Green resistance inducer for controlling fruit storage diseases based on components of citrus and preparation method of green resistance inducer

Abstract

The invention discloses a green resistance inducer for controlling fruit storage diseases based on citrus components and a preparation method of the green resistance inducer, which are specially used for controlling diseases of fresh citrus fruits during low-temperature refrigeration and maintaining the fruit storage quality so as to prolong the storage period and the supply schedule of the fresh citrus fruits. The green resistance inducer is prepared by the following steps: extracting genomic DNA (deoxyribonucleic acid) from citrus fruits, fragmenting the genomic DNA to 250-750bp sections by a non-contact ultrasonic DNA breaking instrument, diluting the genomic DNA to 20mg / L to obtain the green resistance inducer based on the components of the citrus fruits, and spraying the fresh citrus fruits which are selected in advance and subjected to surface disinfection by using the inducer. And after ventilating and airing for 1 hour, sub-packaging in packaging boxes, and storing at low temperature of 4 + / -1 DEG C. When the fresh citrus fruits treated by the green resistance inducer are stored to 32 w, the high quality and the low rotting rate can be kept. The green resistance inducer provided by the invention is extracted from components of citrus fruits, does not need to use allogenic substances, can be extracted and prepared by taking citrus pomace, citrus fresh fruit processing wastes and the like as raw materials, and has both economical efficiency and safety. The green resistance inducer provided by the invention is simple and convenient to use, can be directly sprayed on the surfaces of fruits, and is easy to popularize and apply.

Description

Technical Field

[0001] This invention relates to the preservation and anti-corrosion process of fresh citrus fruits during post-harvest storage and transportation, and in particular to the preparation and application technology of a citrus fruit ribonucleic acid fragment as a preservative, belonging to the field of fruit preservation technology. Technical Background

[0002] Citrus [Citrus spp.] is an evergreen small tree belonging to the Rutaceae family and the Citrus genus. It is an important and advantageous fruit industry in my country. Wanzhou red tangerines are a typical example, rich in citric acid, various vitamins, minerals, and trace elements, as well as functional components such as hesperidin, flavonoids, and limonoids. They possess both excellent processing characteristics and outstanding nutritional value. Wanzhou red tangerines generally ripen and are marketed in autumn and winter, and are often stored at low temperatures after harvest to extend the supply period. However, due to mechanical damage to the peel or the presence of pathogenic fungal spores latent at the stem end, which remain infectious under low temperatures, Wanzhou red tangerines are highly susceptible to fungal rot during storage, leading to a significant decline in the fruit's marketability.

[0003] Major postharvest fungal diseases affecting citrus fruits include Penicillium mold, green mold, white mold, anthracnose, sour rot, black rot, and brown rot. While chemical fungicides such as prochloraz, carbendazim, imazalil, thiophanate-methyl, and methyl thiophanate can effectively suppress these postharvest diseases, long-term reliance on chemical control often leads to increased resistance and food safety risks due to chemical residues. With increasing consumer concern for food safety and the ecological environment, developing green, safe, and efficient control technologies suitable for postharvest storage and preservation of Wanzhou tangerines has become an inevitable trend in the industry's development.

[0004] By directly inhibiting the growth of pathogenic fungi or inducing resistance in the fruit host through the application of green exogenous elicitors such as chemicals and biology, postharvest decay of fruit can be effectively controlled, providing an effective alternative to chemical fungicides. However, these exogenous preservatives are usually obtained through chemical synthesis or extraction, microbial culture, and the isolation of their secondary metabolites, which also have shortcomings in terms of economy, versatility, and safety. Screening suitable green resistance inducers from the fruit's own components using a simple process offers high safety, low cost, and strong scalability, showing broad development prospects.

[0005] Plant extracellular self-DNA (sDNA) can be considered a damage-related molecular model. Once recognized by plant cell membrane receptor proteins, it can directly activate an immune response, thereby enhancing disease resistance, and thus has the potential to become a novel green preservative. However, the DNA directly released from fruit is relatively long (>10,000 bp), with poor solubility and film-forming properties. Furthermore, fruit has difficulty recognizing complete or long DNA chains. Therefore, it is necessary to stably cleave the fruit DNA chain using appropriate methods to form small DNA fragments that can be recognized by fruit cell membrane receptor proteins, and then employ suitable post-harvest fruit treatment methods to achieve satisfactory fruit preservation results. Recently, with funding from the National Natural Science Foundation of China and the Chongqing Basic and Frontier Research Program, researchers have conducted detailed studies on the resistance induction and anti-corrosion effects of sDNA treatment on fruits such as peaches, grapes, strawberries, and citrus, accumulating relevant data and experience, which has led to this invention patent. Summary of the Invention

[0006] While current methods using physical, chemical, and biological elicitors to inhibit pathogen growth or induce resistance in fruit hosts can alleviate the problem of excessive application of chemical fungicides to some extent, these control strategies still require exogenous substances as elicitors, raising issues of economy, versatility, and safety for the promotion of related technologies. This invention aims to propose a method for preparing and using a green resistance inducer based on the components of citrus fruit itself to improve the utilization rate of citrus fruit resources and provide a safe and efficient preservation technology.

[0007] The technical solution of this invention is to provide a green resistance inducer for controlling storage diseases of fresh citrus fruit. By applying it after harvest, it can inhibit the growth of pathogens, reduce the occurrence of storage diseases, reduce the decay rate, and maintain the storage quality of the fruit, thereby achieving the ultimate goal of extending the low-temperature storage period of fresh citrus fruit.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a green resistance inducer for controlling storage diseases in citrus fruits based on their own components includes the following steps:

[0010] (1) Selection of fresh citrus fruits: Harvest fresh citrus fruits that have reached commercial maturity, remove inferior fruits with mechanical damage and pests, retain fruits that are uniform in size, have a smooth and light yellow peel, regular appearance, and a transverse diameter between 6 cm and 8 cm, wash away the dust with tap water, soak in 75% ethanol solution for 5 min, and air dry at room temperature.

[0011] (2) Extraction of genomic DNA: Remove the pericarp of the fresh citrus fruit treated in step (1), extract the genomic DNA of the citrus fruit using the CTAB method, and detect the purity and integrity of the genomic DNA using a NanoDrop micro spectrophotometer and agarose gel electrophoresis.

[0012] (3) Fragmentation of genomic DNA: The extracted genomic DNA was fragmented to 250-750 bp using a non-contact ultrasonic DNA fragmenter, and the fragmentation products were confirmed by agarose gel electrophoresis.

[0013] (4) Dilution of genomic DNA: The concentration of the fragmented DNA obtained in step (3) was determined using a NanoDrop micro spectrophotometer and diluted with sterile water to 20 mg / L to prepare the green resistance inducer for fresh citrus fruit, which was then frozen and stored.

[0014] The method of using the above-mentioned green resistance inducer includes the following steps:

[0015] (1) Resistance inducer treatment: The prepared green resistance inducer was evenly sprayed onto the fresh citrus fruit that had been disinfected with ethanol until the surface of the fruit was completely moistened.

[0016] (2) Packaging and storage: After processing, the citrus fruits are ventilated and air-dried for 1 hour before being packaged in boxes and stored at low temperature for 32 hours.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) Green and safe: sDNA is derived from the plant or fruit itself, is green and non-toxic, environmentally friendly and highly safe. This invention uses a green resistance inducer prepared from sDNA extracted from citrus fruit components to control storage diseases of the fruit, which can avoid the excessive application of traditional chemical fungicides such as imazalil, carbendazim, and thiamethoxam, thus alleviating food safety and environmental pollution problems.

[0019] (2) High efficiency and economy: At present, most of the exogenous elicitors used in the field of postharvest preservation are chemical or biological agents, which have high application costs. The preparation of a green resistance inducer involved in this invention is based on the components of citrus fruit itself, without the need for exogenous substances, and can be extracted and prepared from fruit pomace, fresh fruit processing waste, etc. At the same time, when this resistance inducer is applied to fresh citrus fruit, it can effectively delay the occurrence of diseases during the low-temperature storage of fruit, extend the storage period to 32 weeks, and maintain the postharvest quality of fruit, which is both economical and efficient.

[0020] (3) Easy to operate and easy to promote: Compared with the complicated physical or chemical treatment methods such as hot air, chemical fumigation, modified atmosphere packaging, and radiation irradiation, the treatment method of spraying fresh citrus fruit with green resistance inducer in this invention has the advantages of being simple and easy to implement, requiring less supporting equipment, and requiring no additional training for agricultural technicians. It has the potential to be promoted to the post-harvest processing line of fresh citrus fruit.

[0021] (4) Novel technology and broad application prospects: The green resistance inducer prepared based on the present invention is a new type of elicitor or preservative. It can induce resistance responses in various fruits such as peaches, grapes, strawberries, and citrus, thereby inhibiting the infection of pathogenic microorganisms, reducing the occurrence of diseases, reducing the fruit rot rate, and maintaining storage quality. It has the potential to be developed into a resistance elicitor for post-harvest fruit preservation and has broad commercial application prospects. Detailed Implementation

[0022] To fully disclose the preparation and application method of a green resistance inducer based on the components of citrus fruit itself according to the present invention, the following examples are provided for illustration. However, the present invention is not limited to the following embodiments.

[0023] Commercially mature "Wanzhou Red Tangerine" fruits cultivated at a citrus plantation in a region of Southwest China were harvested and transported back to the laboratory within 2 hours. The fruits were spread out and pre-cooled under natural wind at 20°C to dissipate field heat. Diseased, damaged, and immature fruits were removed, retaining only fresh citrus fruits of uniform size, oval shape, smooth, light yellow skin, and a diameter greater than 6 cm and less than 8 cm. After washing away surface dust with tap water, the fruits were soaked in a 75% ethanol solution for 5 minutes, then removed and laid flat to air dry under natural wind until ready for use.

[0024] Ten kg of fresh citrus fruit were harvested, and the outer peel was removed. Genomic DNA was extracted using the classic CTAB method. The purity and integrity of the extracted genomic DNA were assessed using a NanoDrop micro-spectrophotometer and 1.2% agarose gel electrophoresis. Subsequently, the genomic DNA was fragmented in batches using a non-contact ultrasonic DNA fragmenter. The fragmentation parameters were set as follows: 2 s sonication, 6 s interval, 5 min total sonication time, and 70 W power. The fragmented genomic DNA products were collected and subjected to 1.2% agarose gel electrophoresis to confirm that the product segments were within the 250-750 bp range. Next, the concentration of the 250-750 bp fragmented genomic DNA products was again measured using a NanoDrop micro-spectrophotometer. The fragments were diluted with sterile water to 20 mg / L to prepare the green resistance inducer of this embodiment, which was then dispensed into sprayers for later use.

[0025] The fresh citrus fruits, after being air-dried, were randomly divided into two groups: 1) Treatment group: Diluted green resistance inducer was evenly sprayed onto the surface of the fresh citrus fruits at a rate of 2 liters per 100 kg, and spraying was stopped when the fruit surface was completely wet. 2) Control group: The same volume of sterile distilled water was sprayed, and the spraying process was the same as the treatment group. After spraying, the fruits were laid flat to dry in a natural wind at 20 °C. After drying, the fresh citrus fruits from both groups were packaged separately in polyethylene terephthalate (PET) boxes with a thickness of 0.3 mm and dimensions of 20 cm × 16 cm × 10 cm. Each box had 6 circular ventilation holes with a diameter of 1 mm pre-drilled at the top. Each treatment involved approximately 1000 fresh citrus fruits, with 4 fruits packed into each box, for a total of 250 boxes. These boxes were stacked and stored in a cold storage facility equipped with temperature and humidity control at 4±1 ℃ and 80-90% relative humidity for 32 weeks. Samples were taken every 8 weeks before treatment (0 weeks) and during the cold storage period after treatment to determine the decay rate and quality parameters. All parameter data are averages from three replicates.

[0026] The fragmented products of the extracted genomic DNA were collected and analyzed using a NanoDrop micro-spectrophotometer. The results of three replicate measurements are shown in Table 1 below, where A 260 / A 280 A 260 / A 230 A is the absorbance value. 260 / A 280 Slightly greater than 1.8, A 260 / A 2300 A value between 2.0 and 2.2 indicates that the purity meets the requirements. Meanwhile, the concentration of the genomic DNA fragmentation product was approximately 200 ng / μL, which can be subsequently diluted 10-fold to 20 mg / L.

[0027] Table 1 Results of NanoDrop detection of genomic DNA

[0028]

[0029] The experimental results of the effects of green resistance inducer treatment on the decay rate and quality of citrus fruits during cold storage are shown in Table 2 below. The results showed that after 32 weeks of storage at 4 ℃, the decay rate of fresh citrus fruits in the control group reached 80.33%. At the end of cold storage, the hardness, soluble solids, titratable acid, vitamin C content, total sugar, total phenols, and total flavonoids of the control fruits decreased by 52.49%, 19.10%, 19.77%, 38.10%, 26.44%, 65.10%, and 39.35%, respectively, compared with before storage. The fruits had basically lost their edible value and marketability. However, after treating fresh fruit with a green resistance inducer prepared from citrus component sDNA, the decay rate after 32 weeks of storage was reduced by 69.54% compared to the control fruit. Simultaneously, the fruit's firmness, soluble solids, titratable acid, vitamin C content, total sugar, total phenols, and total flavonoids were significantly higher (P < 0.05) than the control fruit, with increases of 41.84%, 14.90%, 12.66%, 30.38%, 16.61%, 49.32%, and 26.56%, respectively. These results indicate that treatment with the green resistance inducer based on citrus components as described in this invention maintains a very low decay rate of fresh citrus fruit after 32 weeks of storage, while the decrease in fruit firmness, soluble solids, titratable acid, vitamin C content, total sugar, total phenols, and total flavonoids is also relatively small. Therefore, this treatment can maintain a low decay rate and high commercial value of citrus fruit during cold storage.

[0030] Table 2. Effects of green resistance inducer treatment on rot rate and quality of citrus fruit during cold storage.

[0031]

Claims

1. A method for preparing a green resistance inducer for controlling storage diseases of citrus fruits based on components of the citrus itself, characterized by, The method comprises the following steps: (1) selection of fresh citrus fruits: picking fresh citrus fruits reaching commercial maturity, removing inferior fruits with mechanical injuries and pests and diseases, and retaining fruits with uniform size, smooth and light yellow peel, regular appearance, and horizontal diameter of 6-8 cm; after washing off floating dust with tap water, the fruits are soaked in 75% ethanol solution for 5 min and dried at room temperature; (2) extraction of genomic DNA: removing the epicarp of the fresh citrus fruits treated in step (1), and extracting the genomic DNA of the citrus fruits by the CTAB method; the purity and integrity of the genomic DNA are detected by NanoDrop microspectrophotometer and agarose gel electrophoresis; (3) fragmentation of the genomic DNA: fragmenting the extracted genomic DNA to 250-750 bp by a non-contact ultrasonic DNA disruptor, and confirming the fragmentation product by agarose gel electrophoresis; (4) dilution of the genomic DNA: determining the concentration of the fragmented DNA obtained in step (3) by NanoDrop microspectrophotometer, and diluting the genomic DNA to 20 mg / L with sterile water to prepare a green resistance inducer for fresh citrus fruits, which is stored in a frozen state.

2. The method for preparing a green resistance inducer based on components of citrus fruits itself according to claim 1, characterized by In the actual application, the epicarp of the fresh citrus fruits treated in step (1) can also be retained, and the genomic DNA can be extracted from citrus fruit residues, citrus fruit processing waste and the like.

3. The method for preparing a green resistance inducer based on components of citrus fruits itself according to claim 1, characterized by The CTAB method in step (2) is a classic CTAB method.

4. The method for preparing a green resistance inducer based on components of citrus fruits according to claim 1, characterized by The agarose gel electrophoresis in step (2) is 1.2% agarose gel electrophoresis.

5. The method of preparing a green resistance inducer based on components of citrus fruits according to claim 1, characterized by The parameter settings of the non-contact ultrasonic DNA disruptor in step (3) are as follows: ultrasonic time 2 s, interval 6 s, total ultrasonic time 5 min, and power 70 W.

6. The method of preparing a green resistance inducer based on components of citrus fruits according to claim 1, characterized by The concentration unit of the fragmented DNA obtained in step (3) determined by the NanoDrop microspectrophotometer in step (4) is ng / μL, which is equivalent to mg / L.

7. The method of preparing a green resistance inducer based on components of citrus fruits according to claim 1, characterized by The storage in a frozen state in step (4) is storage at-20°C.

8. The method of using the green resistance inducer based on components of citrus fruits themselves for controlling storage diseases of fresh citrus fruits according to any one of claims 1 to 7, characterized in that The method comprises the following steps: (1) treatment of the resistance inducer: uniformly spraying the prepared green resistance inducer on the ethanol-disinfected fresh citrus fruits until the surface of the fruits is completely wetted; (2) packaging and storage: after the treatment, the fresh citrus fruits are ventilated and placed for 1 h, then packaged in packaging boxes, and stored at low temperature for 32 w.

9. The method of using the green resistance inducer based on the components of citrus itself to control the storage diseases of the fruit of citrus according to claim 8, characterized in that In step (1), the prepared green resistance inducer is uniformly sprayed on the ethanol-disinfected fresh citrus fruits, and the specific operation is as follows: the prepared 20 mg / L green resistance inducer is packaged in a sprayer, and uniformly sprayed on the ethanol-disinfected fresh citrus fruits, with a spraying amount of 2 L / 100 kg and a spraying frequency of once every 32 w; the actual application can be scaled up proportionally.

10. The method for using the green resistance inducer for controlling the storage diseases of citrus fresh fruits based on the components of the citrus fruits themselves according to claim 8, characterized in that The packaging box in step (2) is a polyethylene terephthalate (PET) packaging box with a length of 20 cm, a width of 16 cm, a height of 10 cm, and a thickness of 0.3 mm. Each packaging box has 6 circular ventilation holes with a diameter of 1 mm at the top. Each box contains 4 fruits, and the fruits are stacked and stored at 4 ± 1 ℃ and a relative humidity of 80-90% for 32 w.

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