A method for controlling postharvest diseases and storing and preserving eggplant using Bacillus velez

Abstract

The invention belongs to the technical field of biological control, and specifically relates to a method for controlling postharvest diseases of eggplant using Bacillus Velez. The method comprises the following steps: activating and culturing Bacillus Velez to obtain a culture solution, centrifuging to obtain bacterial cells, and diluting the culture solution with sterile water to obtain 1×10 8 cells / mL of bacterial suspension; after drilling holes on the eggplant surface, inject the bacterial suspension, and after standing, inject an equal volume of carrot soft rot pectin Bacillus suspension to achieve the control of post-harvest soft rot of eggplant; or spray the Velez Bacillus suspension evenly on the surface of untreated eggplant and dry it naturally to achieve storage and preservation of eggplant. The present invention can effectively control post-harvest soft rot and natural decay of eggplant, and has no significant adverse effect on the main quality indicators of eggplant after harvest; it is easy to operate, safe and environmentally friendly, and has significant economic and social benefits.

Description

Technical Field

[0001] The invention belongs to the field of biological control of postharvest diseases of fruits and vegetables, and particularly relates to a method for controlling postharvest diseases of eggplants and storing and preserving eggplants using Bacillus Velezii. Background Art

[0002] Eggplant, scientific name Solanum melongena L., is a plant of Solanaceae, also known as dwarf melon and Kunlun melon. Eggplant is rich in vitamins, trace elements such as phosphorus, calcium, potassium and various alkaloids. It has a high content of vitamin P, which can maintain blood vessel resistance and protect blood pressure. It is one of the vegetables with multiple functions that is widely favored by consumers.

[0003] There are many kinds of eggplant diseases, and bacterial diseases mainly include soft rot, bacterial wilt, bacterial brown spot, etc. Among them, soft rot caused by Pectobacterium carotovorum is one of the common and high-incidence postharvest bacterial diseases of eggplant. It develops quickly and spreads widely, causing serious economic losses. Therefore, it is necessary and urgent to adopt appropriate and effective methods to control postharvest diseases of eggplant.

[0004] Traditional control methods for post-harvest diseases of fruits and vegetables mainly include physical methods and chemical methods. Physical methods include heat treatment, refrigeration, controlled atmosphere storage, ionizing radiation, ultraviolet radiation, etc. Physical methods have the advantages of being pollution-free to the environment and harmless to the health of consumers, but they often have high requirements for equipment and costs, limited control effects, and can easily lead to a decline in the sensory quality of fruits and vegetables. The method of using chemical fungicides to control post-harvest diseases of fruits and vegetables is currently the most widely used and most common prevention and control method due to its advantages such as high efficiency, low cost, and ease of use. However, chemical fungicides are easy to remain on fruits and vegetables, causing harm to the health of consumers, and are easy to pollute the environment and cause disease resistance in pathogens. Therefore, finding a safe and effective method to prevent and control post-harvest diseases of fruits and vegetables has become an urgent problem to be solved.

[0005] Biological control refers to the use of antagonistic microorganisms of pathogens to control fruit and vegetable diseases. Biological control methods have the advantages of being safe and non-toxic, environmentally friendly, and not causing pathogens to develop drug resistance. Antagonistic microorganisms discovered so far include bacteria, molds, and yeasts. According to relevant literature reports, Rashid et al. screened a strain of Bacillus subtilis, which had a good control effect on the soft rot of potatoes caused by carrot soft rot pectin bacteria. Qi et al. found that Lactobacillus paracasei can effectively control the soft rot of green peppers and significantly control the occurrence of soft rot diseases.

[0006] However, there are few reports on the use of antagonistic bacteria to control postharvest diseases of eggplant, especially the control of postharvest soft rot, and the control effect needs to be improved. Therefore, screening antagonistic bacteria with good control effects on postharvest diseases of eggplant and extending the storage and shelf life of the fruit has important practical application value. Summary of the invention

[0007] In view of the defects and shortcomings of the prior art, the present invention provides an antagonistic bacterium isolated from the soil around the roots of eggplant - Bacillus Velez, which can effectively control the occurrence of post-harvest soft rot and natural rot of eggplant, reduce the losses caused by post-harvest diseases, and has potential application value.

[0008] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] The Bacillus velezensis for preventing and controlling post-harvest diseases of eggplant provided by the present invention was isolated and purified in this laboratory, and is currently preserved in the China Type Culture Collection (CCTCC) of Wuhan University, Wuhan City, Hubei Province, China, with a preservation number of CCTCCNO: M 2022571, a preservation date of May 9, 2022, and a recommended classification name of Bacillus velezensis T3; the Bacillus velezensis used in the present invention refers to Bacillus velezensis T3.

[0010] The method for using Bacillus Velez for postharvest disease control and storage and preservation of eggplant is carried out according to the following steps:

[0011] (1) First, Bacillus velezensis was inoculated into LB medium for the first activation culture to obtain an activation solution; then, the activation solution was transferred into a new LB medium at an inoculum amount of 1-2% (v / v) to perform a second activation culture to obtain a bacterial culture solution, which was centrifuged to obtain a bacterial sludge, which was washed with sterile saline and prepared into 1×10 8 cells / mL of Bacillus Velezii suspension;

[0012] (2) eggplants without diseases, mechanical damage, uniform color and size are selected, disinfected, and then rinsed with running water. The washed eggplants are placed in a sterilized plastic basket and air-dried; wounds of uniform size and depth are formed in the middle of the air-dried eggplants, and the Bacillus Velez suspension prepared in step (1) is added to each wound. After standing for a period of time, the inoculation concentration is 1×10 6 cells / mL of carrot soft rot pectinobacterium suspension was naturally dried and sealed with plastic wrap, and stored in a constant temperature and humidity incubator, which verified that Bacillus Velezii had a good control effect on postharvest soft rot of eggplant.

[0013] (3) Alternatively, eggplants with no disease, no mechanical damage, uniform color, and similar size were selected and kept in their natural state without any disinfection. 1.0×10 8 The eggplants are evenly sprayed with a suspension of Bacillus Velezii at a concentration of 1 cells / mL, and the fruit is placed in a sterilized plastic basket. After the eggplants are naturally dried, the plastic baskets are sealed with plastic wrap and stored in a constant temperature and humidity incubator, thereby achieving the prevention and control of postharvest eggplant diseases and storage and preservation.

[0014] Preferably, the temperature of the first and second activation cultures in step (1) is 28° C., and the culture time is 12-16 hours.

[0015] Preferably, the centrifugation conditions in step (1) are: 4°C, 8000 rpm, and time 10-15 min.

[0016] Preferably, the disinfection treatment in step (2) is performed by immersing the eggplant in an aqueous solution containing a sodium hypochlorite solution with a volume concentration of 2% for disinfection for 1-3 minutes.

[0017] Preferably, the plastic baskets in step (2) are disinfected by cleaning and drying them, and then spraying them with 75% alcohol for disinfection.

[0018] Preferably, the diameter of the holes punched in step (2) is 3-5 mm, and the depth is 3-5 mm; and the standing period is 2 hours.

[0019] Preferably, in the step (2), the volume ratio of the Bacillus velezensis suspension to the carrot soft rot pectinobacterium suspension added to each wound is 1:1.

[0020] Preferably, in step (2), the culture temperature in the constant temperature and humidity incubator is 20° C. and the humidity is 90%.

[0021] Preferably, the LB medium (based on 1 L) is: 5 g yeast extract, 10 g sodium chloride, 8 g peptone, distilled water to 1000 mL, natural pH, sterilized at 115° C. for 20 min.

[0022] Preferably, the amount of the Bacillus Velez suspension sprayed on the surface of the eggplant in step (3) is 1-2 mL / 250-300 cm 2 .

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] (1) The Bacillus Velezii used in the present invention is a bacterium isolated and screened from the soil around the roots of eggplants. It is an internationally recognized practically non-toxic bacterium and has no harm to human health.

[0025] (2) The Bacillus Velezii used in the present invention can effectively control the post-harvest soft rot of eggplant, thereby reducing the losses caused by the post-harvest soft rot of eggplant.

[0026] (3) The Bacillus Velezii used in the present invention can reduce the natural decay of eggplant after harvest, and has no significant adverse effect on the main quality indicators of eggplant (hardness, titratable acid, soluble solids and vitamin C content).

[0027] (4) The Bacillus Velez subtilis used in the present invention has not been reported to be used in the prevention and treatment of postharvest diseases of eggplant, and is original.

[0028] (5) The present invention uses Bacillus Velezii, which can replace chemical fungicides to prevent and control post-harvest soft rot and natural decay of eggplant, reduce the potential harm of chemical fungicides to human body and environment, and reduce the economic and energy burden caused by physical storage and preservation, thus having significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The control effect of different concentrations of Bacillus Velez on eggplant soft rot; CK is the control group, eggplant treated with sterile saline; 10 6 , 10 7 , 10 8 and 10 9 Respectively, the concentration is 1.0×10 6 , 1.0×10 7 , 1.0×10 8 and 1.0×10 9 cells / mL of antagonistic bacteria treated eggplant; the concentration of carrot soft rot pectinobacterium suspension used was 1.0×10 6 cells / mL; different lowercase letters represent significant differences (P<0.05).

[0030] Figure 2 The control effect of Bacillus Velez on the natural decay of eggplant; CK is the control group, eggplant treated with sterile saline; B is 1.0×10 8 cells / mL of eggplant treated with Bacillus Velezii suspension; different lowercase letters represent significant differences (P<0.05). DETAILED DESCRIPTION

[0031] The present invention is explained in more detail by means of the following embodiments. The following embodiments are merely illustrative and the present invention is not limited to these embodiments.

[0032] The culture procedure of Bacillus Velez is:

[0033] (1) First activation: Pick 2 rings of Bacillus Velezii preserved on the slant, inoculate them into a conical flask containing 50 mL of LB medium, and culture them at 28°C and 180 rpm for 12 h to perform the first activation and obtain an activated solution;

[0034] (2) Secondary activation in liquid: Use a sterilized pipette tip to suck 1 mL of activation solution into another 50 mL bottle of LB medium, and culture at 28°C and 180 rpm for 12 h to obtain bacterial culture solution;

[0035] (3) Centrifugation and resuspension: The bacterial culture solution in (2) was centrifuged at 4°C and 8000 rpm for 10 min, and washed twice with sterile saline to remove the culture matrix to obtain bacterial slurry. Finally, the culture solution was resuspended with sterile saline and finally adjusted to the desired bacterial concentration to obtain a Bacillus Velezii suspension.

[0036] The pathogen used in the present invention, Pectobacterium carotovorum, is a common infectious bacterium, which was isolated and screened from susceptible eggplant. The pathogen was inoculated into LB medium and cultured at 28°C for 12 hours, centrifuged and resuspended to a final concentration of 1×10 6 cells / mL.

[0037] Embodiment 1:

[0038] Bacillus Velez is used to control postharvest soft rot in eggplant;

[0039] 1. Experimental plan

[0040] Select eggplants that are disease-free, mechanically undamaged, uniform in color, and equal in size, rinse the impurities on their surface with clean water, and then soak them in 2% sodium hypochlorite for 3 minutes. After taking them out, rinse the surface of the eggplants with running clean water to remove the residual sodium hypochlorite. Put the washed eggplants in a sterilized plastic basket to dry.

[0041] A 3 mm × 3 mm (diameter × depth) wound was made in the middle of the dried eggplant using a sterile punch, and 10 μL of Bacillus Velez suspension was injected into each of the four experimental groups. The concentration of Bacillus Velez suspension was 1×10 6 , 1×10 7 , 1×10 8 and 1×10 9 After standing for 2 h, 10 μL of 1×10 6 cells / mL of carrot soft rot pectinobacterium suspension. Eggplants treated with sterile saline instead of Bacillus Velez suspension were used as the control group.

[0042] The treated eggplants were naturally dried, sealed with plastic wrap, and placed in a constant temperature and humidity incubator at 20°C (relative humidity 90%) for 5 days before counting the decay rate and decay diameter of the eggplants. Each treatment was repeated three times, with 12 fruits each time. The entire experiment was repeated twice.

[0043] Rot rate = number of rotten eggplant fruits / total number of eggplant fruits × 100%

[0044] 2. Test results

[0045] Depend on Figure 1 Figure A shows that during the entire storage period, 1×10 6 , 1×10 7 , 1×10 8 and 1×10 9 The rot rates of eggplants treated with cells / mL Velez Bacillus suspension were 31.25%, 21.86%, 12.5% ​​and 3.03%, respectively, which were significantly lower than those of the control group (84.38%). Moreover, there were significant differences in the rot rates of eggplants treated with different concentrations of Velez Bacillus suspension (p<0.05). Figure 1 As shown in Figure B, the rot diameter of eggplants treated with different concentrations of antagonistic bacteria suspension was significantly lower than that of the control group (62.36 mm) (p < 0.05), but the concentration of 1×10 8 and 1×10 9 There was no significant difference in the rot diameter of eggplants treated with antagonistic bacteria of 1×10 cells / mL. 8 cells / mL of Bacillus Velez subtilis was used to prevent and control post-harvest soft rot of eggplant. The rot rate of eggplant soft rot treated with antagonistic bacteria at this concentration was only 12.5%, and the rot diameter was 9.56 mm, which were respectively reduced by 71.88% and 84.67% compared with the control group, indicating that the Bacillus Velez subtilis provided by the present invention has achieved very significant effect in preventing and controlling post-harvest soft rot of eggplant.

[0046] Embodiment 2:

[0047] Use of Bacillus Velez for storage and preservation of eggplant;

[0048] 1. Experimental plan

[0049] Eggplants with no disease, no mechanical damage, uniform color, and similar size were selected without any disinfection treatment to maintain the natural condition of the eggplants. 8 cells / mL of Bacillus Velez suspension was evenly sprayed on the surface of eggplant (the dosage was 1-2mL / 250-300cm 2). After spraying, the eggplants were placed in sterilized plastic baskets. After the eggplants were naturally dried, the plastic baskets were sealed with plastic wrap and placed in a constant temperature and humidity incubator at 20°C (relative humidity 90%) for 14 days. The incidence of eggplants was counted, and samples were taken to determine the eggplant hardness, titratable acid, vitamin C and soluble solids content. Eggplants sprayed with sterile saline were used as blank controls. Each treatment was repeated three times, with 10 fruits each time. The entire experiment was repeated twice.

[0050] Rot rate = number of rotten eggplant fruits / total number of eggplant fruits × 100%

[0051] The specific determination methods of quality indicators are as follows:

[0052] 1. Hardness: The hardness of eggplant was measured using a TA-XT2i texture analyzer. The probe diameter was 4 mm, the test depth was 10 mm, and the running speed of the texture analyzer probe was 1.0 mm / s before, during, and after the test. The maximum resistance (in N) encountered when the probe was inserted into the eggplant was defined as the hardness of the fruit.

[0053] 2. Weight loss rate: Use an electronic balance to weigh the initial weight of eggplant before storage and the weight after storage.

[0054] Weight loss rate = (initial weight - weight after storage) / initial weight × 100%

[0055] 3. Soluble solids: Take an appropriate amount of fruit sample and grind it into a homogenate under low temperature conditions, centrifuge it at 4°C and 8000 rpm for 5 minutes, and absorb the supernatant. Use a handheld refractometer to determine the content of soluble solids in eggplant.

[0056] 4. Vitamin C: Determine by potassium iodate titration. Weigh 1g eggplant tissue sample and place it in a mortar, add an appropriate amount of 2% hydrochloric acid solution, grind it into a homogenate under ice bath conditions, transfer it to a 50mL centrifuge tube, rinse the mortar with 2% hydrochloric acid solution, and pour it into a centrifuge tube. Centrifuge at 4℃, 8000rpm for 10min, and take the supernatant for use. Take 0.5mL 10g / LKI solution, 2.0mL 5g / L starch solution, 5mL extract and 2.5mL distilled water and place them in a conical flask and mix them well. Use 1mmol / L KIO3 solution to drip and titrate. Titrate until the light blue color does not fade as the end point. At the same time, use 5.0mL 2% hydrochloric acid solution as a blank and titrate in the same way. According to the consumption of KIO3 titrant, calculate the ascorbic acid content in fruits and vegetables, expressed as the mass of ascorbic acid contained in every 100g sample (fresh weight), that is, mg / 100g.

[0057] Ascorbic acid content (mg / 100g) = (V×(V1-V0)×0.088) / (Vs×m)×100

[0058] Where V is the total volume of the sample extraction solution (mL), V1 is the volume of KIO3 consumed in the sample titration (mL), 0.088 is the mass of ascorbic acid equivalent to 1 mL of 1 mmol / l KIO3 solution (mg), V0 is the volume of KIO3 consumed during the titration blank (mL), Vs is the volume of the sample solution taken during the titration (mL), and m is the sample mass (g).

[0059] 2. Test results

[0060] 1×10 8 After eggplant treated with cells / mL Bacillus Velez suspension was stored at 20℃ for 14 days, its main quality indicators were measured. The results are shown in Table 1.

[0061] Table 1 Effect of Bacillus Velez on storage quality of eggplant

[0062]

[0063] Note: CK: eggplant treated with sterile saline; B: 1.0×10 8 cells / mL eggplant treated with Bacillus Velez.

[0064] As can be seen from the table, there was no significant difference in the hardness, weight loss rate, soluble solids and vitamin C content of eggplant fruits treated with Bacillus Velez during storage. This result shows that Bacillus Velez treatment has no significant adverse effect on the quality of eggplant.

[0065] Depend on Figure 2 It can be seen that 1×10 8 After the eggplants treated with cells / mL Velez subtilis suspension were stored at 20°C for 14 days, the incidence rate was only 31.5%, which was 40.75% lower than that of the control group (72.25%). The results show that Velez subtilis has a significant control effect on the natural decay of post-harvest eggplants, but has no significant adverse effect on their quality. Therefore, the Velez subtilis suspension of the present invention can be used for the storage and preservation of post-harvest eggplants.

[0066] 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 this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for controlling postharvest diseases and preserving eggplant using Bacillus Velezii, characterized in that: The specific steps are as follows: (1) First, Bacillus velezensis was inoculated into LB medium for the first activation culture to obtain an activation solution; then, the activation solution was transferred into a new LB medium at an inoculum amount of 1-2% (v / v) to perform a second activation culture to obtain a bacterial culture solution, which was centrifuged to obtain a bacterial sludge, which was washed with sterile saline and prepared into 1×10 8 cells / mL of Bacillus Velezii suspension; (2) eggplants without diseases, mechanical damage, uniform color and size are selected, disinfected, and then rinsed with running water. The washed eggplants are placed in a sterilized plastic basket and air-dried; wounds of uniform size and depth are formed in the middle of the air-dried eggplants, and the Bacillus Velez suspension prepared in step (1) is added to each wound. After standing for a period of time, the inoculation concentration is 1×10 6 cells / mL of carrot soft rot pectinobacterium suspension was naturally dried and sealed with plastic wrap, and stored in a constant temperature and humidity incubator, which verified that Bacillus Velezii had a good control effect on postharvest soft rot of eggplant. (3) Alternatively, eggplants with no disease, no mechanical damage, uniform color, and similar size were selected and kept in their natural state without any disinfection. 1.0×10 8 cells / mL of Bacillus Velez suspension is evenly sprayed on the surface of eggplants, and the eggplants are placed in a sterilized plastic basket. After the eggplants are naturally dried, the plastic baskets are sealed with plastic wrap and stored in a constant temperature and humidity incubator, thereby achieving the prevention and control of postharvest eggplant diseases and storage and preservation. The Bacillus Velezii is deposited in the China Type Culture Collection of Wuhan University, Wuhan City, Hubei Province, China, with a deposit number of CCTCC NO: M 2022571.

2. The use according to claim 1, characterized in that The temperature of the first and second activation cultures in step (1) is 28° C., and the culture time is 12-16 hours.

3. The use according to claim 1, characterized in that The centrifugal conditions in step (1) are: 4°C, 8000 rpm, and time 10-15 min.

4. The use according to claim 1, characterized in that The disinfection treatment in step (2) is as follows: the eggplant is immersed in an aqueous solution containing a sodium hypochlorite solution with a volume concentration of 2% for disinfection for 1-3 minutes.

5. The use according to claim 1, characterized in that: The plastic baskets in step (2) are disinfected by cleaning and drying them, and then spraying them with 75% alcohol for disinfection.

6. The use according to claim 1, characterized in that: The diameter of the holes punched in step (2) is 3-5 mm, and the depth is 3-5 mm; the standing period is 2 hours.

7. The use according to claim 1, characterized in that In the step (2), the volume ratio of the Bacillus velez suspension to the carrot soft rot pectin bacteria suspension added to each wound is 1:

1.

8. The use according to claim 1, characterized in that In the step (2), the culture temperature in the constant temperature and humidity incubator is 20° C. and the humidity is 90%.

9. The use according to claim 1, characterized in that The components of the LB medium are as follows: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, distilled water to 1000 mL, natural pH, and sterilized at 115° C. for 20 min.

10. The use according to claim 1, characterized in that The amount of the Bacillus Velez suspension sprayed on the eggplant surface in step (3) is 1-2 mL / 250-300 cm 2 .

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