Bacillus velezensis d44 and its application as a biological preservative

Abstract

The application discloses Bacillus velezensis D44, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 20221701. The application also discloses application of the Bacillus velezensis D44 in a biological preservative, application in antagonizing brown rot fungi, rhizopus, penicillium, fusarium, botrytis, syncephalastrum, and pink syncephalastrum, and application in preventing and treating peach brown rot and soft rot. The biological preservative is extracted from a morchella esculenta endophyte, can inhibit the propagation and mycelium growth of pathogenic bacteria through antagonism, can enhance the storage resistance of fruits, and has the characteristics of a new biological preservative, such as safety and broad-spectrum sterilization. The biological preservative has a significant antagonistic effect on peach postharvest brown rot and soft rot bacteria under sublow temperature and low temperature conditions.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fruit preservation, and more particularly relates to Bacillus velezensis D44 and application thereof as a biological preservative. BACKGROUND

[0002] Peaches are prone to spoilage and deterioration when they are ripe, during the high-temperature and high-humidity season in Shanghai. Low temperature can extend the shelf life of the fruit, but long-term low-temperature storage leads to problems such as flesh browning, loss of flavor, reduced disease resistance, and shortened shelf life, resulting in a loss rate of 20% to 30% and reducing the willingness of consumers to purchase. In addition, the post-harvest commercialization process and the application of preservation and storage technology are not standardized, and the loss rate in some production areas is as high as 50%, which seriously affects the value of the peach industry chain. Senescence and softening, as well as the proliferation and infection of exogenous pathogenic bacteria, are the main causes of peach postharvest fruit rot and flavor imbalance. The development of preservatives and preservation and storage technologies has become an important link in reducing losses, increasing efficiency, enhancing product competitiveness, and promoting the sustainable development of the peach industry. Our team has found that peach fruit is mainly infected by six types of mold during postharvest softening and storage, namely Phomopsis sp., Botrytis cinerea, Colletotrichum siaense, Rhizopus sp., Fusarium sp., and Aspergillus sp. Brown rot is the main disease of postharvest peach fruit. The main means of controlling peach postharvest diseases is cold storage combined with physical sterilization, reduced pressure storage, irradiation combined preservation, modified atmosphere combined preservation, heat treatment combined preservation, calcium and NO, and 1-MCP, which can extend the shelf life of the fruit by inhibiting the ethylene release rate and the proliferation of postharvest pathogens. These methods have been applied to some extent in the industry, but there are still problems such as short shelf life at room temperature after long-term cold storage, fruit rot (reduced disease resistance and easy infection by exogenous microorganisms), and reduced flavor, which may be related to the inhibition of fruit softening by low-concentration endogenous ethylene and the reduction of fruit disease resistance by excessively low-concentration ethylene.

[0003] Chemical preservatives have good effect on postharvest preservation of fruits and vegetables, but they have microbial resistance and toxic side effects. The development of new preservatives with high efficiency and safety is an urgent need in production practice. Biological preservation aims to use biological characteristics and environmental interaction and antagonism (antagonism refers to the phenomenon that a microorganism produces certain metabolic products or changes other conditions during its life activities, thereby inhibiting the growth and reproduction of other microorganisms, and even killing other microorganisms). It is the main trend of future development to essentially control the reproduction of pathogenic bacteria and enhance the storage resistance of fruits. Although there are many studies on biological control agents for postharvest diseases in China, few of them can be used in large quantities in the market. The biological control agents used in production practice are only Aspire, Shemer, Candifruit and other products. Therefore, it is of great significance to screen and develop biological control agents that can be widely used. Most of the biocontrol agents show significant inhibition effect at temperatures above 20℃. With the decrease of environmental temperature, the activity gradually inactivates. Low-temperature refrigeration is the main means to extend the preservation period and shelf life of fruits. Cold chain transportation is also becoming more and more widespread in China. How to make biocontrol agents maintain activity and play an inhibitory role under low-temperature conditions, and how to screen low-temperature-resistant biocontrol agents are the focus of future research. Bacillus velezensis D44 is a strain screened from Morchella esculenta endophytic bacteria. The biocontrol strain has strong inhibitory effect on the mycelial growth of pathogenic fungi and strong blocking effect on the infection cycle of pathogenic fungi. It has a wide range of antibacterial properties and has the potential to prevent and control peach brown rot and soft rot. Through experimental verification, the strain has significant antagonistic effect on peach postharvest brown rot and soft rot bacteria under normal temperature (25℃), sublow temperature (12-15℃) and low temperature (1-3℃), and is expected to become a new type of peach fruit biological preservative. SUMMARY

[0004] 1. Objectives.

[0005] The present application screens a strain of Bacillus velezensis D44, which can be used as a biological preservative.

[0006] 2. Technical solutions adopted by the present application.

[0007] The Bacillus velezensis D44 of the present application is extracted from Morchella by the present team and was preserved in the China Center for Type Culture Collection (CCTCC) on October 31, 2022, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with a postal code of 430072. The strain is named Bacillus velezensis, and the preservation number is CCTCC NO: M 20221701.

[0008] The basic biological characteristics of the Bacillus velezensis D44 of the application are as follows:

[0009] (1) Colony morphology: observed on the culture medium, D44 forms round, white, shiny, smooth, opaque, easy to pick up the colony;

[0010] (2) Culture characteristics: the culture temperature of D44 is 37℃, the optimum pH value range is usually between 6.5-7.5, the colony can be formed after 24h culture in LB agar medium, and the bacterial suspension state of D44 can be obtained by using the shaking culture method.

[0011] The Bacillus velezensis D44 of the application can still maintain high activity at low temperature, form film quickly, can be combined with different temperature storage environments of peach after harvest, play a synergistic role in inhibiting postharvest fruit rot, can be used as a biological preservative, and has a wide application field.

[0012] The Bacillus velezensis D44 of the application has significant antagonistic effect on the following seven postharvest pathogens of peach fruit: Monilinia fructicola, Rhizopus stolonifer, Penicillium spp., Fusarium spp., Botrytis cinnerea, Alternaria spp., and Trichothecium roseum.

[0013] 105-10 6 The fermentation broth concentration of 105-10 cfu / mL can significantly inhibit the disease spot diameter of nectarine and honey peach fruit stored at room temperature (25℃), sublow temperature (12-15℃), and low temperature (1-3℃) after inoculation of Monilinia fructicola and Rhizopus stolonifer, delay the onset time, and has a higher disease inhibition rate.

[0014] 10 5 -10 6 The fermentation broth concentration of 105-10 cfu / mL can significantly inhibit the disease spot diameter of nectarine and honey peach fruit stored at room temperature (25℃), sublow temperature (12-15℃), and low temperature (1-3℃) after inoculation of Monilinia fructicola and Rhizopus stolonifer, delay the onset time, and has a higher disease inhibition rate.

[0015] The culture method of Bacillus velezensis D44: the preserved D44 strain is evenly coated on LB solid culture medium with an inoculation loop, and is cultured at 37°C overnight for activation. The activated strain is inoculated into 150 mL NA, and is cultured at 37°C, 180 r·min -1 for 12 h, and the concentration of the bacterial liquid (unit volume of viable bacteria) is measured by plate counting method. Generally, the mother liquor concentration of the fermentation bacterial liquid can be controlled at 10 9 ~ 10 10 cfu / mL, and is used after dilution.

[0016] 3, The technical effects produced by the present application.

[0017] 1) After spraying Bacillus velezensis D44 by using an atomizing device, combined with normal temperature (25°C), sub-low temperature (12-15°C), and low temperature (1-3°C) storage, the seven pathogenic fungi of Pythium porphyrea, Penicillium, Fusarium, Botryodiplodia theobromae, Botrytis cinerea, Athelia sp., and Rhizopus have strong antagonistic effect; the disease spot diameter of fruits inoculated with Botryodiplodia theobromae and Rhizopus can be inhibited, the onset time is delayed, and the disease inhibition rate is high; the weight loss rate and rot rate of fruits under different preservation storage conditions can be significantly reduced, the inherent color and quality of fruits are well maintained, the generation of peculiar smell is inhibited, and the preservation period is prolonged.

[0018] 2) The biological preservative is extracted from Morchella esculenta endophytic fungi, and can inhibit the reproduction and mycelial growth of pathogenic bacteria through antagonistic effect, enhance the storage resistance of fruits, and has the characteristics of safety, broad-spectrum sterilization, and other new biological preservatives.

[0019] 3) The biological preservative can still maintain high activity under low temperature, form film quickly (≦12 h), can be well combined with different temperature storage environments of postharvest peaches (normal temperature, sub-low temperature, and low temperature), play a synergistic role in inhibiting postharvest fruit rot, and can meet the storage and transportation requirements under different temperature conditions.

[0020] 4) The biological preservative has significant anti-corrosion and preservation effect on 'Huoli 018' nectarines, 'Hujingmielu' soft solute peaches, and has great market development potential.

[0021] 5) The biological preservative still has strong activity under low temperature conditions, and has significant effect in inhibiting postharvest fruit rot and water loss.

[0022] 6) The PE anti-fog bag with a thickness of 0.025 mm to 0.04 mm and a porosity (air permeability) of 5‰ can effectively maintain the relative humidity in the bag and prevent carbon dioxide damage caused by fruit metabolism in the preservative bag.

[0023] 7) Gradient precooling can significantly improve the film formation and antibacterial effect of fruits, and prevent the accumulation of respiratory heat in the core of fruits caused by instantaneous low temperature.

[0024] 8) Using the preservative effect of Bacillus velezensis D44, the browning disease of 'Huliao 018' and 'Hujing Milu' watermelon fruits inoculated with brown rot fungus was delayed for 2 days, with an inhibition rate of 85%-99% compared with the control group under normal temperature conditions. The soft rot disease of 'Huliao 018' and 'Hujing Milu' watermelon fruits inoculated with Rhizopus was delayed for 3 days, with an inhibition rate of 87.61%-99.82% compared with the control group. The soft rot disease of 'Huliao 018' and 'Hujing Milu' watermelon fruits inoculated with Rhizopus was delayed for 3 days, with an inhibition rate of 87.61%-99.82% compared with the control group. The inhibition effect of D44 on postharvest browning fungus and Rhizopus of peach fruits under normal temperature conditions was not significantly different from that of W10.

[0025] 9) Using the preservative effect of Bacillus velezensis D44, the browning disease of 'Huliao 018' and 'Hujing Milu' watermelon fruits inoculated with brown rot fungus was delayed to the 2nd and 4th days, respectively, 1 day and 2 days later than the control group under sublow temperature conditions, with an inhibition rate of 59.39% and 99.19%, respectively. The fruit treated with W10 showed no significant delay in disease onset, and the watermelon fruit appeared 1 day later than the control group. The disease onset of 'Huliao 018' and 'Hujing Milu' watermelon fruits inoculated with Rhizopus was delayed by 14 days compared with the control group. The inhibition rate of D44 treatment on Rhizopus was significantly higher than that of the W10 treatment group, indicating that the treatment effect of D44 bacterial solution was better under sublow temperature conditions. It is shown that D44 strain still has significant potential to prevent and control peach browning and soft rot under sublow temperature conditions.

[0026] 10) Using the preservative effect of Bacillus velezensis D44, the browning disease of 'Huliao 018' and 'Hujing Milu' watermelon fruits inoculated with brown rot fungus was delayed to the 8th and 14th days, respectively, 8 days and 6 days later than the control group under low temperature conditions, with an inhibition rate of 75.96% and 71.01%, respectively. The fruit treated with W10 showed no significant delay in disease onset, and the watermelon fruit appeared 4 days later than the control group. The disease onset of 'Huliao 018' and 'Hujing Milu' watermelon fruits inoculated with Rhizopus was delayed by 14 days compared with the control group. The inhibition rate of D44 treatment on Rhizopus was significantly higher than that of the W10 treatment group, indicating that D44 strain still has potential to prevent and control peach browning and soft rot under low temperature conditions.

[0027] 11)Bacillus velezensis D44 combined with normal temperature (25℃), sub-low temperature (12-15℃), low temperature (1-3℃) storage or transportation can significantly reduce the weight loss rate and rot rate of fruits during normal temperature express and shelf placement, delay the onset of disease, better maintain the inherent color and quality of fruits, inhibit the generation of off-flavor, and prolong the shelf life.

[0028] 12) Under normal temperature conditions, the safe storage and preservation period and circulation period of 'Huliao 018' fruits treated by D44 can reach 6-8d, which is 4-6d longer than that of the control group, the rot rate is less than 5%, the weight loss rate is less than 4.5%, and the decrease rate of fruit soluble solids is controlled within 8%; the safe storage and preservation period and circulation period of 'Hujing Milu' juicy peach fruits can reach 4-6d, which is 2-4d longer than that of the control group, the rot rate is less than 8%, the weight loss rate is less than 8%, and the decrease rate of fruit soluble solids is controlled within 4%, with high commodity and edible value.

[0029] 13) Under sub-low temperature conditions, the safe storage and preservation period and circulation period of 'Huliao 018' fruits treated by D44 can reach 16-20d, which is 8-12d longer than that of the control group, the rot rate is less than 5%, the weight loss rate is less than 7%, and the decrease rate of fruit soluble solids is controlled within 5%, the fruit skin hardness is 1.5kg·cm -2 , the flesh tissue hardness is about 1kg·cm -2 , still with high transportation, sales and edible value.

[0030] 14) Under low temperature conditions, the safe storage and preservation period and circulation period of 'Huliao 018' fruits treated by D44 can reach 24-30d, which is 12-18d longer than that of the control group, no rotting occurs, the weight loss rate is less than 8%, and the decrease rate of fruit soluble solids is controlled within 5%, the fruit skin hardness is 4-5kg·cm -2 , the flesh tissue hardness is 2-3kg·cm -2 , the fruits can normally ripen and soften during normal temperature shelfing, with high transportation, sales and edible value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the antagonistic effect diagram of biocontrol bacteria D44, A9 and W10 on postharvest pathogens of peach;

[0032] Figure 2 is the initial screening of non-drug damage concentration;

[0033] Figure 3 is the effect of D44 and W10 treatment on the diameter and inhibition rate of peach brown rot fungus lesion (normal temperature);

[0034] Figure 4Effect of D44 and W10 treatment on peach Monilinia fructicola lesion diameter and inhibition rate (sublow temperature) ;

[0035] Figure 5 Effect of D44 and W10 treatment on peach Monilinia fructicola lesion diameter and inhibition rate (low temperature) ;

[0036] Figure 6 Inhibition effect of biocontrol bacteria D44 and W10 on peach Monilinia fructicola lesion diameter (room temperature) ;

[0037] Figure 7 Changes of ‘Hu you peach 018’ fruit weight loss rate, rot rate, firmness and soluble solid content during room temperature preservation;

[0038] Figure 8 Changes of ‘Hu you peach 018’ fruit weight loss rate, rot rate, firmness and soluble solid content during sublow temperature preservation (sublow temperature) ;

[0039] Figure 9 Changes of ‘Hu you peach 018’ fruit weight loss rate, rot rate, firmness and soluble solid content during low temperature preservation (low temperature) ;

[0040] Figure 10 Changes of ‘Hujing melu’ watermelon fruit weight loss rate, rot rate, firmness and soluble solid content during room temperature preservation;

[0041] Figure 11 Inhibition effect of D44 and W10 on peach Monilinia fructicola lesion diameter (room temperature) ;

[0042] Figure 12 Inhibition effect of D44 and W10 on peach Monilinia fructicola lesion diameter (room temperature). DETAILED DESCRIPTION

[0043] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0044] The application will be further described with reference to the following examples, but the examples are not intended to limit the application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0045] Experimental methods:

[0046] 1) D44 strain extraction: Take fresh Morel, rinse with sterile water for about 5 minutes. Use filter paper to absorb the surface moisture, rinse twice with sterile water, soak in 75% ethanol for 3 minutes, then rinse with sterile water 3 times; then soak in 3% sodium hypochlorite for 3 minutes, and finally rinse with sterile water 4 times.

[0047] 2) Biocontrol bacteria culture: Use a sterile scalpel to remove the epidermis of the Morel fruiting body, cut the internal tissue into small pieces about 0.5 cm, and inoculate on NB, Gao's No. 1 medium and PDA medium plates, with the last rinse water as a control. 122 strains of Morel endophytic bacteria were isolated by "tissue separation method", and identified as 25 species of bacteria by 16S rDNA gene amplification. Marked as A4, A5, A8, A9, A13, A15, A16, D1, D7, D19, D35, D38, D39, D44, D51, D62, D75, D78, D80, E5, E6, E7, F1, F6, F10 and F13. Then the endophytic strain D44 with high inhibition rate to pathogen was screened out by "plate confrontation method" and cultured in a constant temperature incubator at 37°C, 25°C and 28°C respectively.

[0048] 3) Preparation of bacterial solution: The culture temperature of D44 is 37°C, the optimum pH range is usually between 6.5 and 7.5, and the colony can be formed after 24h culture in LB agar medium. The bacterial suspension state of D44 is obtained by using shaking culture method. The bacterial concentration is 10 9 ~ 10 10 cfu·mL -1 .

[0049] 4) Selection of samples: Select 'Shanghai Oil Peach 018', 'Hujing Meilu' watermelon and 'Jinxiu Huangtao' fruits with uniform size, consistent maturity (80% mature), no pests and no mechanical damage.

[0050] 5) Screening of appropriate concentration: Dilute the fermentation broth mother liquor 10 times (10 8 ~ 10 9 cfu / mL), 100 times (10 7 ~ 10 8 cfu / mL), 1000 times (10 6 ~ 10 7 cfu / mL) and 10000 times (10 5 ~ 10 6 cfu / mL) respectively, and spray on the surface of the fruits. Observe whether there will be pesticide damage. The concentration of 10 7 ~ 10 10 cfu / mL will cause pesticide damage to the fruits (yellowing of the fruit skin and rust block). The appropriate treatment concentration is 105 ~ 10 6 cfu / mL.

[0051] 6) Sample processing: The modulated and mixed B. velezensis D44 bacterial solution was sprayed on the surface of peaches using an atomizing device, with an effective spraying concentration of 10 5 ~ 10 6 cfu / mL. Then, it was placed under a blower at room temperature (25°C) for 2 h, and then at room temperature for another 4 h, until no water mist appeared on the surface of the fruit, so that the bacterial solution could rapidly proliferate and form a film.

[0052] 7) Inoculation: After disinfecting the surface of the peach fruit with 75% ethanol and drying, a puncher was used to cut a 3 mm deep and 6 mm diameter piece of peel from the fruit surface, and 50 μL of the biocontrol bacteria was inoculated into the hole, with sterile water and B. licheniformis W10 as controls. After 1 day, a 6 mm diameter piece of peach brown rot fungus or Rhizopus mycelium was inoculated. The test temperature was 25°C, and the relative humidity (RH) was 80%-95%.

[0053] 8) Packaging in preservative bags: In order to better maintain the relative humidity in the bag, reduce the weight loss rate of the fruit, and prevent the harm caused by carbon dioxide generated by the metabolism of the fruit in the preservative bag, the dried fruit was placed in a plastic basket lined with a 0.02 mm-0.04 mm thick, 5‰ porosity (air permeability) PE anti-fog bag, and was placed in a single layer with the opening exposed, which better maintained the relative humidity in the bag. The anti-fog bag with a thickness of 0.04 mm had the best effect.

[0054] 9) Pre-cooling: The packaged fruit was placed in an environment with a temperature of 10±1°C for pre-cooling for 10-12 h, so that the bacterial solution could further proliferate and form a film; then it was placed in an environment with a temperature of 1±0.5°C for pre-cooling for 8-10 h, and then was sealed with a seal. Pre-cooling the peach fruit at two temperature gradients can significantly improve the film-forming effect and prevent the accumulation of respiratory heat in the core caused by instantaneous low temperature.

[0055] 10) Storage and transportation: The pre-cooled fruit was stored or transported at room temperature (25°C), sub-low temperature (12-15°C), and low temperature (1-3°C), which can significantly reduce the weight loss rate and rot rate of the fruit during room temperature express and shelf placement, delay the onset of disease, better maintain the inherent color and quality of the fruit, inhibit the generation of off-flavors, and prolong the shelf life.

[0056] 11) Safe storage period: B. velezensis D44 bacterial solution treatment can significantly reduce the weight loss rate and rot rate of the fruit during room temperature express and shelf placement, delay the onset of disease, better maintain the inherent color and quality of the fruit, inhibit the generation of off-flavors, and prolong the shelf life. It is suitable for storage or transportation under different temperature conditions (room temperature, sub-low temperature, and low temperature).

[0057] The safe storage and circulation period of the D44-treated 'Hulvyou 018' fruits at room temperature can reach 6-8 days, which is 4-6 days longer than the control group, the rot rate is less than 5%, the weight loss rate is less than 4.5%, and the soluble solid content of the fruits decreases by less than 8%. The safe storage and circulation period of the D44-treated 'Hujingmilu' fruits at room temperature can reach 4-6 days, which is 2-4 days longer than the control group, the rot rate is less than 8%, the weight loss rate is less than 8%, and the soluble solid content of the fruits decreases by less than 4%, which has high commercial and edible value.

[0058] Under the condition of sub-low temperature, the safe storage and circulation period of the D44-treated 'Hulvyou 018' fruits at room temperature can reach 16-20 days, which is 8-12 days longer than the control group, the rot rate is less than 5%, the weight loss rate is less than 7%, the soluble solid content of the fruits decreases by less than 5%, the fruit skin hardness is 1.5 kg·cm -2 , and the flesh tissue hardness is 1 kg·cm -2 , which still has high transportation, sales and edible value.

[0059] Under the condition of low temperature, the safe storage and circulation period of the D44-treated 'Hulvyou 018' fruits at room temperature can reach 24-30 days, which is 12-18 days longer than the control group, no rot occurs, the weight loss rate is less than 8%, the soluble solid content of the fruits decreases by less than 5%, the fruit skin hardness is 4-5 kg·cm -2 , and the flesh tissue hardness is 2-3 kg·cm -2 , the fruits can normally ripen and soften during room temperature storage, and have high transportation, sales and edible value.

[0060] Example 1

[0061] The biocontrol bacteria Bacillus licheniformis W10 strain was isolated from the rhizosphere soil of tomatoes by the team of Yangzhou University and has been commercialized as a mature product. Bacillus subtilis A9 and Bacillus velezensis D44 were isolated from the endophytic fungi of Morchella by the team. Peach pink polyphialidium, penicillium, fusarium, brown rot fungus, botrytis, alternaria, and rhizopus were isolated from the rotten peach fruits during storage by the laboratory. The seven pathogenic fungi were inoculated in the center of PDA plates, and the biocontrol bacteria were inoculated in four areas 2.5 cm away from the center of the plates by streaking method.

[0062] The biocontrol bacteria Bacillus licheniformis W10 strain was isolated from the rhizosphere soil of tomatoes by the team of Yangzhou University and has been commercialized as a mature product. Bacillus subtilis A9 and Bacillus velezensis D44 were isolated from the endophytic fungi of Morchella by the team. Peach pink polyphialidium, penicillium, fusarium, brown rot fungus, botrytis, alternaria, and rhizopus were isolated from the rotten peach fruits during storage by the laboratory. The seven pathogenic fungi were inoculated in the center of PDA plates, and the biocontrol bacteria were inoculated in four areas 2.5 cm away from the center of the plates by streaking method. Figure 1As shown in Table 1, *Bacillus velezensis* D44 and *Bacillus licheniformis* W10 exhibit significant antagonistic effects against seven pathogens: *Pseudomonas pulmonarius*, *Penicillium*, *Fusarium*, *Brown rot*, *Botrytis*, *Alternaria*, and *Rhizopus*. *Bacillus subtilis* A9 only shows significant antagonistic effects against *Pseudomonas pulmonarius*, with weaker antagonistic effects against other pathogens. This indicates that *Bacillus velezensis* D44 and *Bacillus licheniformis* W10 have the potential to control major postharvest pathogens of peaches, especially *Brown rot* and *Rhizopus*. The next step will be to conduct in vivo functional verification and preservation storage experiments using strains D44 and W10 as test materials.

[0063] Table 1. Antagonistic effects of three biocontrol bacteria against pathogens.

[0064] Pathogenic bacteria Latin name W10 D44 A9 Pink syncephalastrum Trichothecium roseum √ √ √ Penicillium spp. Weak √ √ Fusarium spp. Weak Monilinia fructicola √ √ Weak Botrytis cinnerea Weak √ √ Alternaria spp. Weak Weak √ √ Rhizopus stolonifer Weak Figure 2 √ Figure 2 Figure 3 Figure 3 Figure 4 √ √ Figure 4

[0065] Example 2 Screening of pesticide-free treatment concentrations

[0066] Dilute the mother liquor 10 times with double-distilled water (10... 8 ~10 9 cfu / mL), 100 times (10 7 ~10 8 cfu / mL), 1000 times (10 6 ~10 7 cfu / mL) and 10000 times (10 5 ~10 6 Prepare the bacterial solution (cfu / mL) for later use. Take healthy nectarines and spray the surface of the fruit with an 80% ethanol solution for disinfection. Let it stand for 5 minutes, then rinse with water for 10 seconds. Afterward, use a misting device to spray different concentrations of bacterial solution onto the surface of healthy nectarines and peaches, blow dry or air dry, and place at room temperature (25℃) for 6 hours to observe whether any chemical damage occurs.

[0067] Depend on Figure 5 It can be known that the concentration is 10. 7 ~10 10 Treatment at concentrations of CFU / mL all caused significant phytotoxicity to the fruit (discoloration of the fruit skin and the appearance of rust spots), with a concentration of 10% showing the best results. 5 ~10 6 Treatment with cfu / mL can preserve the fruit's inherent color. Next, 10... 5 ~10 6 Further experiments were conducted using a concentration of cfu / mL.

[0068] Example 3: Inhibitory effect of D44 on postharvest brown rot fungi and Rhizopus fungi in peach fruit under different temperature storage conditions

[0069] Based on preliminary experiments screening three biocontrol bacteria concentrations, 'Huyoutao 018' and 'Hujing Milu' peaches were used as test materials, with 10... 5 ~10 6 The bacterial concentrations (cfu / mL) were analyzed and compared. After disinfecting the peach surface with 75% ethanol and air-drying, 3mm deep and 6mm diameter pieces of peel were cut from the fruit surface using a punch. 50μL of biocontrol bacteria were inoculated into each hole, with sterile water and Bacillus licheniformis W10 as controls. One day later, 6mm diameter mycelial blocks of *Prunella vulgaris* and *Rhizopus* were inoculated. The inhibitory effects on postharvest *Prunella vulgaris* and *Rhizopus* were observed under normal temperature (25℃), sub-low temperature (12–15℃), and low temperature (1–3℃) conditions. The inhibition results against *Prunella vulgaris* are shown in Table 2. Inhibition rate (%) = [(control fruit spot area - treated fruit spot area) / control fruit spot area] × 100, fruit spot area = π(d / 2)², where d is the average diameter of the fruit lesion.

[0070] Table 2 shows the effects of D44 and W10 bacterial suspension treatments on the inhibition rate and onset time of peach brown rot.

[0071]

[0072] (1) Inhibitory effects of D44 and W10 on brown rot fungi in nectarines and peaches (at room temperature)

[0073] Depend on Figure 6 , Figure 6 As shown in Table 2, after inoculation with brown rot fungus and storage at room temperature (25℃) for 3 days, the diameters of lesions on the control group 'Huyoutao 018' and 'Hujing Milu' peaches were 6.47 cm and 4.38 cm, respectively, indicating that 'Huyoutao 018' was most susceptible to brown rot fungus. Treatment with D44 and W10 bacterial solutions significantly inhibited the diameter of brown rot lesions, but the inhibitory effect weakened with prolonged storage. There was no significant difference in the onset time of brown rot in the D44 and W10 treated groups. The onset time of brown rot in both 'Huyoutao 018' and 'Hujing Milu' peaches sprayed with D44 and W10 bacterial solutions was 3 days, 2 days later than the control group, with an inhibition rate as high as 85%–99%, demonstrating significant efficacy. This further illustrates the potential of D44 and W10 strains to control peach brown rot.

[0074] (2) Inhibitory effects of D44 and W10 on brown rot fungi in nectarines and peaches (sub-low temperature)

[0075] Depend on Figure 7As shown in Table 2, after inoculation with brown rot fungus and storage at sub-low temperature (12-15℃) for 3 days, the diameters of lesions on the control group 'Huyoutao 018' and 'Hujing Milu' peaches were 3.19 cm and 1.32 cm, respectively, further indicating that 'Huyoutao 018' is susceptible to brown rot fungus. Both D44 and W10 bacterial suspensions significantly inhibited the diameter of brown rot lesions, but the inhibitory effect weakened with prolonged storage. The onset time of disease in the D44 and W10 treated peaches was 1 day and 2 days, respectively. D44 treatment delayed the onset of disease in inoculated peaches by 1 day and significantly improved the inhibition rate. The onset time of disease in the D44 and W10 treated peaches was 3 days and 4 days, respectively, delayed by 2 days and 1 day compared to the control group. The inhibition rate of the D44 treated fruit reached 99.19%, showing a significant effect. This indicates that strain D44 still has the potential to control peach brown rot under sub-low temperature conditions.

[0076] (3) Inhibitory effects of D44 and W10 on brown rot fungi in nectarines and peaches (low temperature)

[0077] Depend on Figure 7 As shown in Table 2, after inoculation with brown rot fungus and storage at low temperature (1–3℃) for 8 days, the diameter of lesions on the 'Huyoutao 018' and 'Hujing Milu' peaches in the control group was 1.08 cm and 0.12 cm, respectively. Low temperature inhibited the incidence rate of brown rot. The onset time of the disease in the D44 and W10 treatment groups was 8 days and 4 days, respectively. The D44 treatment delayed the onset of the disease in the brown rot-inoculated peaches by 4 days and significantly improved the inhibition rate of the fruit. The onset time of the disease in the D44 and W10 treatment groups was 14 days and 12 days, respectively, which was 6 days and 4 days later than the control group.

[0078] (4) Inhibitory effect of biocontrol bacteria D44 and W10 on Rhizopus pyrifolia (room temperature)

[0079] Depend on Figure 8 , Figure 8 As shown in Table 3, after inoculation with Rhizopus and storage at room temperature (25℃) for 6 days, the diameters of lesions on the control group 'Huyoutao 018' and 'Hujing Milu' peaches were 5.49 cm and 2.87 cm, respectively. This indicates that 'Huyoutao 018' was most susceptible to Rhizopus infection, while 'Hujing Milu' peaches were more resistant to soft rot caused by Rhizopus. Both D44 and W10 bacterial solutions significantly delayed the incidence of postharvest soft rot in the three types of peaches. D44 bacterial solution showed better and more stable control effects on soft rot in the three types of peaches, delaying the onset of soft rot in 'Huyoutao 018' and 'Hujing Milu' peaches by 3 days compared to the control group, with a disease inhibition rate as high as 87.61%–99.82%.

[0080] As shown in Table 3, after inoculation with Rhizopus, under sub-low temperature storage conditions, the fruits in the control group developed disease on day 14, while the fruits (nectarines and peaches) in the D44 and W10 bacterial solution treatment groups developed disease 14 days later than the control group. The inhibition rate of Rhizopus by the D44 treatment was significantly higher than that of the W10 treatment group, indicating that the treatment effect of D44 bacterial solution was better under sub-low temperature conditions.

[0081] As shown in Table 3, after inoculation with Rhizopus, under low-temperature storage conditions, the fruits in the control group developed the disease on day 21, while the fruits (nectarines and peaches) in the D44 and W10 bacterial solution treatment groups developed the disease 14 days later than the control group. Moreover, the inhibition rate of Rhizopus by the D44 treatment group was significantly higher than that of the W10 treatment group, indicating that the D44 bacterial solution can still play a better role in inhibiting Rhizopus under low-temperature conditions.

[0082] Table 3. Effects of D44 and W10 bacterial suspension treatments on the inhibition rate and disease onset time of *Rhizopus sylvestris*.

[0083]

[0084] Example 4: Effects of D44 and W10 treatments on the preservation effect of 'Huyoutao 018' fruit under different temperature storage conditions

[0085] (1) Effects of D44 and W10 treatments on weight loss, decay rate, fruit firmness and soluble solids content of 'Huyoutao 018' fruit (at room temperature)

[0086] Depend on Figure 8 It was found that during the entire storage period, the weight loss and decay rates of the control group 'Hu You Tao 018' fruit showed a sharp upward trend, indicating that water loss and decay were the main problems for nectarines on a room-temperature shelf. Both D44 and W10 treatments significantly inhibited post-harvest water loss and decay, but there was no significant difference in the effect of D44 and W10 bacterial solutions on fruit weight loss. In the later stages of storage (4-8 days), the decay rate of the fruit in the D44 bacterial solution treatment group was significantly lower than that in the W10 treatment group and the control group. By day 6 of storage, no decay had occurred in the D44 treatment group; by day 8, the decay rate of the fruit in the D44 treatment group was only 5.56%, significantly lower than the 44.44% of the control group.

[0087] Depend on Figure 9 It can be seen that during the entire storage period, the firmness of the fruit with peel and the firmness of the pulp tissue both showed a sharp downward trend, and there was no significant difference between the different treatment groups. After 2-6 days of storage, the soluble solids content of the fruit in the D44 bacterial solution treatment group was significantly higher than that of the control group, while there was no significant difference in the soluble solids content of the fruit in the W10 treatment group, the control group, and the D44 treatment group.

[0088] (2) Effects of D44 and W10 treatments on the weight loss, decay rate, fruit firmness and soluble solids content of 'Huyoutao 018' fruit (sub-low temperature)

[0089] Depend on ​ It was found that during the entire storage period, the weight loss and rot rates of the control group 'Hu You Tao 018' fruit showed a sharp upward trend, indicating that water loss and rot remained the main problems in the sub-low temperature storage of nectarines. Both D44 and W10 treatments significantly inhibited postharvest water loss and rot, with D44 bacterial solution having a more significant effect on fruit weight loss and rot rates. In the later stages of storage (8-16 days), the rot rate of the fruit in the D44 bacterial solution treatment group was significantly lower than that in the W10 treatment group and the control group. By day 16 of storage, no rot occurred in the D44 treatment group, with a weight loss rate of 3.4%, significantly lower than that in the control group and the W10 treatment group. By day 20, the rot rate of the fruit in the D44 treatment group was still below 5%, significantly lower than the 22.73% of the control group.

[0090] Depend on ​ It can be seen that during the entire storage period, the firmness of the fruit with peel and the firmness of the pulp tissue both showed a sharp downward trend, and there was no significant difference between the different treatment groups; the soluble solids content of the fruit in the D44 bacterial solution treatment group was not significantly different from that of the control group and the W10 treatment group.

[0091] (3) Effects of D44 and W10 treatments on the weight loss, decay rate, fruit firmness and soluble solids content of 'Huyoutao 018' fruit (low temperature)

[0092] Depend on ​ It was found that during the entire storage period, the weight loss and rot rates of the control group 'Hu You Tao 018' fruit showed a sharp upward trend, indicating that water loss and rot remained the main problems in low-temperature storage of nectarines. Both D44 and W10 treatments significantly inhibited postharvest water loss and rot, with D44 bacterial solution having a more significant effect on fruit rot rate. In the later stages of storage (12-24 days), the rot rates of the D44 and W10 bacterial solution treatment groups were significantly lower than those of the control group. By day 24 of storage, no rot occurred in the D44 treatment group, significantly lower than that in the control and W10 treatment groups. The weight loss rates of the D44 and W10 bacterial solution treatment groups were significantly lower than those of the control group, with no significant difference between the two groups.

[0093] Depend on ​ It was found that during low-temperature storage (12-30 days), the firmness of the fruit with skin and the firmness of the pulp tissue in the D44 bacterial solution treatment group were significantly higher than those in the control group and the W10 treatment group. Furthermore, the W10 treatment accelerated the softening rate of the fruit during cold storage. There was no significant difference in the soluble solids content of the fruit among the different treatment groups.

[0094] Example 5

[0095] (1) Effects of D44 and W10 on weight loss and decay rate of 'Lakeview Honey Dew' peaches

[0096] Depend on ​ It can be seen that on the 4th day of shelf life, the weight loss rate and decay rate of the control group fruit were as high as 13.24% and 22.22%, respectively. The D44 and W10 treatments can significantly reduce the weight loss rate and decay rate of the fruit, especially the decay rate. (2) Effects of D44 and W10 on the firmness and soluble solids content of peach fruit

[0097] Depend on ​ It can be seen that the D44 and W10 bacterial solutions had no significant effect on the firmness of the fruit with skin and the firmness of the pulp tissue, and could effectively inhibit the decrease in the soluble solids content of the fruit in the later stage of storage (4-6 days).

[0098] The embodiments described above are merely preferred embodiments provided to better explain the present invention. Therefore, the above embodiments are not intended to limit the present invention. Modifications, improvements, and substitutions made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Bacillus belye (Bacillus velezensis) D44 antagonizes Rhizopus in peaches Rhizopus stoloniferous and brown rot fungi Monilinia fructicola, And its application in extending the post-harvest storage and preservation time of peaches, including Bacillus belye Bacillus velezensis The preservation number of D44 is: CCTCC NO:M 20221701. The peaches are stored at sub-low temperature or low temperature after harvesting. The peaches are nectarines or white peaches.

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