A biocontrol serratia marcescens and its use for postharvest disease control and storage preservation of pear fruit

Abstract

The present application belongs to the technical field of postharvest disease control, and particularly relates to a biocontrol Serratia marcescens and its use for postharvest disease control and storage of pears. The steps are as follows: the Serratia marcescens is activated and cultured, and the bacterial cells are obtained by centrifugation after the culture; the bacterial cells are resuspended in sterile physiological saline to obtain a bacterial suspension; the bacterial suspension is injected into the holes punched on the surface of the pears, and then an equal volume of Penicillium expansum spore suspension is injected, so that the effective control of the Penicillium disease caused by Penicillium expansum is achieved; or the bacterial suspension of the Serratia marcescens is uniformly sprayed on the surface of the pears, and then naturally dried, so that the use for postharvest disease control and storage of the pears is achieved; the present application can significantly reduce the occurrence of the postharvest Penicillium disease of the pears, and reduce the natural rot rate of the pears after harvest, and is safe and environmentally friendly, and not only has no significant adverse effect on the main quality indicators of the postharvest pears, but also can maintain the storage quality of the pears, and achieves a significant technical effect, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for postharvest diseases, specifically relating to a method for using Serratia marcescens to control and preserve pear fruit after harvesting Penicillium rot. Background Technology

[0002] Pears are a globally popular fruit, considered a superior nutritious fruit and often referred to as the "king of fruits." However, during post-harvest storage and transportation, pears are susceptible to spoilage due to pathogenic microorganisms, significantly reducing their commercial value and causing substantial economic losses. *Penicillium expansum* is the main pathogen causing post-harvest pear mold disease. It not only causes fruit rot but also secretes patulin (PAT), which has carcinogenic, teratogenic, and mutagenic effects, endangering the health of consumers. Therefore, it is necessary to research a safe and effective method to control the occurrence of post-harvest pear mold disease.

[0003] Currently, the main methods for controlling postharvest diseases of fruits and vegetables include physical methods, chemical methods, and biological control methods. Physical methods, such as heat treatment, refrigeration, and controlled atmosphere storage, can inhibit the activity of pathogens and the accumulation of toxins to varying degrees; however, they require sophisticated equipment, are difficult to industrialize, and have significant limitations in practical application. Chemical methods typically use chemical fungicides to control postharvest diseases of fruits and vegetables, and are currently the most common method. This method is low-cost, fast-acting, and simple to operate, but long-term use can lead to drug resistance in pathogens and cause environmental pollution. Biological control utilizes antagonistic microorganisms of pathogens to control fruit and vegetable diseases. Compared to physical and chemical methods, it has advantages such as safety and environmental friendliness. However, the use of antagonistic microorganisms to control fruit and vegetable diseases often faces the problem of unsatisfactory results. Moreover, research on using antagonistic bacteria to control postharvest diseases of pears is rarely reported. Summary of the Invention

[0004] To address the shortcomings and deficiencies of existing technologies, this invention provides a strain of bacteria—Serratia rubidaea—isolated and purified in our laboratory. The strain's accession number is CCTCC NO: M 20241377, and it is deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China. The deposit date is June 26, 2024. The suggested classification name is Serratia rubidaea B11.

[0005] The *Serratia marcescens* strain in this invention was obtained through self-screening and identification. Currently, it has not been used in the prevention and control of postharvest diseases in pears. Through research, it has been found that it has strong antagonistic efficacy. This bacterium can effectively control the occurrence of postharvest blue mold and natural rot in pears, reduce the rot rate, and reduce losses caused by postharvest diseases. Furthermore, it has no significant adverse effects on the quality of pears and can slow down the quality deterioration of pears during storage, thereby achieving the effect of storage and preservation, providing a theoretical basis for its application in postharvest preservation.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The *Serratia marcescens* strain provided by this invention was isolated and purified in our laboratory and is currently deposited at the China Center for Type Culture Collection (CCTCC) with the strain accession number CCTCC NO: M 20241377.

[0008] The application of *Serratia marcescens* for postharvest disease control in pear fruit, provided by this invention, is carried out according to the following steps:

[0009] (1) First, *Serratia marcescens* was inoculated into LB medium for the first activation culture to obtain the activated solution; then, the activated solution was transferred to a new LB medium for the second activation to obtain the bacterial culture broth. After centrifugation, bacterial sludge was obtained, washed with sterile physiological saline, and then prepared into a *Serratia marcescens* bacterial suspension with a concentration of 1×10⁻⁶. 6 ~1×10 9 cells / mL, ready for use;

[0010] (2) Select pears that are free from disease and mechanical damage and have uniform color and size, disinfect them, rinse them with running water, and put them in a disinfected plastic basket to dry. Create wounds of uniform size and depth on the equatorial part of the dried pears, add the Serratia marcescens suspension prepared in step (1) to each wound, let it stand for a period of time, inoculate with Penicillium spore suspension, let it dry naturally, put it in a disinfected plastic basket, seal it with plastic wrap, and place it in a constant temperature and humidity incubator to achieve the control of postharvest Penicillium rot on pears by Serratia marcescens.

[0011] Alternatively, select pears that are free from disease and mechanical damage, have uniform color, and are similar in size. Without any disinfection treatment, keep the pears in their natural state and evenly spray the Serratia marcescens suspension prepared in step (1) on the surface of the pears. After air-drying, the pears can be used for post-harvest disease prevention and control and storage preservation.

[0012] Preferably, the LB culture medium (in 1L) mentioned in step (1) is: 10g tryptone, 5g yeast extract, 10g sodium chloride, distilled water to a final volume of 1000mL, natural pH, sterilized at 115℃ for 20min.

[0013] Preferably, the culture conditions for the first and second activation cultures in step (1) are: 37℃ for 16-18h; and the centrifugation conditions are: 4℃, 8000rpm, for 10-15min.

[0014] Preferably, the inoculum amount of the activation solution transferred in step (1) is 1-2% (v / v); the concentration of *Serratia marcescens* is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0015] Preferably, the disinfection process in step (2) is as follows: the pears are immersed in water containing 0.2% sodium hypochlorite solution for 2-4 minutes for disinfection; the plastic baskets are disinfected by rinsing them with clean water and drying them, and then spraying them evenly with 75% alcohol to obtain disinfected plastic baskets.

[0016] Preferably, the diameter of the hole in step (2) is 5mm and the depth is 4mm.

[0017] Preferably, in step (2), the volume ratio of bacterial suspension to Penicillium spore suspension added to each wound is 1:1; the concentration of the Penicillium spore suspension is 1×10⁻⁶. 5 spores / mL; the standing time is 2 hours.

[0018] Preferably, the temperature of the constant temperature and humidity incubator in step (2) is 20-25℃ and the relative humidity is 90%.

[0019] Preferably, in step (2), the Serratia marcescens suspension is sprayed evenly on the surface of the pear fruit, with a specific dosage of 0.02 to 0.05 mL of suspension per square centimeter.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] (1) The Serratia marcescens used in this invention was isolated and purified in our laboratory and verified as a non-toxic bacterium by acute toxicity test in mice, and is safe and harmless to the human body.

[0022] (2) The Serratia marcescens used in this invention can effectively control postharvest blue mold and natural rot of pears. It requires a small amount and has a significant effect. At the same time, it has no adverse effect on the quality of pears during postharvest storage, such as hardness, soluble solids and ascorbic acid content. It can also slow down the quality deterioration during storage, thereby controlling the decline in tomato quality and facilitating the storage and preservation of pears.

[0023] (3) There are currently no reports on the application of the deep red Serratia in the prevention and control of postharvest diseases of pear fruit in this invention, which is original. It has good biocontrol effect and can replace or reduce the use of chemical fungicides to control postharvest diseases of pear fruit. At the same time, it reduces the harm of chemical fungicides to consumers' health and the environment. It has the advantages of being green and environmentally friendly and has good market application prospects. Attached Figure Description

[0024] Figure 1 The study investigated the control effect of different concentrations of *Serratia marcescens* on postharvest Penicillium mold in pear fruit; (A) Figure shows the pear fruit rot rate, and (B) Figure shows the rot diameter; CK is the control group, consisting of pear fruit treated with sterile physiological saline; 10^6, 10^7, 10^8, and 10^9 represent the concentrations of 1×10^6, 10^7, 10^8, and 10^9, respectively. 6 1×10 7 1×10 8 and 1×10 9 Pears treated with Serratia marcescens at CFU / mL; the concentration of pathogen used was 1×10⁻⁶. 5 CFU / mL; different lowercase letters indicate significant differences (p<0.05).

[0025] Figure 2 (A) shows the effect of *Serratia marcescens* on the natural decay of pear fruit; (B) shows firmness; (C) shows soluble solids; (D) shows titratable acid; (E) shows ascorbic acid; and (F) shows browning degree. Note: CK is the control group, pear fruit treated with sterile physiological saline; B represents 1×10⁻⁶ pears. 8 Pears treated with CFU / mL Serratia marcescens suspension; "*" indicates significant difference (P<0.05). Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] The *Serratia marcescens* strain provided by this invention was isolated and purified in our laboratory; it is currently deposited at the China Center for Type Culture Collection (CCTCC), with the strain accession number CCTCC NO: M 20241377.

[0029] The culture procedure for Serratia marcescens is as follows:

[0030] (1) First activation: Pick 2 loops of Serratia deep red from the LB slant and inoculate them into 50 mL of LB medium. Incubate at 37℃ and 180 rpm for 16 h to carry out the first activation and obtain the activated solution.

[0031] (2) Liquid secondary activation: Use a sterilization pipette tip to draw 1 mL of activation solution into another bottle of 50 mL LB medium, and incubate at 37℃ and 180 rpm for 16 h to obtain bacterial culture solution;

[0032] (3) Centrifugation and resuspension: The bacterial culture broth from (2) above was centrifuged at 4℃ and 8000 rpm for 10 min, and washed twice with sterile physiological saline to remove the culture medium, obtaining bacterial sludge. Finally, it was resuspended with sterile physiological saline and adjusted to the required bacterial concentration (1×10⁻⁶). 8 (CFU / mL)

[0033] Safety of Serratia marcescens;

[0034] I. Test Plan

[0035] Preparation of *Serratia marcescens* bacterial sludge: *Serratia marcescens* was inoculated into Erlenmeyer flasks containing 50 mL of LB medium and cultured at 37°C for 16 h on a shaker. The sludge was then obtained by centrifugation, washed, and diluted with pure water to a concentration of 500 mg / mL. The bacterial solution administered to mice via gavage was derived from the dilution of fresh bacterial sludge.

[0036] The test species was SPF-grade KM mice. Twenty KM mice, weighing between 18g and 22g, were selected, with half males and half females. Prior to the experiment, the mice were pre-fed in a laboratory barrier-controlled animal facility for 3 days to acclimatize to the environment and for quarantine observation, and were fasted for 4 hours before the experiment. The test substance was administered once by gavage at a volume of 20mL / kg body weight, followed by observation for 14 days. Throughout the experiment, the mice's activity, any abnormal behaviors, and signs of poisoning or death were comprehensively observed and recorded. If any abnormalities or deaths occurred, the time, degree, and duration of the changes were recorded, and potential target organs for toxicity were assessed. Animal weight was weighed and recorded at the start of the experiment and weekly during the observation period.

[0037] II. Test Results

[0038] Following the above experimental procedures, the safety test results of Serratia marcescens are as follows:

[0039] Mice administered Serratia marcescens by gavage did not die within 14 days, and no poisoning or other abnormalities were observed during the entire experiment. Table 1 shows that the acute oral LD50 of this strain in both female and male KM mice was greater than 10000 mg / kg·bw. According to the acute toxicity classification standard, Serratia marcescens is considered a safe and non-toxic bacterium, suitable for use in the control of postharvest diseases and storage preservation of pear fruit.

[0040] Table 1 Results of acute oral toxicity test of Serratia marcescens in mice

[0041]

[0042] As shown in Table 1, the animal acute toxicity test confirmed that it is safe and non-toxic.

[0043] Example 1:

[0044] Serratia marcescens is used to control postharvest blue mold in pears;

[0045] I. Test Plan

[0046] Select disease-free, mechanically undamaged pears of uniform color and size. Soak them in a 0.2% sodium hypochlorite solution for 2 minutes, then rinse with running water. Place the rinsed pears in a sterilized plastic basket to air dry (the basket is sterilized by rinsing with water, air-drying, and then spraying evenly with 75% alcohol). Create 5mm × 4mm (diameter × depth) wounds at the equatorial region of the dried pears using a sterile punch. Add 30μL of *Serratia marcescens* bacterial suspension (1×10⁻⁶) to each wound. 8 CFU / mL). After incubation at room temperature for 2 hours, inject 30 μL of Penicillium spore suspension (1×10⁻⁶ CFU / mL) into the wound. 5 (CFU / mL). After air-drying at room temperature, store in a constant temperature and humidity incubator (25℃, 90% relative humidity) for 3-4 days, and record the incidence rate and diameter of lesions on the pear fruits. Pear fruits treated with sterile physiological saline instead of Serratia marcescens suspension served as controls. Each treatment was repeated in triplicate, with 12 pear fruits per replicate.

[0047] Rot rate = (Number of rotten pores / Total number of pores) × 100%

[0048] II. Test Results

[0049] Depend on Figure 1 As shown in Figure (A), after 3 days of storage, the decay rate of the control group had reached 100%, 1×10 6 1×10 7 1×10 8 and 1×10 9The incidence rates of Penicillium rot in pears treated with CFU / mL *Serratia marcescens* were 52.38%, 25.40%, 15.87%, and 15.87%, respectively, all significantly lower than the control group. After 4 days of storage, the rot rate of pears treated with different concentrations of *Serratia marcescens* was significantly lower than the control group, and the control efficacy increased with increasing concentration. Specifically, the control efficacy was highest at 1×10⁻⁶ CFU / mL. 8 and 1×10 9 The pears treated with CFU / mL of Serratia marcescens had rot rates of 38.09% and 20.63%, respectively, which were significantly lower than those of the control group (p<0.05).

[0050] Depend on Figure 1 As shown in Figure (B), similar to the results of the decay rate, on the 3rd day of storage, 1×10 6 1×10 7 1×10 8 and 1×10 9 The diameter of rotten pears treated with CFU / mL *Serratia marcescens* was significantly lower than that of the control group (15.38 mm) (p<0.05); on the 4th day of storage, the diameter of rotten pears treated with 1×10⁻⁶ *Serratia marcescens* was significantly lower than that of the control group (15.38 mm) (p<0.05); 8 and 1×10 9 The diameters of decayed pears treated with CFU / mL were 11.76 mm and 11.55 mm, respectively. At this point, the control group and the 1×10⁻⁶ CFU / mL treatment... 6 and 1×10 7 The rot diameters of pears treated with CFU / m concentrations were 21.71 mm, 14.21 mm, and 13.14 mm, respectively, showing significant differences.

[0051] Considering that different concentrations of *Serratia marcescens* can significantly reduce the occurrence of postharvest Penicillium rot in pears, taking into account the cost in actual use, and combining the rot diameter index, a concentration of 1×10⁻⁶ was selected. 8 CFU / mL of Serratia marcescens can be used for the control of Penicillium purpureus, and further experiments will be conducted.

[0052] Example 2:

[0053] Serratia marcescens is used for controlling postharvest natural decay and for storage and preservation of pear fruit;

[0054] I. Test Plan

[0055] Select disease-free, mechanically undamaged, and uniformly colored and sized pears. Do not disinfect them. Keep the pears in their natural state and spray them directly and evenly on the surface of the pears with (1) sterile physiological saline, denoted as CK in the figure; (2) Serratia marcescens suspension (1×10⁻⁶). 8(CFU / mL), indicated by the letter B in the figure; the spraying standard was 0.05 mL of bacterial suspension per square centimeter (the volume of bacterial suspension used per pear was statistically estimated to be 1-1.5 mL); after spraying, the pears were placed in sterilized plastic baskets (sterilized by rinsing with clean water, drying, and then spraying evenly with 75% alcohol). After the pears were allowed to air dry naturally, the plastic baskets were sealed with plastic wrap and stored in a constant temperature and humidity incubator (relative humidity 90%) for different number of days (0, 13, 26, 39, 52 days). The natural decay rate of the pears was recorded, and the firmness, soluble solids content, titratable acid content, ascorbic acid content, and browning degree of the pears were measured. Each treatment was performed in triplicate, with 12 pears per replicate.

[0056] Natural decay rate (%) = (Number of rotten fruits / Total number of fruits) × 100%

[0057] The specific methods for measuring quality indicators are as follows:

[0058] 1. Hardness: The hardness of pears was determined using a TA-XT2i physical property tester. A P5 probe was selected, with a testing speed of 5 mm / s and a testing depth of 5 mm. Three equidistant points along the equator of the pear were chosen for testing. The maximum resistance encountered when the probe was inserted into the pear was recorded as the hardness (N).

[0059] 2. Soluble solids content: 5.0g of fruit sample was ground into a homogenate under low temperature conditions, centrifuged at 4℃ and 4000rpm for 10min, and the supernatant was collected. The soluble solids content of the pear fruit was determined using a handheld refractometer.

[0060] 3. Titratable acid content: Determined by sodium hydroxide titration. Weigh approximately 10g of sample, add an appropriate amount of distilled water, and grind into a homogenate in a mortar. Transfer to a 50mL centrifuge tube and let stand for 30 minutes, shaking once every 10 minutes. Centrifuge at 8000rpm for 10 minutes at 4℃. Transfer the supernatant to a 25mL volumetric flask and dilute to the mark. Transfer 10mL of the sample solution to an Erlenmeyer flask, add 3 drops of 1% phenolphthalein, and titrate with 0.1mol / L NaOH until the solution initially turns a pale pink color that does not fade within 0.5 minutes. Distilled water serves as a blank control. Results are expressed as mass fraction (%).

[0061] Titratable acid content (%) = (V×c×(V1-V0)×0.067) / (Vs×m)×100%

[0062] Where V is the total volume of the sample extract (mL), c is the NaOH concentration (mol / L), V1 is the volume of sodium hydroxide solution consumed in the titration of the sample solution (mL), V0 is the volume of sodium hydroxide solution consumed in the titration of distilled water (mL), Vs is the volume of sample taken during titration (mL), m is the sample mass (g), and 0.067 is the malic acid conversion factor (g / mmol).

[0063] 5. Ascorbic acid content: Determined by 2,6-dichlorophenolindophenol titration. Weigh approximately 10g of pear fruit tissue sample and place it in a mortar. Add an appropriate amount of 1% oxalic acid solution and grind into a homogenate in the mortar under light-protected ice bath conditions. Transfer to a 100mL volumetric flask and dilute to volume with oxalic acid. Shake well, let stand at 4℃ for 10min, then centrifuge and collect the filtrate. Pipette 10mL of the filtrate into an Erlenmeyer flask and titrate with standardized 2,6-dichlorophenolindophenol solution until a light red color appears and does not fade within 15s. Simultaneously, use 10mL of 20g / L oxalic acid solution as a blank for titration to zero the solution. Perform three replicates per group. Calculate the ascorbic acid content in the pear fruit based on the amount of 2,6-dichlorophenolindophenol used, expressed as the mass of ascorbic acid per 100g sample, i.e., mg / 100g.

[0064] Titration T = (c × V) / (V1 - V0)

[0065] Where c is the mass concentration of the ascorbic acid standard solution (mg / mL), V is the volume of the ascorbic acid standard solution taken (mL), V1 is the volume of 2,6-dichlorophenolindophenol solution consumed when titrating the standard liquid (mL), and V0 is the volume of 2,6-dichlorophenolindophenol solution consumed when titrating the blank (1% oxalic acid) (mL).

[0066] Ascorbic acid content (mg / 100g) = (V×(V1-V0)×T) / (Vs×m)×100

[0067] Where V is the total volume of the sample extraction solution (mL), V1 is the volume of dye consumed in the sample titration (mL), V0 is the volume of dye consumed in the blank titration (mL), Vs is the volume of the sample solution taken during titration (mL), and m is the sample mass (g).

[0068] 6. Browning degree

[0069] Take 10g of pear fruit sample and homogenize it with 20mL of distilled water. After centrifugation, take 10mL of the supernatant and mix it with 15mL of 95% ethanol. Centrifuge again and measure the absorbance of the supernatant at 420nm to indicate the degree of browning.

[0070] II. Test Results

[0071] Depend on Figure 2 As can be seen in Figure (A), after 1×108 Pears treated with CFU / mL *Serratia marcescens* showed no signs of rotting after 13 days of storage at 20℃, similar to the control group. On day 26, the rotting rate in the treatment group was 0.00%, while the control group had a rotting rate of 9.09%. On day 39, the rotting rate in the treatment group remained at 0.00%, while the control group reached 18.18%. On day 52, the rotting rates in the treatment group and the control group were 9.09% and 31.82%, respectively. The natural rotting rate in the *Serratia marcescens* treatment group was significantly lower than that in the control group on days 26, 39, and 52 (p<0.05). This demonstrates that *Serratia marcescens* has a significant controlling effect on postharvest natural rotting of pears. Therefore, this application demonstrates that the *Serratia marcescens* strain has excellent efficacy in preventing postharvest natural rotting of pears.

[0072] Depend on Figure 2 As shown in Figure (B), the firmness of pears exhibited a consistent trend during post-harvest storage, decreasing continuously with increasing storage time. However, the firmness of the *Serratia marcescens* treatment group remained consistently higher than that of the control group, with a significant difference observed on day 13 of storage.

[0073] Depend on Figure 2 As shown in Figure (C), during postharvest storage, the soluble solids content of pears generally exhibited a trend of first increasing, then decreasing, and then increasing again. On days 13 and 39 of storage, the soluble solids content in the treatment group was higher than that in the control group, and the difference was significant on day 13.

[0074] Depend on Figure 2 As shown in Figure (D), during the post-harvest storage of pears, the titratable acid content of the treatment group showed a trend of first decreasing and then increasing. The trends of the treatment group and the control group were basically the same, and there was no significant difference between the two.

[0075] Depend on Figure 2 As shown in Figure (E), the ascorbic acid content of pears initially increased and then decreased during postharvest storage. Throughout the storage period, the ascorbic acid content of pears treated with *Serratia marcescens* was slightly higher than that of the control group, but there was no significant difference between the two.

[0076] Depend on Figure 2 (F) It can be seen that throughout the entire post-harvest storage process, the browning degree of pears treated with *Serratia marcescens* was consistently lower than that of the control group. At day 52, there was a significant difference in browning resistance between pears treated with *Serratia marcescens* and the control group, indicating that treatment with *Serratia marcescens* can slow down browning.

[0077] In conclusion, Serratia marcescens has a good effect on preventing natural rot of pear fruit, while having no significant adverse effects on the quality of pear fruit. It can also alleviate the deterioration of pear fruit quality to a certain extent, and can be used for the storage and preservation of pear fruit after harvest.

[0078] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, 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. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. The use of a biocontrol agent, *Serratia marcescens*, for postharvest disease control and storage preservation of pear fruit, characterized in that... The biocontrol strain of Serratia marcescens is Serratia marcescens (… Serratia rubidaea B11, deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M 20241377.

2. The use of the biocontrol agent *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of pear fruit, characterized in that... The specific steps are as follows: (1) First, Serratia marcescens was inoculated into LB medium for the first activation culture to obtain the activated solution; then, the activated solution was transferred to a new LB medium for the second activation to obtain the bacterial culture medium. After centrifugation, the bacterial sludge was obtained, washed with sterile physiological saline, and then prepared into a Serratia marcescens bacterial suspension with a concentration of 1×10⁻⁶. 6 ~1×10 9 cells / mL; (2) Select pears that are free from disease and mechanical damage and have uniform color and size, disinfect them, rinse them with running water, and put them in a disinfected plastic basket to dry. Make wounds of uniform size and depth at the equatorial part of the dried pears, add the Serratia marcescens suspension prepared in step (1) to each wound, let it stand for a period of time, inoculate with Penicillium spore suspension, let it dry naturally, put it in a disinfected plastic basket, seal it with plastic wrap, and place it in a constant temperature and humidity incubator to achieve the control of postharvest Penicillium rot in pears by Serratia marcescens. Alternatively, select pears that are free from disease and mechanical damage, have uniform color, and are similar in size. Without any disinfection treatment, keep the pears in their natural state and spray the Serratia marcescens suspension prepared in step (1) evenly on the surface of the pears. After air-drying, the pears can be used for post-harvest disease prevention and control as well as storage and preservation.

3. The use of the biocontrol agent *Serratia marcescens* according to claim 2 for postharvest disease control and storage preservation of pear fruit, characterized in that... The LB medium described in step (1) consists of 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and distilled water to a final volume of 1000 mL. The pH is set to natural, and the medium is sterilized at 115°C for 20 min.

4. The use of the biocontrol agent *Serratia marcescens* according to claim 2 for postharvest disease control and storage preservation of pear fruit, characterized in that... The centrifugation conditions in step (1) are: 4℃, 8000 rpm, and 10~15 min; the conditions for the first and second activation cultures are: 37℃ and 16~18 h.

5. The use of the biocontrol agent *Serratia marcescens* according to claim 2 for postharvest disease control and storage preservation of pear fruit, characterized in that... In step (1), the inoculum amount for the activation solution transfer is 1-2% (v / v); the concentration of *Serratia marcescens* is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

6. The use of the biocontrol agent *Serratia marcescens* according to claim 2 for postharvest disease control and storage preservation of pear fruit, characterized in that... The disinfection process in step (2) is as follows: immerse the pears in water containing 0.2% sodium hypochlorite solution for 2-4 minutes; disinfect the plastic baskets by rinsing them with clean water, drying them, and then spraying them evenly with 75% alcohol to obtain disinfected plastic baskets.

7. The use of the biocontrol agent *Serratia marcescens* according to claim 2 for postharvest disease control and storage preservation of pear fruit, characterized in that... In step (2), the pore size of the wound is 5 mm and the depth is 4 mm; 30 μL of *Serratia marcescens* suspension and *Penicillium spore suspension* are added to each wound; the settling time is 2 h; the concentration of the *Penicillium spore suspension* is 1 × 10⁻⁶. 5 CFU / mL.

8. The use of the biocontrol agent *Serratia marcescens* according to claim 2 for postharvest disease control and storage preservation of pear fruit, characterized in that... In step (2), the temperature of the constant temperature and humidity incubator is 20-25℃ and the relative humidity is 90%.

9. The use of the biocontrol agent *Serratia marcescens* according to claim 2 for postharvest disease control and storage preservation of pear fruit, characterized in that... In step (2), the Serratia marcescens suspension is sprayed evenly on the surface of the pear fruit. The specific dosage is 0.02~0.05 mL of suspension per square centimeter.

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