CN116076509A - Biological agent for preventing and treating gray mold of tomatoes and preparation method thereof - Google Patents
Biological agent for preventing and treating gray mold of tomatoes and preparation method thereof Download PDFInfo
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- CN116076509A CN116076509A CN202310243839.7A CN202310243839A CN116076509A CN 116076509 A CN116076509 A CN 116076509A CN 202310243839 A CN202310243839 A CN 202310243839A CN 116076509 A CN116076509 A CN 116076509A
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- A—HUMAN NECESSITIES
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- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
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- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/157—Inorganic compounds
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses a biological agent for preventing and treating gray mold of tomatoes and a preparation method thereof, wherein the biological agent is prepared by compounding an epsilon-polylysine solution serving as a main agent and a chitosan solution and a ferrous sulfate solution serving as auxiliary agents. The mass ratio of chitosan to epsilon-polylysine in the biological agent is 1.3-7.5:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.1-0.85:1; wherein the mass concentration of epsilon-polylysine is 0.3-2.4 g/L. The biological agent is prepared by compounding an epsilon-polylysine solution serving as a main agent and a chitosan solution and a ferrous sulfate solution serving as auxiliary agents, and the three active components are synergistic mutually, so that the problem of unstable effect of the chitosan gray mold control preparation in the prior art is solved. Through optimizing the epsilon-polylysine content in the biological preparation and optimizing the mass ratio of the three effective components, the biological preparation plays a good role in inhibiting botrytis cinerea, and is suitable for preventing and treating the gray mold of tomatoes.
Description
Technical Field
The invention relates to the technical field of biological agents, in particular to a biological agent for preventing and treating gray mold of tomatoes and a preparation method thereof.
Background
The fruits and vegetables are easy to decay, nutrition and moisture loss and the like during the storage and transportation after picking, and huge economic loss is caused. Fruit and vegetable postharvest diseases can be divided into two main categories, one category is physiological diseases, and the quality loss is mainly caused by abiotic factors; the other is an infectious disease caused by a pre-harvest pathogenic bacterium latent infection and a post-harvest exogenous infectious pathogenic bacterium. Of these, the infectious diseases are the most leading causes of quality loss after harvest of fruits and vegetables, and about 25% of fresh fruits are lost to the infectious diseases every year worldwide.
According to different infection sources, the infectious diseases can be classified into fungal diseases, bacterial diseases and viral diseases, wherein the losses caused by the fungal diseases are most common and serious. Gray mold in common fungal infection diseases is a common fungal disease which is difficult to control in fruit and vegetable crops, and the fruit and vegetable gray mold is mainly caused by the infection of botrytis cinerea. Tomatoes are important commercial crops, and how to control the gray mold of tomatoes has important research value and economic value.
The prior art shows that chitosan is prepared by deacetylation of chitin, and chitin is widely existing in the exoskeleton of crustaceans and the cell walls of fungi, and is natural polysaccharide with the earth content inferior to that of cellulose; the chitosan is healthy and nontoxic, has antioxidant and antibacterial activities, and therefore is considered to have good application prospect in the aspect of controlling postharvest diseases of fruits and vegetables. However, it is notable that the stability of the control effect of a single chitosan product for tomato gray mold is poor.
Disclosure of Invention
Aiming at the problems, the invention provides a biological agent for preventing and treating the gray mold of tomatoes and a preparation method thereof, wherein the biological agent comprises chitosan, epsilon-polylysine and inorganic compound ferrous sulfate, and the biological agent has excellent inhibition effect on the gray mold of tomatoes and has stable and good prevention and treatment effect on the gray mold of tomatoes.
Specifically, on one hand, the biological agent for preventing and treating the gray mold of the tomato is prepared by compounding an epsilon-polylysine solution serving as a main agent and a chitosan solution and a ferrous sulfate solution serving as auxiliary agents.
The biological agent contains epsilon-polylysine and inorganic compound ferrous sulfate besides chitosan. The epsilon-polylysine has a cationic surfactant with broad-spectrum antibacterial effect, has activity on various microorganisms such as gram-positive bacteria, gram-negative bacteria, yeast and fungi, is safe and nontoxic, has good water solubility and high heat stability, and is popularized and used as a food-grade preservative all the time; the ferrous sulfate is one of main fertilizer varieties for supplementing iron elements in agricultural production and flower planting, and is often used as an auxiliary component for placing plant invasive diseases besides the function of supplementing nutrients, so that the biological preparation is prepared by adopting the 3 effective components, and the biological preparation with good tomato gray mold control effect is prepared by taking epsilon-polylysine solution as a main agent and chitosan solution and ferrous sulfate solution as auxiliary agents.
Further, the mass ratio of chitosan to epsilon-polylysine in the biological agent is 1.3-7.5:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.1-0.85:1; wherein the mass concentration of epsilon-polylysine is 0.3-2.4 g/L. The control effect of the chitosan for controlling gray mold is not stable, and in addition, if the ferrous sulfate solution is applied to plants, excessive sulfur and effective iron components in soil can cause plant poisoning. Therefore, the content and the mutual proportion of the 3 effective components are optimized, and the synergistic effect of the 3 components is fully exerted, so that a better tomato gray mold control effect is achieved.
Furthermore, the mass concentration of epsilon-polylysine is 0.6-1.2 g/L.
Further, the mass concentration of epsilon-polylysine is 1.2g/L, and the inventor researches show that the biological preparation with the proportion has the best inhibition effect on Botrytis cinerea.
Further, the mass ratio of chitosan to epsilon-polylysine in the biological agent is 2-4:1, preferably 2:1.
Further, the mass ratio of ferrous sulfate to epsilon-polylysine in the biological agent is 0.5-0.7:1, preferably 0.5:1.
In another aspect, the invention provides a method for preparing a biological agent for controlling botrytis cinerea, comprising the following steps:
(1) Preparing a main agent: preparing a required epsilon-polylysine water solution by using sterile water;
(2) Preparing an auxiliary agent: 2.1 preparing chitosan solution: weighing chitosan powder, placing the chitosan powder into a conical flask, adding sterile water and acetic acid, oscillating by a shaking table, and adjusting the pH value after the chitosan is completely dissolved to obtain a chitosan auxiliary agent; 2.2 preparing ferrous sulfate solution: adding acetic acid into sterile water, uniformly mixing, adding ferrous sulfate, stirring and dissolving, and regulating the pH value to obtain a ferrous sulfate auxiliary agent;
(3) Preparing the biological agent by using a main agent and an auxiliary agent according to the content of epsilon-polylysine, chitosan and ferrous sulfate in the biomass;
wherein the mass ratio of chitosan to epsilon-polylysine in the biological agent is 1.3-7.5:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.1-0.85:1; wherein the mass concentration of epsilon-polylysine is 0.3-2.4 g/L.
Further, the mass concentration of epsilon-polylysine is 0.6-1.2 g/L, preferably 1.2g/L.
Further, the mass ratio of chitosan to epsilon-polylysine in the biological agent is 2-4:1, preferably 2:1.
Further, the mass ratio of chitosan to epsilon-polylysine in the biological agent is 2-4:1, preferably 2:1.
Compared with the prior art, the invention has the beneficial effects that:
the biological agent is prepared by compounding an epsilon-polylysine solution serving as a main agent and a chitosan solution and a ferrous sulfate solution serving as auxiliary agents, and the three active components are synergistic mutually, so that the problem of unstable effect of the chitosan gray mold control preparation in the prior art is solved. Through optimizing the epsilon-polylysine content in the biological preparation and optimizing the mass ratio of the three effective components, the biological preparation plays a good role in inhibiting botrytis cinerea, and is suitable for preventing and treating the gray mold of tomatoes.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 shows the inhibitory effect of the present invention on Botrytis cinerea growth;
FIG. 2 shows the effect of the biological agents of the present invention on leakage of Botrytis cinerea cell content;
FIG. 3 shows the effect of the biological agents of the present invention on the integrity of the botrytis cinerea membrane;
FIG. 4 shows the control effect of the biological agent of the present invention on tomato gray mold;
FIG. 5 shows the inhibitory effect of the biological agent of the present invention on the diameter of gray mold lesions of tomato.
Detailed Description
In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to preferred embodiments thereof. It should be understood that these examples are for the purpose of more detailed description only and should not be construed as limiting the invention in any way, i.e., not intended to limit the scope of the invention.
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive concepts pertain. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The epsilon-polylysine used in the invention is purchased from Tianjin Hindesi Biochemical technology Co., ltd, and the purity is 98%; the nano-solution (natamycin which can be directly dissolved in water after being developed by embedding technology) is purchased from Zhejiang new silver elephant bioengineering company; phenyllactic acid was purchased from Shanghai source leaf biotechnology Co., ltd and had a purity of 98%. Each microbial source bacteriostat was formulated with sterile water to the desired concentration prior to use and sterilized with a 0.22 μm microporous membrane.
The tomatoes used in the invention are commercial mature cherry tomatoes purchased in farmer markets near schools, and are transported back to a laboratory immediately after purchase. The fruit has uniform size, and no physiological and pathological diseases. Before the fruit is used, 75% alcohol is sprayed for disinfection, and then tap water is used for washing and air drying.
In addition, botrytis cinerea is derived from diseased tomatoes, separated and purified by a laboratory, and stored in a refrigerator at-80 ℃. Before the test, botrytis cinerea was activated on PDA plates and incubated at 25℃for 7d. After 7d of culture, sterile water was added to the petri dish, and the conidium was gently scraped off with a sterile tip to prepare a Botrytis cinerea spore suspension. The concentration of the spore suspension was determined by a hemocytometer and adjusted to 1.0X106 CFU/mL.
Example 1: inhibition of Botrytis cinerea growth by the biological agent of the invention
In the embodiment, spore suspension is mixed in PDA culture medium (namely potato dextrose agar culture medium) at about 45 ℃ to prepare a bacteria-containing flat plate, 6 and 7 oxford cups are uniformly placed on the flat plate, and 200 mu L of bacteriostatic agent solution with different concentrations is added into the oxford cups; then, the cells were cultured at 25℃for 7 days, and the diameter of the inhibition zone was observed. Each treatment included three replicates and the entire experiment was repeated twice.
Further, the influence of biological agents with different component ratios on the growth of Botrytis cinerea mycelium is judged by measuring the diameters of bacterial colonies, and the biological agents with different component concentrations are added into an unset PDA culture medium so as to prepare the biological agents with the final mass concentrations of epsilon-polylysine of 0, 0.5, 1, 1.5 and 2g/L, wherein the mass ratio of chitosan to epsilon-polylysine is 2:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.5:1.
The effect of different component concentrations of the biological agents on Botrytis cinerea growth on the plates is shown in FIG. 1. As can be seen from FIG. 1, after the same culture time, a clear inhibition zone appears around the oxford cup with the biological agent of the invention on the Botrytis cinerea flat plate, and the inhibition effect on the Botrytis cinerea growth is stable.
Example 2: effect of the biological Agents of the invention on Botrytis cinerea cell leakage
In this example, 3 pieces of 1-week-old Botrytis cinerea cake were shake-cultured in PDB medium at 25℃for 3d at a rotation speed of 150 rpm; then washing twice by adopting sterile distilled water, taking 2g of mycelium, re-suspending in the biological preparation containing 0, 0.6 and 1.2g/L epsilon-polylysine, wherein the mass ratio of chitosan to epsilon-polylysine is 2:1, the mass ratio of ferrous sulfate to epsilon-polylysine is 0.5:1, and culturing for 0, 1, 2, 3 and 4 hours at 25 ℃ and 150rpm in an oscillating way. The mycelia were then filtered and the relative conductivity, soluble protein and nucleic acid leakage were measured with an aqueous solution.
The conductivity in the solution was measured using a conductivity meter in units of μscm-1, relative conductivity (%) = (L1-L0)/(L lethal-L0) ×100%. The soluble protein content was determined using the Bradford method at a wavelength of 595 nm. The nucleic acid leakage was measured by detecting the optical density at 260nm (OD 260). Each treatment contained three replicates and the experiment was repeated twice.
As shown by the results in FIG. 2, the treatment of the biological agent of the present invention increases the permeability of the Botrytis cinerea membrane, resulting in leakage of intracellular electrolytes, soluble proteins and nucleic acids out of the cell.
Example 3: effect of the biological agents of the invention on the integrity of Botrytis cinerea membranes
In the embodiment, 2g of mycelium is suspended in the biological preparation containing 0, 0.6 and 1.2g/L epsilon-polylysine, wherein the mass ratio of chitosan to epsilon-polylysine is 2:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.5:1; the culture was performed at 25℃and 150rpm for 6 hours with shaking. The treated mycelia were washed three times with PBS (100 mmol L-1, pH 7.4) buffer, stained with 50mg L-1 Propidium Iodide (PI) at 37℃for 20min in the absence of light, washed with PBS buffer to remove residual stains, and observed under an overhead fluorescence microscope. Three fields were randomly selected for each treatment and the experiment was repeated twice.
PI is a commonly used dye that can stain damaged cells red. As shown in FIG. 3, the hyphae were observed under a forward fluorescence microscope, red fluorescence was hardly seen in the untreated control group, and intense red fluorescence was detected in the 0.6 or 1.2g/L epsilon-polylysine biological agent treated group, and there was no significant difference between the biological agent treated groups with 0.6g/L epsilon-polylysine and 1.2g/L epsilon-polylysine content. This indicates that the biological agent treatment of the present invention severely disrupts cell membrane integrity.
Example 4: the biological agent has the control effect on the gray mold of tomatoes
In this example, a hole of 3mm in depth and 2mm in width was punched on both sides of each fruit with a sterile bamboo stick, and 10. Mu.L of spore suspension at a concentration of 1.0X106 CFU/mL was inoculated to each wound and air-dried at room temperature for 1 hour. Then 10. Mu.L of various concentrations of epsilon-polylysine biologic was added to each wound and air dried at room temperature for 1 hour with sterile water as a control. The treated fruits were placed in a constant temperature and humidity incubator at 25℃and 95% humidity, and the spot diameter was measured after 7 days.
In the embodiment, biological agents with mass concentration gradients of 0, 0.6, 1.2 and 2.4g/L of epsilon-polylysine are arranged, wherein the mass ratio of chitosan to epsilon-polylysine is 2:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.5:1; each treatment included 3 replicates of 15 fruits each and the experimental results are shown in figures 4 and 5.
As can be seen from FIG. 4, when the concentration of epsilon-polylysine in the biological preparation reaches 2.4g/L, the infection degree of Botrytis cinerea on cherry tomatoes is significantly lighter than that of the control group. Further, the lesion diameter was measured. As shown in FIG. 5, the diameters of lesions in the biological preparation treatment groups with mass concentration of 0.6, 1.2 and 2.4g/L epsilon-polylysine were 13.73, 10.65 and 4.96mm, respectively, which were 70.96%, 55.04% and 36.13% of those in the control group (19.35 mm). The result shows that the inhibition effect of the biological agent on gray mold in fruits and vegetables is enhanced along with the increase of the concentration.
The foregoing is a further detailed description of the invention in connection with specific embodiments, and it is not intended that the invention be limited to such description. It will be apparent to those skilled in the art that several simple modifications and adaptations of the invention can be made without departing from the spirit of the invention and are intended to be within the scope of the invention.
Claims (10)
1. A biological agent for preventing and treating gray mold of tomatoes is characterized in that the biological agent is prepared by compounding epsilon-polylysine solution serving as a main agent and chitosan solution and ferrous sulfate solution serving as auxiliary agents.
2. The biological agent for preventing and treating gray mold of tomatoes according to claim 1, wherein the mass ratio of chitosan to epsilon-polylysine in the biological agent is 1.3-7.5:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.1-0.85:1; wherein the mass concentration of epsilon-polylysine is 0.3-2.4 g/L.
3. The biological agent for controlling botrytis cinerea according to claim 2, wherein the mass concentration of epsilon-polylysine is 0.6-1.2 g/L.
4. The biological agent for controlling botrytis cinerea according to claim 2, wherein the mass concentration of epsilon-polylysine is 1.2g/L.
5. Biological agent for controlling botrytis cinerea according to claim 2, characterized in that the mass ratio of chitosan to epsilon-polylysine in the biological agent is 2-4:1, preferably 2:1.
6. Biological agent for controlling botrytis cinerea according to claim 2, characterized in that the mass ratio of ferrous sulphate to epsilon-polylysine in the biological agent is 0.5-0.7:1, preferably 0.5:1.
7. The preparation method of the biological agent for preventing and treating the gray mold of the tomatoes is characterized by comprising the following steps:
(1) Preparing a main agent: preparing a required epsilon-polylysine water solution by using sterile water;
(2) Preparing an auxiliary agent: 2.1 preparing chitosan solution: weighing chitosan powder, placing the chitosan powder into a conical flask, adding sterile water and acetic acid, oscillating by a shaking table, and adjusting the pH value after the chitosan is completely dissolved to obtain a chitosan auxiliary agent; 2.2 preparing ferrous sulfate solution: adding acetic acid into sterile water, uniformly mixing, adding ferrous sulfate, stirring and dissolving, and regulating the pH value to obtain a ferrous sulfate auxiliary agent;
(3) Preparing the biological agent by using a main agent and an auxiliary agent according to the content of epsilon-polylysine, chitosan and ferrous sulfate in the biomass;
wherein the mass ratio of chitosan to epsilon-polylysine in the biological agent is 1.3-7.5:1, and the mass ratio of ferrous sulfate to epsilon-polylysine is 0.1-0.85:1; wherein the mass concentration of epsilon-polylysine is 0.3-2.4 g/L.
8. The method for producing a biological agent according to claim 7, wherein the mass concentration of epsilon-polylysine is 0.6-1.2 g/L, preferably 1.2g/L.
9. The method for preparing a biological agent according to claim 7, wherein the mass ratio of chitosan to epsilon-polylysine in the biological agent is 2-4:1, preferably 2:1.
10. The method for preparing a biological agent according to claim 7, wherein the mass ratio of ferrous sulfate to epsilon-polylysine in the biological agent is 0.5-0.7:1, preferably 0.5:1.
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