A strain of Bacillus japonensis for promoting wound healing of postharvest potato tubers and its application

Abstract

The present invention provides a strain of Bacillus toyonensis that promotes callus of post-harvest potato tubers, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on May 6, 2024, with a deposit number of CGMCC No. 30494. The Bacillus toyonensis CGMCC No. 30494 strain of the present invention is separated from the callus potato tissue, has the advantages of fast reproduction, easy collection of strains, easy processing of post-harvest tubers, and accelerated generation speed of the tuber callus epidermal sealing layer (i.e., wound periderm, which is mainly composed of a cell layer of polyphenol suberin, polyester suberin and lignin), thereby reducing the weight loss rate of the tuber and the disease index of Fusarium infection.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus orientalis for promoting the wound healing of post-harvest potato tubers and an application thereof. Background Art

[0002] Potato (Solanum tuberosum L.) is an important food and vegetable crop in my country. The tuber is rich in a variety of nutrients, including starch, vitamins, dietary fiber, minerals, polyphenols and flavonoids, which are beneficial to the balanced nutrition of the human body. It is grown in 158 countries and regions around the world, and at least 1 billion people use it as their main food source. my country is the largest producer of potatoes. With the continuous expansion of potato planting area, potatoes have occupied an increasingly important position in my country's agricultural production. However, the surface periderm of freshly harvested potato tubers is extremely fragile and tender, and it is very easy to suffer mechanical damage during harvesting and post-harvest storage and transportation. The damaged surface not only accelerates the loss of water in the tuber tissue, but also provides a channel for infection for external pathogenic microorganisms, resulting in a decline in the quality of the tubers during storage and an increase in post-harvest losses.

[0003] Although the potato tuber epidermis can self-repair after mechanical damage, forming a protective wound periderm to inhibit water loss and infection by pathogenic microorganisms. However, it takes a long time under natural conditions. During the long wound self-healing process, the probability of pathogenic microorganisms infecting the tubers increases, which increases the post-harvest rot rate. At the same time, in commercial production, potato tubers need to be stored as soon as possible after harvesting to reduce the space and labor required for turnover. As a result, there is a lack of conditions for tuber self-healing after harvesting, the healing effect is poor, and the risk of rot during storage is increased. The defect of slow self-healing of the tuber epidermis greatly affects the harvesting of potato tubers and the management of post-harvest storage and transportation. Although there are some reports that high temperature and chemical treatment can promote the formation of wound periderm for healing of injured parts of potato tubers, excessively high temperatures can easily increase the physiological metabolism of post-harvest tubers and accelerate quality deterioration, and chemical treatment will be accompanied by the problem of drug residues. Therefore, the development of bio-based healing agents has considerable advantages in solving the problem of slow natural healing of tubers in the development of the potato industry. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a Bacillus toyonensis strain for promoting the callus of post-harvest potato tubers. Accordingly, the present invention also provides the use of the strain in preparing a product for promoting the callus of post-harvest potato tubers.

[0005] The first object of the present invention is to provide a strain of Bacillus toyonensis that promotes post-harvest potato tuber callus, wherein the Bacillus toyonensis is the Bacillus toyonensis S5770 strain, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 6, 2024, with a deposit number of CGMCC No. 30494.

[0006] Bacillus toyonensis is a plant growth promoting bacterium (PGPR) purified and separated from the damaged potato tuber epidermis stored in a potato ventilation warehouse in Huichuan Town, Weiyuan County, Dingxi City, Gansu Province. It can effectively reduce the harm caused by various pathogens to plants, provide necessary nutrients for plant growth, and effectively promote the digestion and absorption of nutrients, and has the ability to promote plant growth. The inventor of the present application unexpectedly discovered that the Bacillus toyonensis CGMCC No. 30494 of the present invention separated from the wound of potato tubers can accelerate the formation and thickening of the wound periderm of potato tubers after harvest.

[0007] The second object of the present invention is to provide a bacterial solution of Bacillus toyonensis, which is obtained by expanding and culturing the above-mentioned Bacillus toyonensis CGMCC No.30494 strain.

[0008] Preferably, the expanded culture is specifically: inoculating the Bacillus toyonensis CGMCC No. 30494 strain into a culture medium, and culturing it at 37±1° C. on a shaking platform.

[0009] Preferably, the culture medium is LB liquid culture medium.

[0010] The third object of the present invention is to provide the use of the above-mentioned Bacillus toyonensis or the bacterial solution of Bacillus toyonensis in the preparation of a product for promoting healing of post-harvest potato tubers.

[0011] Preferably, the method of promoting wound healing of harvested potato tubers comprises accelerating the formation and thickening of wound periderm of harvested potato tubers with damaged surface periderm.

[0012] The fourth object of the present invention is to provide the use of the above-mentioned Bacillus toyonensis or the bacterial solution of Bacillus toyonensis in the preparation of products for inhibiting Fusarium. Tricinctum.

[0013] A fifth object of the present invention is to provide a method for promoting wound healing of post-harvest potato tubers, which comprises applying or spraying the above-mentioned Bacillus toyonensis strain or a bacterial solution of Bacillus toyonensis onto the surface of potato tubers.

[0014] In production practice, the concentration of Bacillus toyonensis in the range of 10 3 ~10 6 cells / mL, and the bacterial solution was sprayed directly onto the surface of all fresh potato tubers that had just been harvested through a sprayer, and then stored at room temperature in the dark after drying. Since potatoes are harvested and transported in large quantities, the treatment efficiency will be higher if the Bacillus oyonensis bacterial solution is directly sprayed onto the surface of all fresh potato tubers, rather than picking out the damaged potato tubers and spraying the damaged potato tubers separately.

[0015] Preferably, the potato tubers are potato tubers harvested within 24 hours or potato tubers with damaged skin.

[0016] Preferably, after coating or spraying the surface of potato tubers, the potato tubers are naturally dried and stored at room temperature in the dark.

[0017] The sixth object of the present invention is to provide a potato epidermis biogenic healing agent, the active ingredient of which includes the above-mentioned Bacillus toyonensis strain or Bacillus toyonensis bacterial liquid.

[0018] The Bacillus toyonensis CGMCC No.30494 strain of the present invention is separated from the wounded potato tissue, has the advantages of fast reproduction, easy collection of strains, easy processing of tubers after harvest, and accelerated generation speed of the wound epidermis closure layer (i.e., wound periderm, which is mainly composed of a cell layer of polyphenol suberin, polyester suberin and lignin), thereby reducing the weight loss speed of the tubers (see Figure 2 ) and the disease index of Fusarium infection (see Figure 3 ).

[0019] The present invention applies or sprays a suspension of Bacillus toyonensis within 24 hours after potato tubers are harvested: the harvested potato tubers are rinsed, soaked and disinfected with sodium hypochlorite, rinsed with deionized water and dried; the target bacterial suspension is evenly applied or sprayed on the surface of artificially damaged potato tubers, naturally dried and placed in a perforated fresh-keeping bag, and wound healing is performed under room temperature and dark conditions. The Bacillus toyonensis used in the present invention is separated from the naturally healed epidermis of damaged potato tubers, is not genetically modified, and is identified as a wild-type Bacillus toyonensis, which can promote the formation of periderm of wounds after potato tubers are harvested and accelerate the healing process. The present invention can solve the problem of slow healing speed caused by the long time required for natural repair of epidermal wounds after potato tubers are harvested, thereby reducing the infection of pathogenic microorganisms and reducing the post-harvest rot rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 This is a colony morphology diagram of the Bacillus toyonensis CGMCC No.30494 strain of the present invention.

[0022] Figure 2 The present invention shows the influence of Bacillus toyonensis CGMCC No. 30494 on the weight loss rate of potato tubers during the wound healing period.

[0023] Figure 3 The invention discloses the influence of Bacillus toyonensis CGMCC No.30494 on the disease index caused by Fusarium thaliana during the callus period of potato tubers.

[0024] Figure 4 The figure shows the effect of Bacillus toyonensis CGMCC No. 30494 of the present invention on the deposition of suberin polyphenols (SPP) (Figure A) and the thickness of the cell layer (Figure B) during the callus period of potato tubers.

[0025] Figure 5 The figure shows the effect of Bacillus toyonensis CGMCC No. 30494 of the present invention on the deposition of polyester suberin (SPA) (Figure A) and the thickness of the cell layer (Figure B) during the callus period of potato tubers.

[0026] Figure 6The figure shows the effect of Bacillus toyonensis CGMCC No.30494 of the present invention on the deposition of lignin (Figure A) and the thickness of the cell layer (Figure B) during the callus period of potato tubers. DETAILED DESCRIPTION

[0027] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0028] Example 1

[0029] The Bacillus toyonensis strain in the present invention is purified and separated from the damaged potato tuber epidermis stored in a potato ventilated warehouse in Huichuan Town, Weiyuan County, Dingxi City, Gansu Province, and is named Bacillus toyonensis S5770. It is not genetically modified and belongs to the wild type Bacillus toyonensis. It was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection on May 6, 2024. The address of the depository unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30494.

[0030] The screening method of Bacillus toyonensis CGMCC No.30494 is as follows: potato tubers stored in a ventilated warehouse in Huichuan Town, Weiyuan County, Dingxi City, Gansu Province are collected, tubers with damaged epidermis and natural healing are selected, and are put into fresh-keeping bags, transported to the laboratory at a low temperature of 4°C, washed with tap water, disinfected with sodium hypochlorite and naturally dried, and the damaged epidermis is scraped off with a blade under sterile conditions, and 1mm to 2mm tuber tissue under the skin is taken and cut into about 1mm 2 Weigh 1g of small pieces and dilute to 10 in 9mL of sterile water. 3 ~10 6 cells / mL, 1 mL was spread on LB medium and cultured at 37°C. Single colonies were selected for further streaking isolation until pure strains were isolated and molecular biological identification was performed. One of the strains was Bacillus toyonensis S5770.

[0031] The composition of LB medium is as follows: tryptone (peptone) 10 g / L, yeast extract (yeast powder) 5 g / L, NaCl 5 g / L, agar 15 g / L, pH 7.0. Sterilize at 121°C for 25 min before use.

[0032] The Bacillus toyonensis CGMCC No.30494 of the present invention has the following biological characteristics:

[0033] 1. Morphological characteristics: Bacillus toyota colonies are milky white, round, smooth and slightly convex, with a diameter of 1.0-1.5 mm and a viscous texture. Figure 1 .

[0034] Figure 1 This is a colony morphology diagram of the Bacillus toyonensis CGMCC No.30494 strain of the present invention.

[0035] 2. Genetic characteristics (strain 16srRNA sequence): The present invention measured the 16srRNA sequence of Bacillustoyonensis CGMCC No.30494 strain, which is as follows, with a total length of 1488 bp:

[0036]

[0037]

[0038] Example 2 Experimental study on the performance of Bacillus toyonensis CGMCC No.30494 in promoting potato tuber healing

[0039] 1. Bacillus toyonensis CGMCC No.30494 promotes potato tuber healing

[0040] Preparation of Bacillus toyonensis CGMCC No.30494 bacterial suspension: 100 mL of LB liquid medium was placed in a 250 mL conical flask, and one loop of Bacillus toyonensis was inoculated. The suspension was cultured at 37°C and a shaking speed of 180 r / min for 24 h to obtain a seed solution. The suspension was centrifuged at 4°C and 8000 r / min for 10 min to obtain the precipitate as the bacterial cells. The collected bacterial cells were resuspended in sterile water to prepare 10 7 cells / mL concentration of bacterial suspension.

[0041] The LB medium consists of 5 g / L peptone, 1 g / L yeast extract powder, 5 g / L NaCl, and pH 7.0.

[0042] Potato tuber treatment method: Using the 'Atlantic' potato variety, the potato tubers were first rinsed with tap water, then soaked and disinfected in sodium hypochlorite with a volume concentration of 1% for 2 minutes, and finally rinsed with sterile water and dried; a sterile stainless steel knife was used to artificially injure the superficial epidermis of the tubers to simulate the wound formed after harvesting, and the wound area was about 1cm×2cm. In production, it is recommended to treat the potato tubers within 24 hours after harvesting. In this experiment, the tubers were coated immediately after being damaged. The experimental group used a sterile applicator to evenly apply the prepared Bacillus toyonensis CGMCC No.30494 bacterial suspension to the surface of the potato tuber wounds, and after natural air drying, they were placed in a perforated fresh-keeping box and healed at room temperature in the dark. The sterile water treatment without bacterial suspension was used as the control treatment.

[0043] 2. Determination of weight loss rate during potato tuber callus:

[0044] The weight loss rate of potato tubers during storage was determined by the gravimetric method. Potato tubers treated with sterile water and bacterial solution were placed in dark conditions at room temperature for callus. The tuber weight was measured on callus 0 day, callus 1 day, callus 3 days, callus 5 days and callus 7 days, and the weight loss rate during callus was calculated according to the following formula. The experiment was repeated 3 times.

[0045] Calculation of weight loss rate:

[0046] Weight loss rate (%) = [(W1-W2) / W1] × 100

[0047] Where W1 is the weight of the tuber before callus (g);

[0048] W2 is the weight of the tuber after wound healing (g).

[0049] The experimental results are shown in Figure 2 .

[0050] Figure 2 The present invention shows the influence of Bacillus toyonensis CGMCC No.30494 on the weight loss rate of potato tubers during the wound healing period.

[0051] Depend on Figure 2 It can be seen that at the time of measuring points after callus, the weight loss rate of potato tubers in the control group and the group treated with Bacillus orientalis showed an increasing trend. Except for the first day, the weight loss rate of tubers in the group treated with Bacillus orientalis was significantly lower than that of the control. On the seventh day of callus, it was 15.2% lower than that of the control.

[0052] 3. Determination of the diameter of lesions during potato tuber callus:

[0053] An appropriate amount of sterile water was added to the culture dish of Fusarium.Tricinctum cultured for 7 days. The spores were evenly scraped off with a sterile applicator and filtered into a 50 mL sterile centrifuge tube with gauze. The tube was shaken for 15 seconds. The concentration of the spore suspension was adjusted to 1×10 by counting with a hemocytometer. 6 CFU·mL -1 The prepared spore suspension was evenly applied to the surface wound of potato tubers at 0, 1, 3, 5, and 7 days after injury, and after drying, it was placed in a black perforated polyethylene bag and stored away from light. The disease index was observed after 7 days.

[0054] Calculation of disease index:

[0055] Disease index = ∑ (number of wounds at each disease level × disease level) / (total number of wounds × highest disease level).

[0056] The disease grade standards in the formula are: grade 4, the entire wound is diseased; grade 3, 3 / 4 of the wound area is diseased; grade 2, 1 / 2 of the wound area is diseased; grade 1, 1 / 4 of the wound area is diseased; grade 0, the wound is not diseased.

[0057] The experimental results are shown in Figure 3 .

[0058] Figure 3 The invention discloses the influence of Bacillus toyonensis CGMCC No.30494 on the disease index caused by Fusarium thaliana during the callus period of potato tubers.

[0059] Depend on Figure 3 It can be seen that after 7 days of callus, the group treated with Bacillus ymosus delayed the infection rate of potato tubers by Fusarium solani compared with the control group, and the disease index was significantly lower than that of the control treatment group, which was 13.68% and 15.12% lower than the control on the 3rd and 5th days respectively.

[0060] 4. Staining observation of polyphenol suberin SPP in the periderm of damaged potato tubers and determination of cell layer thickness:

[0061] Potato tubers at 0d, 1d, 3d, 5d, and 7d of callus were taken respectively, and the wound tissue was cut into thin slices (about 1cm in length and width, 0.2-0.3mm in thickness) with a stainless steel blade perpendicular to the wound surface. First, rinse with sterile water 6-8 times to wash away the starch, stain with 0.1% berberine for 45 minutes, rinse with sterile water 2-3 times, rinse with 75% ethanol 2-3 times, and then rinse with 95% ethanol 1-2 times to remove the dye, and then re-stain with 0.25% toluidine blue for 1.5 minutes, and finally rinse with sterile water and 75% ethanol to remove the dye. The SPP is then stained and photographed under a microscope for fluorescence observation. The thickness of the SPP cell layer was measured using IS Capture image software. The experimental results are shown in Figure 4 .

[0062] Figure 4 The figure shows the effect of Bacillus toyonensis CGMCC No. 30494 of the present invention on the deposition of suberin polyphenols (SPP) (Figure A) and the thickness of the cell layer (Figure B) during the callus period of potato tubers.

[0063] Depend on Figure 4 It can be seen that after treatment with Bacillus japonensis, the generation rate of SPP in the damaged epidermis of potato tubers was faster than that of the control treated with sterile water, and the thickness of the cell layer showed statistically significant differences from the first day after wound healing.

[0064] 5. Observation of the staining of polyester suberin SPA in the periderm of damaged potato tubers and determination of cell layer thickness:

[0065] Take potato tubers at 0d, 1d, 3d, 5d, and 7d of callus, and use a stainless steel blade to cut the wound tissue into thin slices (about 1 cm in length and width, 0.2-0.3mm in thickness) perpendicular to the wound surface. First, rinse with sterile water 6-8 times to wash away the starch. Stain with 0.05% toluidine blue for 45 minutes and then wash off the dye. Rinse with sterile water 2-3 times, rinse with 75% ethanol 2-3 times, and then rinse with 95% ethanol 1-2 times to remove the dye. Then re-stain with 1% neutral red, and finally wash off the dye with sterile water and 75% ethanol. The SPA can be stained and observed and photographed under a fluorescence microscope. The thickness of the SPA cell layer was measured using IS Capture image software. The experimental results are shown in Figure 5 .

[0066] Figure 5 The figure shows the effect of Bacillus toyonensis CGMCC No. 30494 of the present invention on the deposition of polyester suberin (SPA) (Figure A) and the thickness of the cell layer (Figure B) during the callus period of potato tubers.

[0067] Depend on Figure 5It can be seen that after treatment with Bacillus orientalis, the generation rate of SPA in the damaged epidermis of potato tubers was faster than that of the control treated with sterile water, and the thickness of the cell layer showed statistically significant differences from the first day after wound healing.

[0068] VI. Observation of lignin staining in the periderm of damaged potato tubers and determination of cell layer thickness:

[0069] Take the potato tubers of callus 0d, 1d, 3d, 5d, and 7d respectively, and cut the wound tissue into thin slices (about 1cm in length and width, 0.2-0.3mm in thickness) with a stainless steel blade perpendicular to the wound surface. Add 2-3 drops of 5% phloroglucinol to stain the tissue slices, and then add 1-2 drops of concentrated hydrochloric acid, the lignin will be dyed red, and the lignin will be photographed and observed under an optical microscope. The thickness of the SPA cell layer was measured using ISCapture image software, and the experimental results are shown in Figure 6 .

[0070] Figure 6 The figure shows the effect of Bacillus toyonensis CGMCC No.30494 of the present invention on the deposition of lignin (Figure A) and the thickness of the cell layer (Figure B) during the callus period of potato tubers.

[0071] Depend on Figure 6 It can be seen that after treatment with Bacillus orientalis, the production rate of lignin in the damaged epidermis of potato tubers was faster than that of the control treated with sterile water, and the thickness of the cell layer showed statistically significant differences from the first day after wound healing.

[0072] It can be concluded from the accumulation of SPP, SPA and lignin that after being treated with the Bacillus toyonensis CGMCC No.30494 of the present invention, the speed of wound periderm formation on the surface of potato tubers can be accelerated by 2 to 3 days, thereby promoting the healing of potato tubers.

[0073] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus japonensis that promotes wound healing of postharvest potato tubers Bacillus toyonensis , characterized in that: The Bacillus orientalis is Bacillus orientalis Bacillus toyonensis The S5770 strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 30494.

2. Bacillus japonensis Bacillus toyonensis The bacterial liquid is characterized by: The Bacillus sp. of claim 1 Bacillus toyonensis The S5770 strain was obtained by expanded culture.

3. The Bacillus sp. according to claim 2 Bacillus toyonensis The bacterial liquid is characterized by: The enlarged culture specifically comprises: Bacillus toyonensis The S5770 strain was inoculated into the culture medium and cultured at 37±1°C in a shaking incubator.

4. The Bacillus sp. according to claim 3 Bacillus toyonensis The bacterial liquid is characterized by: The culture medium is LB liquid culture medium.

5. The Bacillus sp. of claim 1 Bacillus toyonensis Or the Bacillus sp. according to any one of claims 2 to 4 Bacillus toyonensis Application of bacterial liquid in preparing products for promoting healing of post-harvest potato tubers.

6. The use according to claim 5, characterized in that: The method of promoting wound healing of harvested potato tubers specifically includes accelerating the formation and thickening of wound periderm of harvested potato tubers with damaged surface periderm.

7. A method for promoting healing of postharvest potato tubers, characterized in that: The Bacillus sp. of claim 1 Bacillus toyonensis S5770 strain or the Toyo Bacillus according to any one of claims 2 to 4 Bacillus toyonensis The bacterial solution is spread or sprayed onto the surface of potato tubers.

8. The method according to claim 7, characterized in that: The potato tubers are potato tubers harvested within 24 hours or potato tubers with damaged skin; and / or After applying or spraying on the surface of potato tubers, the potato tubers are dried naturally and stored at room temperature in the dark.

9. A potato epidermis biogenic healing agent, characterized in that: Its active ingredients include the Bacillus japonensis of claim 1 Bacillus toyonensis S5770 strain or the Toyo Bacillus according to any one of claims 2 to 4 Bacillus toyonensis Bacterial liquid.

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