阅读说明：本技术 解脂亚罗酵母应用于芦笋采后病害防治及贮藏保鲜的用途 (Application of yarrowia lipolytica in preventing and treating postharvest diseases of asparagus and storing and refreshing asparagus ) 是由 张晓云 闫雪莉 张红印 胡婉营 赵利娜 于 2019-10-30 设计创作，主要内容包括：本发明属于采后病害生物防治领域,具体涉及一种解脂亚罗酵母应用在芦笋采后病害防治及贮藏保鲜的用途；步骤为：将解脂亚罗酵母进行活化、培养,离心得到菌体,用无菌生理盐水洗涤、离心,得到的菌体用无菌生理盐水稀释成浓度为1×10<Sup>7</Sup>cells/mL的菌悬液,直接均匀喷洒在芦笋表面,自然风干；即可实现防治芦笋采后病害和贮藏保鲜；本发明可以有效控制芦笋采后根腐病和自然腐烂,从而降低芦笋采后病害所造成的损失；同时对采后芦笋主要品质指标可溶性糖、维生素C和叶绿素含量无不良影响,且安全环保,可以代替化学杀菌剂防治芦笋采后病害,减少化学杀菌剂对人体及环境的潜在危害。(The invention belongs to the field of postharvest disease biological control, and particularly relates to application of yarrowia lipolytica in postharvest disease control and storage and preservation of asparagus; the method comprises the following steps: activating yarrowia lipolytica, culturing, centrifuging to obtain thallus, washing with sterile normal saline, centrifuging, diluting with sterile normal saline to 1 × 10 concentration 7 Directly and uniformly spraying cell/mL bacterial suspension on the surface of the asparagus, and naturally drying; the disease control and storage and fresh keeping of the asparagus after picking can be realized; the method can effectively control the root rot and the natural rot of the picked asparagus, thereby reducing the loss caused by the disease of the picked asparagus; simultaneously, the method has no adverse effect on the main quality indexes of the picked asparagus, such as the content of soluble sugar, vitamin C and chlorophyll, is safe and environment-friendly, and can replace chemical bactericides to prevent and treat the asparagusThe postharvest diseases reduce the potential harm of chemical bactericides to human bodies and the environment.)

1. The application of Yarrowia lipolytica in disease control and storage and fresh-keeping of asparagus after picking is characterized in that the Yarrowia lipolytica WSL-01 is used for controlling root rot and natural rot of asparagus after picking and is used for storage and fresh-keeping of asparagus.

2. The application of yarrowia lipolytica for postharvest disease control and storage and freshness preservation of asparagus according to claim 1 is characterized by comprising the following specific steps:

activating yarrowia lipolytica, culturing yarrowia lipolytica in NYDB culture medium, centrifuging to obtain thallus, washing with sterile physiological saline, centrifuging, repeating the washing and centrifuging steps for several times, and diluting the thallus with sterile physiological saline to 1 × 10⁷cell/mL of bacterial suspension; directly and uniformly spraying the bacterial suspension on the surface of the asparagus, and naturally drying; then the mixture is put into a plastic basket, sealed by a preservative film and stored under the condition of room temperature or in an incubator.

3. The application of yarrowia lipolytica in the postharvest disease control and storage and preservation of asparagus as claimed in claim 1, wherein the condition of yarrowia lipolytica cultured in NYDB medium is: the temperature is 28-30 ℃, the rotating speed is 180-200 rpm, and the time is 24-28 h.

4. The application of yarrowia lipolytica in preventing and treating postharvest diseases of asparagus as well as storing and refreshing the asparagus as claimed in claim 1, wherein the centrifugation conditions are as follows: the rotating speed is 7000g, and the time is 10-15 min.

5. The application of yarrowia lipolytica in asparagus postharvest disease control and storage fresh-keeping according to claim 1 is characterized in that the temperature of the incubator is set to be 20-25 ℃ and the humidity is set to be 90%.

6. The use of yarrowia lipolytica for postharvest disease control and storage and freshness preservation of asparagus according to claim 1, wherein the NYDB medium is: 5g of yeast extract, 10g of glucose, 8g of beef extract and distilled water are added to a constant volume of 1000mL, the pH is natural, and the mixture is sterilized at 115 ℃ for 20 min.

Technical Field

The invention belongs to the field of biological prevention and control of postharvest diseases of vegetables, and particularly relates to application of yarrowia lipolytica (yarrowia lipolytica) in prevention and control of postharvest diseases of asparagus and storage and freshness preservation.

Background

Asparagus, the academic name of Asparagus (Asparagus officinalis), also known as Asparagus, is a perennial herb of the Asparagus genus of the liliaceae family. The green asparagus contains chlorophyll, amino acid, multivitamins, carbohydrate, calcium, phosphorus and other mineral substances and various active ingredients, has higher nutritional value, is considered as one of the medicine and food dual-purpose plants with the best nutritional balance in the world, and has curative effects on various diseases such as hypertension, hyperlipidemia, arteriosclerosis, heart disease, hepatitis, liver cirrhosis, nephritis, edema, cystitis and the like. The long-term eating of the health-care food can help digestion, enhance appetite, improve immunity of the human body, reduce toxicity of harmful substances, inhibit vitality of cancer cells, prevent generation of the cancer cells and have good anticancer effect.

At present, China has become the first world-wide Luzhou bamboo shoot production and export country, the sowing area and the export amount exceed 50% of the world total amount, and the annual output value reaches hundreds of billions of yuan. However, the harvested asparagus tissue is fragile and tender and is easily damaged mechanically and physiologically, and further infected by various pathogenic bacteria such as Fusarium (Fusarium), Alternaria (Alternaria) and Botrytis (Botrytis). After the picked asparagus is infected by pathogenic bacteria, the physiological and metabolic activities of the asparagus are deteriorated to different degrees, the nutritional value and the eating quality of the asparagus are greatly influenced, and huge economic loss is caused. Therefore, appropriate methods must be taken to control the postharvest disease.

At present, the control methods for postharvest diseases of fruits and vegetables at home and abroad are mainly divided into a physical control method, a chemical control method and a biological control method. The physical control method has the advantages of safety, harmlessness and no adverse effect on human bodies and environment. However, most physical control methods have high requirements on equipment, the cost is increased to a certain extent, improper condition control is extremely easy to have adverse effects on sensory quality of the picked asparagus, for example, the asparagus is frozen due to too low temperature, the hardness of the asparagus is increased due to too high temperature, the edible rate is reduced, the degradation of nutrient substances and the deterioration of flavor are accelerated, and the like. Since 1960, the use of chemical bactericides has become the most important means for controlling postharvest diseases of fruits and vegetables. The chemical bactericide has the advantages of high efficiency, low price, convenient use and the like, and is used in large quantities for a long time, but the method has the defects of causing pathogenic bacteria drug resistance, harming human health, polluting environment and the like. With the concern of people on environment, ecology and health, the policy for chemical sterilization is becoming stricter. Therefore, it is very important to actively find an efficient and safe method for preventing and treating postharvest diseases of fruits and vegetables, which can replace chemical bactericides.

The biological control method can effectively control the postharvest diseases of the fruits and the vegetables, has the advantages of safety, no toxicity, no pollution to the environment, no generation of drug resistance of pathogenic bacteria and the like, gradually becomes a research hotspot for controlling the postharvest diseases of the fruits and the vegetables, and is expected to replace chemical bactericides in the future. However, no biological control method for safely and effectively reducing the postharvest diseases of the asparagus exists at present, and no research report on the use of yarrowia lipolytica for preventing and treating the postharvest diseases of the asparagus is found.

Disclosure of Invention

The invention aims to provide a method for controlling postharvest diseases of asparagus by Yarrowia lipolytica (Yarrowia lipolytica), which can effectively control the occurrence of postharvest root rot and natural rot of asparagus and reduce the disease index of asparagus, thereby achieving the purposes of storing and refreshing asparagus and reducing the loss caused by postharvest diseases and having potential application value.

The technical scheme adopted by the invention

The Yarrowia lipolytica WSL-01 for preventing and treating postharvest diseases of asparagus provided by the invention is disclosed in the patent of the application and the use method of Yarrowia lipolytica in the prevention and treatment of postharvest diseases of grapes (patent number: ZL 201410231417.9); is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.8492, the preservation address is China general microbiological culture Collection center of China microbiological culture Collection management Committee of West Lu No. 1 Hospital, North West Chen, south China, Kyoho, Beijing, and the preservation time is 11 months and 21 days in 2013.

The yarrowia lipolytica WSL-01 is used for controlling the postharvest root rot and natural rot of the asparagus and is used for storing and refreshing the asparagus.

The yarrowia lipolytica is applied to the prevention and control of diseases after the asparagus is picked and the storage and the fresh-keeping of the asparagus, and the specific steps are as follows:

activating yarrowia lipolytica, culturing yarrowia lipolytica in NYDB culture medium, centrifuging to obtain thallus, washing with sterile physiological saline, centrifuging, repeating the washing and centrifuging steps for several times, and diluting the thallus with sterile physiological saline to 1 × 10⁷cell/mL of bacterial suspension; punching the surface of Germinatus Phragmitis with sterilized puncher, injecting appropriate volume of yarrowia lipolytica suspension into each hole, inoculating Fusarium spore suspension with the same volume as the yeast suspension into each hole after a period of time, wherein the concentration is 5 × 10⁴spores/mL; or directly and uniformly spraying the bacterial suspension on the surface of the asparagus; naturally drying; placing in plastic basket, sealing with plastic wrap, and storing at room temperature or in incubator.

Preferably, the culture conditions are: the temperature is 28-30 ℃, the rotating speed is 180-200 rpm, and the time is 24-28 h.

Preferably, the centrifugation conditions are: the rotating speed is 7000g, and the time is 10-15 min.

Preferably, the aperture of the punched hole is 3mm, and the depth is 2 mm; the period of time is 2 hours.

Preferably, the injection volume of the yarrowia lipolytica suspension and the injection volume of the fusarium sporophore suspension into the wells are both 20 μ L.

Preferably, the temperature of the incubator stored in the incubator is set to be 20-25 ℃, and the humidity is set to be 90%.

Preferably, the NYDB culture medium (calculated by 1L) is: 5g of yeast extract, 10g of glucose, 8g of beef extract and distilled water are added to a constant volume of 1000mL, the pH is natural, and the mixture is sterilized at 115 ℃ for 20 min.

The invention has the advantages that:

(1) the yarrowia lipolytica WSL-01 used in the invention can effectively control the postharvest root rot and natural rot of asparagus, thereby reducing the loss caused by postharvest diseases of asparagus.

(2) The yarrowia lipolytica WSL-01 used in the invention has no adverse effect on the content of soluble sugar, vitamin C and chlorophyll which are main quality indexes of the picked asparagus, and has the effect of slowing down the reduction of the quality indexes.

(3) The yarrowia lipolytica WSL-01 used in the invention has no report of application in asparagus postharvest disease control at present, and the yarrowia lipolytica WSL-01 used in the invention can replace a chemical bactericide to control asparagus postharvest diseases, reduce potential harm of the chemical bactericide to human bodies and environment, reduce economic and energy burden generated by physical sterilization and storage, and has remarkable economic and social benefits.

Drawings

FIG. 1 shows the control effect of yarrowia lipolytica WSL-01 with different concentrations on asparagus root rot; wherein: the disease index of asparagus tip part (I) and the disease index of asparagus basal part (II) are shown in the specification; the bar graphs corresponding to different days are CK, A, B, C and D from left to right in sequence;

CK is sterile normal saline treated group, namely a control group; A. b, C, D represent different concentrations of yarrowia lipolytica WSL-01 bacterial suspension (unit: cells/mL), wherein A: 1X 10⁶；B：1×10⁷；C：1×10⁸；D：1×10⁹(ii) a The different lower case letters represent significant differences (P)<0.05)。

FIG. 2 shows the control effect of yarrowia lipolytica WSL-01 on the natural decay of asparagus; wherein: CK is sterile normal saline treated group, namely a control group; y is 1X 10⁷cells/mL yeast suspension treatment group; the different lower case letters represent significant differences (P)<0.05)。

FIG. 3 is a graph showing the effect of yarrowia lipolytica WSL-01 on soluble sugar content in asparagus; wherein: the content of soluble sugar at the tip part of the asparagus (I) and the content of soluble sugar at the base part of the asparagus (II) are shown in the specification; CK is sterile normal saline treated group, namely a control group; y is 1X 10⁷cells/mL yeast suspension treatment group; the different lower case letters represent significant differences (P)<0.05)。

FIG. 4 is a graph showing the effect of yarrowia lipolytica WSL-01 on vitamin C content in asparagus; wherein: the vitamin C content of asparagus tips (I) and the vitamin C content of asparagus basal parts (II) are shown in the specification; CK is sterile normal saline treated group, namely control group(ii) a Y is 1X 10⁷cells/mL yeast suspension treatment group; the different lower case letters represent significant differences (P)<0.05)。

FIG. 5 is the effect of yarrowia lipolytica WSL-01 on the chlorophyll content of asparagus; wherein: the chlorophyll content of the asparagus tips (I) and the chlorophyll content of the asparagus basal parts (II) are shown in the specification; CK is sterile normal saline treated group, namely a control group; y is 1X 10⁷cells/mL yeast suspension treatment group; the different lower case letters represent significant differences (P)<0.05)。

Detailed Description

The invention is explained in more detail by means of the following examples. The following examples are illustrative only, and the present invention is not limited by these examples.

The culture program of yarrowia lipolytica WSL-01 is as follows: (1) solid activation: inoculating the yeast strain to NYDA culture medium, and culturing at 28 deg.C for 48 hr; (2) liquid culture: 50mL of NYDB seed culture medium is filled into a 250mL triangular flask, a loop is inoculated with a loop activated yeast, and the yeast is cultured for 24h at the temperature of 28 ℃ and the speed of 180 rpm; (3) centrifugal separation and resuspension: the yeast culture mixture was centrifuged for 15min at 7000g and washed 2 times with sterile physiological saline to remove the culture medium, and finally resuspended with sterile physiological saline and finally adjusted to the desired yeast cell concentration.

Pathogenic bacteria, Fusarium proliferatum (Fusarium proliferatum) used in the invention is obtained by self-screening in a laboratory, culturing for 7 days at 25 ℃ on a PDA culture medium, scraping hyphae into sterile physiological saline, filtering with sterile eight-layer gauze to obtain spore suspension, and finally adjusting to 5 × 10⁴spores/mL。