Application of edible mushroom polysaccharide in prevention and treatment of postharvest botrytis cinerea of tomato

Abstract

The present application relates to the technical field of plant disease control, in particular to the application of edible mushroom polysaccharide in the prevention and treatment of postharvest botrytis cinerea of tomato. The effective component for preventing and treating postharvest botrytis cinerea of tomato is edible mushroom polysaccharide, the edible mushroom polysaccharide is agaricus bisporus polysaccharide and / or termitomyces polysaccharide, the dosage of the edible mushroom polysaccharide is 25-100 mg / mL, and the use method of the edible mushroom polysaccharide is that the edible mushroom polysaccharide solution is injected into tomato fruits, and the use period is after the tomato fruits are harvested. The application of edible mushroom polysaccharide in the prevention and treatment of botrytis cinerea is proposed for the first time, and it is found that the edible mushroom polysaccharide has the effect of inhibiting the incidence of botrytis cinerea of tomato fruits within a certain concentration range; and the botrytis cinerea is prevented and treated mainly by inducing the increase of defensive enzyme activity in the fruits.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to the application of edible fungi polysaccharides in the control of postharvest gray mold in tomatoes. Background Technology

[0002] Tomatoes are rich in nutrients and have a unique flavor, making them a favorite among consumers. However, due to their high water content and thin skin, tomatoes are easily damaged during transportation and storage. Fungal contaminants, such as Aspergillus niger and Botrytis cinerea, can easily infect tissues through these wounds, causing fungal diseases. Gray mold is a general term for a group of diseases caused by fungi of the genus Botrytis. It is a typical airborne disease that can spread through air, water, and field operations. Once crop plants are infected with gray mold, they will soften and rot, constrict or break, and eventually the seedlings will rot, wither, and die. Once fruits are infected with gray mold, they will soften and rot, making them unstorable.

[0003] Therefore, post-harvest prevention strategies for tomatoes are the most promising way to reduce post-harvest rot. Commonly used methods include spraying with iprodione, primarily a chemical control method, but this is toxic to humans and ingestion could be life-threatening. Therefore, chemical control techniques still have some drawbacks, including potential toxicity, food safety concerns, and adverse effects on human health.

[0004] Edible fungi polysaccharides are a type of natural polysaccharide, mainly extracted from the fruiting bodies of edible fungi. They possess most of the characteristics of polysaccharides, such as good water solubility and outstanding biological activity, and have attracted much attention because they can be prepared into edible films and gels.

[0005] Agaricus bisporus polysaccharides (ABP) and Oudemansiella raphanipies polysaccharides (ORP) are natural polysaccharides found in edible fungi, containing glucose, galactose, and other compounds. They can be extracted from fruiting bodies or waste materials. Their production and processing are simple, they have high biocompatibility, are easily soluble in water, and exhibit good chemical stability. Currently, there is very little research or application of these two polysaccharides in disease control.

[0006] Therefore, traditional synthetic fungicides for controlling postharvest diseases of fruits and vegetables have been gradually replaced by a promising new strategy of inducing resistance. Research on safer biological control in the prevention and control of gray mold is imperative. Among them, the activation of defense-related enzymes and / or the accumulation of resistance-related substances by elicitors are important mechanisms for inducing resistance to postharvest diseases. If the application of edible fungi polysaccharides in the prevention and control of gray mold is effective, it will further improve the means and safety of postharvest gray mold control in tomatoes. Summary of the Invention

[0007] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0008] On the one hand, this application provides the application of edible fungi polysaccharide in the prevention and control of postharvest gray mold in tomatoes, wherein the effective component for preventing and controlling postharvest gray mold in tomatoes is edible fungi polysaccharide.

[0009] Based on the above technical solution, the edible fungi polysaccharide is one or both of Agaricus bisporus polysaccharide and Termitomyces albuminosus polysaccharide.

[0010] Based on the above technical solution, the concentration of the edible fungi polysaccharide is 25-100 mg / mL.

[0011] On the other hand, this application provides a tomato postharvest gray mold inhibitor, characterized in that the active ingredient is edible fungi polysaccharide.

[0012] Based on the above technical solution, the active ingredient, edible fungi polysaccharide, is one or both of Agaricus bisporus polysaccharide and Termitomyces albuminosus polysaccharide.

[0013] The optimal ratio of button mushroom polysaccharide to termite mushroom polysaccharide is 1:1.

[0014] Based on the above technical solution, the concentration of the active ingredient, edible fungal polysaccharide, is 25-100 mg / mL. The optimal preferred solution is a edible fungal polysaccharide concentration of 100 mg / mL.

[0015] Based on the above technical solution, the method of using the inhibitor is as follows: the inhibitor is injected into the tomato fruit, and the application time is after the tomato fruit is harvested.

[0016] Based on the above technical solution, the injection dosage of the inhibitor is 100 μL / unit.

[0017] Optionally, based on the above technical solution, the inhibitor can be used by injecting it into the fruit, soaking the fruit, or spraying it onto the surface of the fruit.

[0018] Optionally, based on the above technical solution, the inhibitor can be used after or before fruit harvesting.

[0019] The present invention has the following advantages:

[0020] 1. This invention is the first to propose the application of edible fungi polysaccharides in the prevention and control of gray mold, and finds that edible fungi polysaccharides can inhibit the incidence of gray mold in tomato fruits within a certain concentration range; the main method of gray mold control is to induce the increase of defensive enzyme activity in the fruit.

[0021] 2. This application also found that tomato fruits treated with edible fungi polysaccharides can maintain good nutritional value for a certain period of time; edible fungi polysaccharide treatment can protect the integrity of cell membranes and maintain normal physiological metabolic activities of cells to a certain extent; edible fungi polysaccharide treatment can inhibit the production of reactive oxygen species to a certain extent, thereby resisting diseases; it increases the ascorbic acid content, effectively improving the tomato's resistance to diseases; edible fungi polysaccharide treatment can promote the increase of PPO and POD activities, and promote the increase of SOD and CAT activities, thereby playing a role in resisting diseases.

[0022] 3. The activities of chitinase (CHI), β-1,3-glucanase (GLU), and phenylalanine ammonia-lyase (PAL) in tomato fruits treated with edible fungi polysaccharides were significantly increased, and the expression levels of disease-related protein genes were significantly upregulated. This indicates that edible fungi polysaccharides significantly inhibited fungal invasion, induced upregulation of disease-related protein gene expression in plants, activated the plant's own disease resistance mechanism, and thus enhanced the disease resistance effect.

[0023] 4. Although the aforementioned edible fungi polysaccharides have existed for a long time, they have played completely different roles in other technical fields. This application is the first to discover the excellent effect of edible fungi polysaccharides in postharvest gray mold of tomato fruits. Moreover, since the component is derived from edible fungi, it is safer and more environmentally friendly to use, and has good application prospects as a gray mold inhibitor. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0025] Figure 1 The effects of different concentrations of edible fungi polysaccharide treatment on the incidence of disease in tomatoes in the embodiments of this invention;

[0026] Figure 2 The effects of edible fungi polysaccharide treatment on tomato soluble protein (A) and reducing sugar (B) in the embodiments of the present invention;

[0027] Figure 3 The effects of edible fungi polysaccharide treatment on the content of malondialdehyde (MDA) (A) and lipoxygenase (LOX) (B) in tomatoes in the embodiments of the present invention;

[0028] Figure 4 In the embodiments of the present invention, the effect of edible fungi polysaccharide treatment on the H2O2 content (A) and O in tomatoes was observed. 2- Effects on production rate (B) and ascorbic acid content (C);

[0029] Figure 5 The effects of edible fungi polysaccharide treatment on tomato peroxidase (POD) (A), polyphenol oxidase (PPO) (B), superoxide dismutase (SOD) (C) and catalase (CAT) (D) in the embodiments of the present invention;

[0030] Figure 6 The effects of edible fungi polysaccharide treatment on chitinase (CHI) (A), β-1,3-glucanase (GLU) (B) and phenylalanine ammonia-lyase (PAL) (C) in tomatoes in the embodiments of the present invention;

[0031] Figure 7 The effects of edible fungi polysaccharide treatment on the regulation of expression levels of six disease-related protein genes in this embodiment of the invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0033] Example 1:

[0034] 1.1 Experimental Materials and Methods

[0035] Experimental materials: Tomatoes were sourced from Weifang, Shandong Province, and the variety was Provence Tomato; Agaricus bisporus polysaccharide and Termitomyces albuminosus polysaccharide were purchased from Xi'an Best Biotechnology Co., Ltd., with a purity of 50%.

[0036] Experimental Methods: Fruit Treatment: Tomato fruits of uniform size, free from disease and mechanical damage, and in the green ripening stage were selected. After disinfection by soaking in 1% sodium hypochlorite for 2 minutes, they were randomly divided into three groups. A 5mm × 3mm hole was punched near the equator of the tomato using a sterile punch, and 20μL of 1×10⁻⁶ sodium chloride solution was injected. 5 The treatment groups were abbreviated as AOPS, consisting of a CFU / mL Botrytis cinerea suspension and 100 μL (25 mg / mL, 50 mg / mL and 100 mg / mL) edible fungi polysaccharide solutions, with distilled water as the control group (CK).

[0037] The treated tomato fruits were stored for 7 days under specific temperature and humidity conditions (25℃, RH 90%), with samples taken at the same time each day. Samples were taken from the peel and 5 mm below the peel, chopped, and flash-frozen in liquid nitrogen. Three biological replicates were used for sampling.

[0038] Preparation method of edible fungus polysaccharide solution: Weigh 2.5g, 5g, and 10g of edible fungus polysaccharide (prepared by mixing button mushroom polysaccharide and termite mushroom polysaccharide in a mass ratio of 1:1), dissolve them in 100mL of distilled water respectively, let them stand overnight, and prepare edible fungus polysaccharide solutions of 25mg / mL, 50mg / mL, and 100mg / mL for later use.

[0039] Preparation method of botrytis cinerea suspension:

[0040] ① Take a PDA plate that has been incubated with Botrytis cinerea at 28℃ for 5 days;

[0041] ② Rinse with sterile water, then transfer to a certain amount of sterile water;

[0042] ③ Count the spores under a microscope using a hemocytometer and calculate the concentration of the bacterial suspension;

[0043] ④ Dilute the stock solution according to the concentration in ③ to ensure that the bacterial suspension concentration is 1×10⁻⁶. 5 CFU / mL.

[0044] 1.2 Analysis of Experimental Results

[0045] Fruit disease incidence rate Figure 1 As shown, the incidence of Botrytis cinerea increased in all experimental groups during storage due to inoculation with the pathogenic fungus suspension. However, the edible fungus polysaccharide solution significantly reduced the incidence of Botrytis cinerea at different concentrations, with the best effect observed at 100 mg / mL. This demonstrates that edible fungus polysaccharides have a significant effect in inhibiting the incidence of postharvest Botrytis cinerea in tomatoes.

[0046] Example 2:

[0047] The experimental materials and methods were the same as in Example 1. For Example 2, the relevant data were measured using the treatment group with edible fungal polysaccharide concentration of 100 mg / mL. The experimental results are analyzed as follows:

[0048] 2.1 Soluble proteins and reducing sugars

[0049] The experimental results for soluble proteins and reducing sugars are as follows: Figure 2 As shown. Soluble proteins ( ) during storage Figure 2 A) and reducing sugars ( Figure 2B) All of these increased over time, but the increase was slow due to disease. However, after treatment with edible fungus polysaccharides, the increase was faster than the control group and significantly higher in the last two days. This indicates that tomatoes can maintain good nutritional value for a short period of time after treatment with edible fungus polysaccharides.

[0050] 2.2 Malondialdehyde (MDA) and lipoxygenase (LOX)

[0051] The experimental results for malondialdehyde (MDA) and lipoxygenase (LOX) are as follows: Figure 3 As shown. By Figure 3 As shown in A, the MDA content gradually decreased during storage, and was even lower after treatment with edible fungus polysaccharides. Since MDA is an indicator of cell membrane damage, a lower content indicates less cell membrane damage; therefore, treatment with edible fungus polysaccharides to some extent protected the integrity of the cell membrane.

[0052] Figure 3 B shows the changes in LOX activity during storage. During storage, enzyme activity was inhibited over time, and the LOX activity in the edible fungus polysaccharide treatment was significantly lower than that in the control group. This indicates that the edible fungus polysaccharide treatment can reduce the peroxidation of unsaturated fatty acids and reduce the production of reactive oxygen species, thereby maintaining normal physiological metabolic activities of cells.

[0053] 2.3H2O2 content, O 2- Production rate and ascorbic acid content

[0054] H2O2 content, O 2- The experimental results for production rate and ascorbic acid content are as follows: Figure 4 As shown. By Figure 4 A and Figure 4 From B, we can know that the H2O2 content and O 2- The production rate decreased over time, indicating that the content of reactive oxygen species was suppressed during storage, thereby reducing cell damage. After treatment with edible fungi polysaccharides, the H2O2 content and O2 content decreased. 2- The more significant decrease in the production rate indicates that polysaccharide treatment of edible fungi can inhibit the production of reactive oxygen species, thereby resisting diseases. Figure 4 As shown in C, the ascorbic acid content showed a trend of first increasing and then decreasing during storage. Furthermore, the ascorbic acid content was consistently higher in the edible fungus polysaccharide treatment than in the control group. As a non-enzymatic defense substance, the increase in its content indicates that the edible fungus polysaccharide treatment effectively improved the tomato's resistance to diseases.

[0055] 2.4 Activities of peroxidase (POD), polyphenol oxidase (PPO), superoxide dismutase (SOD), and catalase (CAT)

[0056] The experimental results for the activities of peroxidase (POD), polyphenol oxidase (PPO), superoxide dismutase (SOD), and catalase (CAT) are as follows: Figure 5 As shown.

[0057] Depend on Figure 5 As shown in A and B, the activities of both POD and PPO increased over time during storage. Furthermore, the activities of both enzymes were higher in the edible fungus polysaccharide treatment group than in the control group. POD can increase the strength of plant cell walls to resist pathogen invasion, while PPO can catalyze the oxidation of phenolic compounds to produce quinine, which has direct antibacterial and cytotoxic activity against pathogens. The increased activities of these two enzymes indicate that edible fungus polysaccharide treatment promotes the increase of PPO and POD activities, thereby resisting pathogen infection.

[0058] Figure 5 Figures C and D illustrate the changes in SOD and CAT. As shown in the figure, the activities of both enzymes showed an increasing trend during storage, but the activity of the control group was higher than that of the edible fungus polysaccharide treatment group in the early stage. This may be due to the accumulation of reactive oxygen species. However, as time went on, the edible fungus polysaccharide treatment promoted the increase of the activities of the two enzymes, thereby promoting the metabolism of reactive oxygen species and thus playing a role in resisting diseases.

[0059] 2.5 Chitinase (CHI), β-1,3-glucanase (GLU), and phenylalanine ammonia-lyase (PAL) activities

[0060] The experimental results for the activities of chitinase (CHI), β-1,3-glucanase (GLU), and phenylalanine ammonia-lyase (PAL) are as follows: Figure 6 As shown.

[0061] Depend on Figure 6 As shown in Figure A, CHI activity gradually increased over time during storage, and the CHI activity in the edible fungus polysaccharide treatment group was significantly higher than that in the control group. CHI is a known disease-resistant defensive enzyme, and its increase has a positive effect on inhibiting diseases. Therefore, the effect of edible fungus polysaccharide treatment is better.

[0062] Depend on Figure 6 As shown in Figure B, GLU activity gradually increased over time during storage, and the GLU activity after treatment with edible fungi polysaccharides was significantly higher than that in the control group. GLU is an enzyme activity related to disease resistance, and treatment with edible fungi polysaccharides significantly inhibited fungal invasion.

[0063] Depend on Figure 6As shown in Figure C, PAL activity gradually increased over time during storage, and the enzyme activity after treatment with edible fungi polysaccharides was significantly higher than that in the control group. PAL is a key enzyme activity in resistance-related metabolic pathways, and the significant increase in enzyme activity after treatment with edible fungi polysaccharides suggests that it may have induced related disease resistance pathways, thereby enhancing the disease resistance effect.

[0064] 2.6 Analysis of disease resistance gene expression levels

[0065] These six genes belong to the category of disease-associated proteins, a class of plant-encoded proteins induced by various stress stimuli, playing a crucial role in plant disease resistance. Analysis of expression changes in these six disease-associated protein genes (PR2, GLU, PAL, CHI, PR5, and PR1) revealed... Figure 7 As shown in AF, the expression levels of these six genes were significantly upregulated with increasing storage time, indicating that treatment with edible fungi polysaccharides can induce an increase in the expression of disease-related proteins within the fruit, thereby enhancing resistance to diseases.

[0066] 2.7 Experimental Conclusions

[0067] Edible fungi polysaccharides can enhance disease resistance by inducing an increase in the activity of defensive enzymes in tomato fruits, inducing related disease resistance pathways in plants, and inducing an upregulation of the expression of disease-related protein genes in plants. ABP has a good control effect on diseases caused by postharvest gray mold in tomatoes.

[0068] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. The application of edible fungi polysaccharides in the control of postharvest gray mold in tomatoes, characterized in that, The effective component for preventing postharvest gray mold in tomatoes is edible fungi polysaccharide, which is prepared by mixing Agaricus bisporus polysaccharide and Termitomyces albuminosus polysaccharide in a mass ratio of 1:1, and the concentration of the edible fungi polysaccharide is 100 mg / mL.

2. A postharvest gray mold inhibitor for tomatoes, characterized in that, The active ingredient is edible fungus polysaccharide, which is prepared by mixing Agaricus bisporus polysaccharide and Termitomyces albuminosus polysaccharide in a mass ratio of 1:1, and the concentration of the edible fungus polysaccharide is 100 mg / mL.

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