZG2 supernatant, and e is the plate with ZG2 heat-killed solution. Figure B shows the effect of different ZG2 treatment solutions on the colony diameter of Alternaria alternifolia. Figure C shows the inhibition rate of different ZG2 treatment solutions on Alternaria alternifolia. Figure 3 This study investigated the in vitro antifungal activity of different concentrations of Bacillus amyloliquefaciens ZG2 fermentation broth. Figure A represents the antifungal effect of different concentrations of ZG2 fermentation broth on Alternaria alterniflora. In the figure, a represents the control plate (CK), and b represents a 1×10⁻⁶ plate. 9 CFU / mL, c = 1×10 8 CFU / mL, d = 1×10 7 CFU / mL, e = 1×10 6CFU / mL, f = 1×10 5 CFU / mL; B represents the effect of different concentrations of ZG2 fermentation broth on the colony diameter of Alternaria alternata; C represents the inhibition rate of different concentrations of ZG2 fermentation broth on Alternaria alternata. Figure 4 To determine the in vivo biocontrol effect of Bacillus amyloliquefaciens strain ZG2, the following results were obtained: A represents the effect of ZG2 fermentation broth on the incidence of black spot disease in Yulu Xiang pear 3–7 days post-harvest; B represents the effect of ZG2 fermentation broth on the disease index of black spot disease in Yulu Xiang pear; and C represents the effect of ZG2 fermentation broth on the control efficacy of black spot disease in Yulu Xiang pear.

[0015] Preservation Instructions Bacillus amyloliquefaciens is Bacillus amyloliquefaciens Bacillus amyloliquefaciens The ZG2 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 11, 2015, and the accession number is CGMCC No. 11868. Detailed Implementation

[0016] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0017] In the test materials of this invention, the pathogenic fungus *Alternaria* is... Alternaria alternata The samples were isolated from diseased Yulu Xiangli pears and preserved at the Institute of Agricultural Product Storage and Preservation, Shanxi Academy of Agricultural Sciences; the Yulu Xiangli pears were collected from the experimental garden of the Fruit Tree Research Institute, Shanxi Academy of Agricultural Sciences; and the Wistar rats (weighing 180–220 g) were obtained from the Experimental Animal Center of Shanxi Medical University.

[0018] Example 1: Biological characteristics and fermentation broth preparation of strain ZG2 ZG2 strain was activated by inoculation into LB agar plates, and then the activated strain was inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h to obtain seed culture. The seed culture was then transferred to fresh LB liquid medium at an inoculation rate of 1 vt% and cultured at 30℃ and 180 rpm to obtain ZG2 fermentation broth. The morphology of the activated strain was observed and Gram staining was performed. The results are as follows: Figure 1 As shown.

[0019] The results showed that ZG2 colonies were Gram-positive short rods that appeared milky white, round, raised, and smooth on LB plates. After prolonged incubation, the colony surface became wrinkled.

[0020] Example 2: Acute oral toxicity test Wistar rats weighing 180–220 g were acclimatized and then divided into 5 groups of 8 rats each (half male and half female) using the Horn method. Each group was administered ZG2 fermentation broth prepared in Example 1 at doses of 464 mg / kg, 1000 mg / kg, 2150 mg / kg, 4640 mg / kg, and 5000 mg / kg of rat body weight via gavage. The gavage volume was 1 mL / 100 g of rat body weight. Symptoms of poisoning and mortality were observed immediately after gavage for 14 days. The median lethal dose (LD50) was calculated based on the number of deaths. 50 The results are shown in Table 1.

[0021] Table 1 Acute oral toxicity of ZG2 fermentation broth to Wistar rats

[0022] The results showed that within the gavage dose range of 464–5000 mg / kg, no rats in any group of ZG2 fermentation broth died, and no poisoning symptoms were observed. Their behavior, activity, and diet remained normal. The LD50 was [not specified]. 50 >5000 mg / kg. This indicates that the ZG2 strain of this invention is practically non-toxic and has extremely high biosafety.

[0023] Example 3: In vitro antifungal test The ZG2 fermentation broth prepared in Example 1 was mixed with melted potato dextrose agar medium to achieve a viable concentration of 1 × 10⁻⁶ Bacillus amyloliquefaciens in the fermentation broth. 8 CFU / mL; using the amount of ZG2 fermentation broth added at this time as the standard volume, standard volumes of ZG2 bacterial suspension, ZG2 supernatant, and ZG2 heat-killed solution were added to melted potato dextrose agar medium, respectively; 5 mm diameter Alternaria mycelium cakes were placed in the center of each plate and incubated at 28°C, with plates containing a standard volume of sterile LB liquid medium added to melted potato dextrose agar medium serving as controls. When the control plates were fully covered with Alternaria colonies, the colony diameter of each plate was measured and the inhibition rate was calculated. The results are as follows: Figure 2 As shown; The ZG2 fermentation broth prepared in Example 1 was mixed with melted potato dextrose agar medium to achieve a viable concentration of 1×10⁻⁶ Bacillus amyloliquefaciens in the fermentation broth. 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5CFU / mL; Alternaria alternata mycelial discs with a diameter of 5 mm were placed in the center of each plate and incubated at 28°C. Plates containing standard volumes of sterile LB broth added to melted potato dextrose agar served as controls. Once the control plates were fully colonized with Alternaria alternata, the colony diameter of each plate was measured and the inhibition rate was calculated. The results are as follows: Figure 3 As shown.

[0024] The results showed that the ZG2 fermentation broth exhibited the best antagonistic effect against Alternaria alternata, with an inhibition rate of 97.5% on Alternaria alternata colony diameter after 7 days of culture. The ZG2 bacterial suspension and ZG2 heat-killed solution also showed significant antifungal activity, with inhibition rates of 84.71% and 83.53%, respectively. The ZG2 supernatant showed a relatively weaker antifungal effect, at 57.65%. This indicates that the ZG2 strain of this invention possesses extremely strong in vitro antifungal activity against pear pathogens. The inhibitory effect of the ZG2 fermentation broth on Alternaria alternata was dose-dependent, with a viable cell concentration of 1×10⁻⁶. 8 CFU / mL and 1×10 9 The ZG2 fermentation broth at CFU / mL showed significant inhibitory effects on Alternaria alterniflora, with an inhibition rate of 88.37%. Considering both control efficacy and cost-effectiveness, subsequent experiments used a live Bacillus amyloliquefaciens concentration of 1×10⁻⁶. 8 ZG2 fermentation broth, CFU / mL.

[0025] Example 4: In vivo biocontrol efficacy determination Two points were selected at the equatorial region of the pear fruit of the Yulu Xiang pear, and wounds 5 mm deep and 5 mm wide were made. Each wound was inoculated with 50 μL of 1×10 8 ZG2 fermentation broth at CFU / mL was inoculated, with an equal volume of sterile water as a control. After drying for 2 hours, 10 μL of 1×10⁻⁶ CFU / mL ZG2 fermentation broth was inoculated into the same wound. 5 Alternaria spore suspension (mL) was used to store pear fruits at 20℃ and 95% relative humidity. Disease occurrence was observed daily, and the disease index was calculated based on lesion diameter. Each treatment consisted of 24 pear fruits, with 3 replicates. Results are shown below. Figure 4 As shown.

[0026] The results showed that, compared with the control treated with sterile water, the diameter of lesions on pears inoculated with ZG2 fermentation broth was significantly smaller. The disease index of the control pears rapidly increased from 38.33 to 86.11 from day 3 to 7, while the disease index of the pears inoculated with ZG2 fermentation broth remained significantly lower, reaching only 40.00 on day 7. Although the control effect of the ZG2 strain decreased slightly with prolonged storage, the control effect remained above 53% at the end of storage. This indicates that the ZG2 strain of the present invention can significantly reduce the severity of black spot disease in Yulu Xiang pears.

[0027] As can be seen from the above embodiments, the present invention provides *Bacillus amyloliquefaciens* and its application in the preparation of products for the prevention and control of pear black spot disease. The *Bacillus amyloliquefaciens* of the present invention... Bacillus amyloliquefaciens The ZG2 strain exhibits biosafety and strong antifungal activity, significantly reducing the incidence of pear black spot disease. This provides safety evidence and a theoretical basis for the preparation of novel, safe, and efficient postharvest biological preservatives for pears.

[0028] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Bacillus amyloliquefaciens, characterized in that, The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens. Bacillus amyloliquefaciens The ZG2 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 11, 2015, and the accession number is CGMCC No. 11868.

2. The use of Bacillus amyloliquefaciens according to claim 1 in the preparation of products for preventing and controlling pear black spot disease.

3. A biological preservative for controlling pear black spot disease, characterized in that, The biological preservative includes Bacillus amyloliquefaciens as described in claim 1.

4. The biological preservative according to claim 3, characterized in that, In the aforementioned biopreservative, the initial viable bacterial concentration of Bacillus amyloliquefaciens is ≥1×10⁻⁶. 8 CFU / mL.

5. The application of Bacillus amyloliquefaciens according to claim 1 or the biological preservative according to any one of claims 3 to 4 in the prevention and control of pear black spot disease.

6. A method for preventing and controlling pear black spot disease, characterized in that, The Bacillus amyloliquefaciens of claim 1 or the biological preservative of any one of claims 3 to 4 is inoculated onto the diseased spots of pear fruit.

7. The method according to claim 5, characterized in that, The pear black spot disease is caused by Alternaria. Alternaria alternata cause.

8. The method according to claim 5, characterized in that, The inoculation amount of the biological preservative is 30–70 μL / site.

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