A preparation process of chitosan oligosaccharide and its application in water-soluble fertilizer

Abstract

The present invention provides a preparation process of chitosan oligosaccharide and its application in water-soluble fertilizer. The preparation process of chitosan oligosaccharide is to obtain chitosan oligosaccharide with more concentrated molecular weight, smaller average molecular weight, easier to dissolve in water and higher physiological activity by subjecting chitosan to combined enzymolysis of chitosanase and chitinase. The obtained chitosan oligosaccharide is compounded with amino acids to obtain water-soluble fertilizer, which is applied to crops such as lettuce, tomato and cucumber, etc., and can significantly promote the growth of crops and improve the quality and disease resistance of crops.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural fertilizers, and relates to the preparation of chitosan oligosaccharides, and in particular to a preparation process of chitosan oligosaccharides and application of the chitosan oligosaccharides in water-soluble fertilizers. Background Art

[0002] Chitosan, also known as deacetylated chitosan, soluble chitosan, and polysaminoglucosamine, has a chemical name of β-(1,4)-2-amino-2-deoxy-D-glucan, which is obtained by deacetylation of chitin. At the C2 position, chitosan is replaced by an acetylamino group, while chitosan is replaced by an amino group. This makes chitosan one of the few alkaline polysaccharides in natural polymers. In addition to the fields of medicine and textiles, chitosan has more and more applications in the agricultural field. For example, chitosan can inhibit the growth of many plant pathogens such as Erwinia, Pseudomonas, Xanthomonas, Agrobacterium tumefaciens, Rhizopus stolonifer, Anthrax, Rhizoctonia solani, Alternaria alternata, and Botrytis cinerea; at the same time, chitosan can also increase the growth of plant biocontrol bacteria, such as spore production, thereby improving the biocontrol effect.

[0003] There are many methods for preparing chitosan, including natural extraction (such as extracting from the shells of crustaceans such as shrimps and crabs), chemical synthesis, biosynthesis, and enzymatic synthesis. However, since chitosan itself is an alkaline polysaccharide, it is insoluble in water and soluble in weak acid liquids, but acidic liquids have a certain inhibitory effect on the growth of roots, stems, and leaves of agricultural plants. How to solve the water solubility problem of chitosan, reduce the adverse effects on plant growth, and do not affect the efficacy of chitosan is a problem that needs to be solved at present. The prior art already has oligosaccharide products obtained by degrading chitosan through special bioenzyme technology, chemical degradation, or microwave degradation technology, which are called chitosan oligosaccharides (also known as chitosan oligosaccharides or oligomeric chitosans). The degree of polymerization of chitosan oligosaccharides is usually between 2 and 20, and the molecular weight does not exceed 3500Da. For example, in CN201611247543.9, papain and chitinase from Paecilomyces lilacinus were used to degrade chitosan to obtain chitosan oligosaccharides, but the enzymatic hydrolysis needs to be further combined with hydrogen peroxide as a catalyst, and a step of removing the remaining hydrogen peroxide is required after the reaction is completed, which has the disadvantages of high energy consumption, low yield and complicated steps. How to prepare chitosan oligosaccharides with stable molecular weight distribution and higher physiological activity efficiently and at low cost is still an urgent problem to be solved.

[0004] Although water-soluble fertilizers containing chitosan or chitosan oligosaccharides are already available on the market, they have limited effects on the growth of crops, the improvement of their quality, and the ability to resist plant pathogens due to the low physiological activity and few effective components of chitosan oligosaccharides. Therefore, there is an urgent need for water-soluble organic fertilizers containing chitosan oligosaccharides with good water solubility and strong functionality and rich nutrients. Summary of the invention

[0005] In view of the problems in the prior art that the preparation process of chitosan oligosaccharide is time-consuming, the molecular weight is high and the distribution is wide, the physiological activity is low, and the water-soluble fertilizer containing chitosan oligosaccharide has low physiological activity, single ingredients, and limited effects, a preparation process of chitosan oligosaccharide and its application in water-soluble fertilizer are provided. In order to solve the above technical problems, the present invention adopts the following technical scheme:

[0006] The present invention provides a preparation process of chitosan oligosaccharide, comprising the following steps:

[0007] 1) adding chitosan to an acidic solution, stirring while adding until the mixture becomes a gel, then adding water to dilute to a certain volume, stirring evenly, and adjusting the pH to obtain a colloidal chitosan solution;

[0008] 2) heating the colloidal chitosan solution to a predetermined temperature, adding chitosanase and chitinase to the colloidal chitosan solution, stirring at a constant temperature, and after a period of reaction time, reaching the reaction end point, and then adding NaOH solution to terminate the reaction to obtain a reaction solution;

[0009] 3) The reaction solution is centrifuged and ultrafiltered to obtain a filtrate, the filtrate is directly dried using a spray dryer, and then crushed and sieved to obtain a chitosan oligosaccharide product.

[0010] Preferably, the ratio of the amount of chitosan in step 1) to the amount of chitosanase and chitinase added in step 2) is 1 g: 60 U: 75 U.

[0011] Preferably, the volume ratio of chitosan to water dilution in step 1) is 100g:1L.

[0012] Preferably, the acidic solution in step 1) is any one of glacial acetic acid, succinic acid or maleic acid. Preferably, the pH value in step 1) is 5.0-6.0, more preferably, the pH value is 6.0, and most preferably, the pH value is 5.5.

[0013] Preferably, the chitosanase in step 2) is derived from Bacillus mycoides, specifically the chitosanase encoded by the gene sequence shown at positions 11174 to 12541 of GenBank accession number QTTY0100044. Preferably, the chitinase in step 2) is derived from Pseudoa l teromonasund ina, specifically the chitinase encoded by the gene sequence shown at positions 291495 to 293117 of GenBank accession number LITK01000002.

[0014] Preferably, the chitosanase and chitinase are obtained by recombinant expression in a common Pichia pastoris expression system.

[0015] Preferably, the Pichia pastoris in the above-mentioned Pichia pastoris expression system is Pichia pastoris GS115.

[0016] Preferably, the expression vector in the above Pichia pastoris expression system is pPI C9K.

[0017] Preferably, the chitosanase and chitinase activities obtained by the above recombinant expression are 4800 U / g and 2700 U / g respectively.

[0018] Preferably, the predetermined temperature in step 2) is 40°C-50°C, preferably, the predetermined temperature is 40°C, more preferably, the predetermined temperature is 50°C, and most preferably, the predetermined temperature is 45°C.

[0019] Preferably, the reaction time in step 2) is 80 min-100 min, preferably, the reaction time is 80 min, more preferably, the reaction time is 100 min, and most preferably, the reaction time is 90 min.

[0020] Preferably, the volume ratio of the amount of NaOH solution added in step 2) to the volume of dilution water in step 1) is 1:100.

[0021] Preferably, the mass fraction of the NaOH solution in step 2) is 2%, expressed in g / 100g.

[0022] The present invention also provides a water-soluble fertilizer comprising the chitosan oligosaccharide, wherein the water-soluble fertilizer comprises the chitosan oligosaccharide, amino acid, alginic acid, humic acid, water-soluble organic carbon, urea and potassium sulfate.

[0023] Preferably, the pH of the water-soluble fertilizer is 7.3.

[0024] Preferably, the contents of the components in the water-soluble fertilizer are: chitosan oligosaccharide 5-15g / L, amino acid 80-100g / L, alginic acid 2-5g / L, humic acid 6-10g / L, water-soluble organic carbon 200-280g / L, urea 10-15g / L, potassium sulfate 20-25g / L, and the solvent is water.

[0025] The present invention also provides application of the water-soluble fertilizer in agriculture.

[0026] Preferably, the application specifically includes improving the yield and quality of vegetables such as lettuce, tomato and cucumber.

[0027] Preferably, the application includes improving the disease resistance of vegetables such as lettuce, tomato and cucumber.

[0028] Preferably, the disease is cucumber mosaic virus.

[0029] Compared with the prior art, the present invention has the following beneficial technical effects:

[0030] 1. By combining specific chitosanase and chitinase to degrade chitosan, the degradation time is shorter, the average molecular weight of the obtained chitosan oligosaccharides is lower, and the molecular weight distribution of chitosan oligosaccharides is narrower.

[0031] 2. It was verified in water-soluble fertilizers that the chitosan oligosaccharides obtained by the above combined degradation have higher physiological activity than those obtained by single enzyme degradation.

[0032] 3. The water-soluble fertilizer provided can significantly increase the yield and quality of crops including lettuce, tomatoes and cucumbers, as well as enhance the plants’ resistance to pathogens. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0034] Unless otherwise specified, the experimental methods involved in the examples are all conventional methods.

[0035] The main reagents and specifications involved in the examples are as follows:

[0036] 1. Chitosan, purchased from Jinan Dongxuan Bioengineering Co., Ltd., food grade (deacetylation degree 92%).

[0037] 2. Other reagents such as Pichia pastoris GS115 and expression vector pPIC9K can be obtained from commercial channels.

[0038] 3. Chitosanase encoded by the gene with the sequence shown at positions 11174 to 12541 of Bacillusmycoides and GenBank accession number QTTY0100044 and chitinase encoded by the gene with the sequence shown at positions 291495 to 293117 of Pseudoa l teromonasund in a GenBank accession number LITK01000002 were obtained by using Pichia pastoris GS115 and expression vector pPI C9K recombinant expression system.

[0039] Chitosanase activity determination: 3,5-dinitrosalicylic acid (DNS) method was used, with glucosamine as the standard. Enzyme activity definition: Under the reaction condition of 40°C, the amount of enzyme required to produce 1 μmol of reducing sugar per minute is defined as one enzyme activity unit (U). The chitosanase activity was measured to be 4800U / g.

[0040] The chitinase activity was determined according to GB / T34799-2017 standard, and the chitinase activity was found to be 2700 U / g.

[0041] According to the above-mentioned chitosanase activity determination method, the orthogonal test was used to determine the reducing sugar content in the reaction solution during the degradation of chitosan by chitosanase to prepare chitosan oligosaccharides, taking into account factors such as substrate, enzyme dosage ratio, reaction temperature, pH and time. The results showed that the optimal ratio of chitosan and chitosanase was 1g:60U, the optimal reaction temperature was 40℃, the optimal reaction pH was 5.0, and the optimal reaction time was 120min.

[0042] According to the above chitinase activity determination method, the chitinase degradation of chitosan to prepare chitosan oligosaccharides was measured by orthogonal test, considering factors such as substrate, enzyme dosage ratio, reaction temperature, pH and time, and the reducing sugar content in the reaction solution was determined. It was found that the optimal ratio of chitosan and chitinase was 1g:75U, the optimal reaction temperature was 50℃, the optimal reaction pH was 6.0, and the optimal reaction time was 140min.

[0043] Example 1 Preparation of chitosan oligosaccharides by degrading chitosan with chitosanase and chitinase 100 g of chitosan was added to 100 mL of glacial acetic acid, stirred while adding, and after the mixture became gelatinous, water was added to dilute it to 1 L, stirred evenly, and the pH was adjusted to 5.0 to obtain a colloidal chitosan solution, which was then heated to a predetermined temperature of 40°C, 6000 U of chitosanase and 7500 U of chitinase were added to the colloidal chitosan solution, stirred at a constant temperature, reacted for 80 min, reached the reaction end point, and then 10 ml of a 2% NaOH solution was added to terminate the reaction to obtain a reaction solution; the reaction solution was centrifuged and ultrafiltered to obtain a filtrate. The filtrate was directly dried with a spray dryer, and then crushed and sieved to obtain a finished product.

[0044] Example 2 Preparation of chitosan oligosaccharides by combining chitosanase and chitinase to degrade chitosan

[0045] 100g of chitosan is added to glacial acetic acid, and stirred while adding until the mixture becomes gelatinous, and then water is added to dilute it to 1L, and stirred evenly, and the pH is adjusted to 5.5 to obtain a colloidal chitosan solution, and then heated to a predetermined temperature of 45°C, 6000U chitosanase and 7500U chitinase are added to the colloidal chitosan solution, and stirred at a constant temperature. After reacting for 90 minutes, the reaction reaches the end point, and then 10ml of a 2% NaOH solution is added to terminate the reaction to obtain a reaction solution; the reaction solution is centrifuged and ultrafiltered to obtain a filtrate. The filtrate is directly dried with a spray dryer, and then crushed and sieved to obtain a finished product.

[0046] Example 3 Preparation of chitosan oligosaccharides by combining chitosanase and chitinase to degrade chitosan

[0047] 100g of chitosan is added to glacial acetic acid, and stirred while adding until the mixture becomes a gel, and then water is added to dilute it to 1L, and stirred evenly, and the pH is adjusted to 6.0 to obtain a colloidal chitosan solution, and then heated to a predetermined temperature of 50°C, 6000U chitosanase and 7500U chitinase are added to the colloidal chitosan solution, and stirred at a constant temperature. After reacting for 100 minutes, the reaction reaches the end point, and then 10ml of a 2% mass fraction NaOH solution is added to terminate the reaction to obtain a reaction solution; the reaction solution is centrifuged and ultrafiltered to obtain a filtrate. The filtrate is directly dried with a spray dryer, and then crushed and sieved to obtain a finished product.

[0048] Comparative Example 1 Preparation of Chitosan Oligosaccharides by Degrading Chitosan with Chitosanase

[0049] Add 100g of chitosan to glacial acetic acid, stir while adding until the mixture becomes gelatinous, then dilute with water to 1L, stir evenly, and adjust the pH to 5.0 to obtain a colloidal chitosan solution, then heat to a predetermined temperature of 40°C, add 6000U chitosanase to the colloidal chitosan solution, stir at a constant temperature, react for 120min, reach the reaction end point, then add 10ml of a 2% NaOH solution by mass to terminate the reaction, and obtain a reaction solution; centrifuge the reaction solution, ultrafilter to obtain a filtrate. The filtrate is directly dried with a spray dryer, and then crushed and sieved to obtain a finished product.

[0050] Comparative Example 2 Preparation of chitosan oligosaccharides by degradation of chitosan by chitinase 100g of chitosan was added to glacial acetic acid, stirred while adding until the mixture became colloid, then diluted to 1L with water, stirred evenly, and adjusted the pH to 6.0 to obtain a colloidal chitosan solution, then heated to a predetermined temperature of 50°C, 7500U of chitinase was added to the colloidal chitosan solution, stirred at a constant temperature, reacted for 140min, reached the end of the reaction, and then 10ml of a 2% by mass NaOH solution was added to terminate the reaction to obtain a reaction solution; the reaction solution was centrifuged and ultrafiltered to obtain a filtrate. The filtrate was directly dried with a spray dryer, and then crushed and sieved to obtain a finished product.

[0051] Test Example 1 Determination of Molecular Weight and Distribution of Chitosan Oligosaccharides

[0052] The average molecular weight and molecular weight distribution of the chitosan oligosaccharides obtained in Examples 1 to 3 and Comparative Examples 1 to 2 were measured using a gel permeation chromatography (GPC) system (Waters Technology (Shanghai) Co., Ltd., specifically including: a GPC aqueous phase system based on ACQUITI Arc, using a Waters Ultrahydrogel chromatographic column, to measure the molecular weight distribution of chitosan, and using Empower software to calculate the relative average molecular weight). The results are shown in Table 1.

[0053] Table 1 Average molecular weight and molecular weight distribution of chitosan oligosaccharides prepared in Examples 1-3 and Comparative Examples 1-2

[0054] Group Molecular weight distribution Average molecular weight Example 1 1100～3500 1200 Example 2 1000～3200 1100 Example 3 1200～3800 1300 Comparative Example 1 1400～7000 2600 Comparative Example 2 1600～6300 2900

[0055] As shown in Table 1, the combined degradation of chitosan by chitosanase and chitinase can not only shorten the degradation time, but also reduce the average molecular weight of chitosan oligosaccharides and narrow the molecular weight distribution of chitosan oligosaccharides.

[0056] Experimental Example 1 Preparation of water-soluble fertilizer containing chitosan oligosaccharide 5 g of chitosan oligosaccharide prepared in Example 2, 80 g of amino acid, 2 g of alginic acid, 6 g of humic acid, 200 g of water-soluble organic carbon, 10 g of urea, and 20 g of potassium sulfate were added to 1 L of aqueous solution in sequence, and the pH of the solution was adjusted to 7.3 to obtain a water-soluble fertilizer.

[0057] Experimental Example 2 Preparation of water-soluble fertilizer containing chitosan oligosaccharide

[0058] 10 g of chitosan oligosaccharide, 90 g of amino acid, 3 g of alginic acid, 8 g of humic acid, 240 g of water-soluble organic carbon, 12.5 g of urea and 22.5 g of potassium sulfate prepared in Example 2 were added to 1 L of aqueous solution in sequence, and the pH of the solution was adjusted to 7.3 to obtain a water-soluble fertilizer.

[0059] Experimental Example 3 Preparation of water-soluble fertilizer containing chitosan oligosaccharide

[0060] 15 g of chitosan oligosaccharide, 100 g of amino acid, 5 g of alginic acid, 10 g of humic acid, 280 g of water-soluble organic carbon, 15 g of urea and 25 g of potassium sulfate prepared in Example 2 were added to 1 L of aqueous solution in sequence, and the pH of the solution was adjusted to 7.3 to obtain a water-soluble fertilizer.

[0061] Comparative Experimental Example 1 Preparation of water-soluble fertilizer

[0062] The difference from Experimental Example 2 is that chitosan oligosaccharide is not added.

[0063] Comparative Experimental Example 2 Preparation of Water-soluble Fertilizer

[0064] The difference from Experimental Example 2 is that the chitosan oligosaccharide prepared in Example 2 is replaced by the chitosan oligosaccharide prepared in Comparative Example 1.

[0065] Comparative Experimental Example 3 Preparation of Water-soluble Fertilizer

[0066] The difference from Experimental Example 2 is that the chitosan oligosaccharide prepared in Example 2 is replaced by the chitosan oligosaccharide prepared in Comparative Example 2.

[0067] Application Example 1

[0068] Divide the lettuce in the greenhouse into 21 equal-sized areas, each with an area of ​​2m 2 , spraying was repeated 3 times. After the lettuce was planted, the water-soluble fertilizers prepared in Experimental Examples 1-3 and Comparative Experimental Examples 1-3 (diluted according to the ratio of 1L water-soluble fertilizer to 250L water) and the clean water group were used for the first spraying, and sprayed once every 7 days (100mL each time), for a total of 3 times. After each area of ​​lettuce grew, the outer leaves were removed and weighed, and the average yield of 3 repetitions was calculated. The results are shown in Table 2.

[0069] Table 2 Average yield of lettuce

[0070]

[0071]

[0072] **Indicates that the difference compared with the control group is significant by t test

[0073] As shown in Table 2, compared with the control group, the control group and the blank control group, the application of the water-soluble fertilizer containing the chitosan oligosaccharide of the present invention can significantly increase the yield of lettuce.

[0074] Application Example 2

[0075] Plastic pots with a diameter of 35cm and a depth of about 25cm were installed in the soil of the tomato greenhouse, and an appropriate amount of soil was installed in each plastic pot. One tomato seedling was planted in each pot, a total of 21 pots, and 3 repetitions were made in each pot. When the tomato seedlings were in the growth period, the water-soluble fertilizers prepared by Experimental Examples 1-3 and Comparative Experimental Examples 1-3 (diluted according to the proportion of 1L water-soluble fertilizer added to 250L water) and the clean water group were sprayed on the leaves respectively, once a week (10mL each time), and sprayed 8 times in total. After the tomatoes matured, they were weighed respectively, and the final yield of each pot of fruit was accumulated to calculate the average final yield of each group. The iodine titration method was used to determine the Vc content, the acid-base titration method was used to measure the total acidity, and the anthrone method was used to determine the soluble sugar. The results are shown in Table 3.

[0076] Table 3 Average yield and quality of tomatoes

[0077]

[0078]

[0079] **Indicates that the difference compared with the control group is significant by t test

[0080] As shown in Table 3, compared with the control group, the control group and the blank control group, the application of the water-soluble fertilizer containing the chitosan oligosaccharide of the present invention can significantly improve the yield and quality of tomatoes, such as the Vc content and the sugar-acid ratio.

[0081] Application Example 3

[0082] When potted cucumber grows to 3-4 leaf stage, after inoculation of cucumber mosaic virus (Cucumber Mosaic Virus, hereinafter referred to as CMV, ATCC NO.PV-29, purchased from Beijing Biobowei Biotechnology Co., Ltd.), the water-soluble fertilizer prepared by Experimental Example 1-3 and Comparative Experimental Example 1-3 (adding 250L water proportional dilution according to 1L water-soluble fertilizer) is sprayed on the leaves, once a week (each time 10mL), spraying 5 times in total. It is divided into 8 groups in total, 20 potted cucumbers in each group, corresponding to embodiment 1-3, comparative experimental example 1-3, clear water+inoculation CMV group, and non-inoculation CMV group. Finally, the cucumber incidence rate of each group is analyzed and counted. The result is as shown in Table 4.

[0083] Table 4 Effect of water-soluble fertilizers on cucumbers against CMV

[0084]

[0085]

[0086] **Indicates that the difference compared with the control group is significant by t test

[0087] It can be seen from Table 4 that, compared with the control group and the blank control group, the application of the water-soluble fertilizer containing the chitosan oligosaccharide of the present invention can significantly improve the resistance of cucumber seedlings to cucumber mosaic virus infection.

[0088] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A process for preparing chitosan oligosaccharide, characterized in that: The steps include: 1) Add chitosan to the acidic solution, stir while adding until the mixture becomes a gel, then dilute with water, stir evenly, and adjust the pH to obtain a colloidal chitosan solution; 2) heating the colloidal chitosan solution to a predetermined temperature, adding chitosanase and chitinase to the colloidal chitosan solution, stirring at a constant temperature, and reaching the reaction end point after a certain reaction time, and then adding NaOH solution to terminate the reaction to obtain a reaction solution; 3) The reaction solution is centrifuged and ultrafiltered to obtain a filtrate, the filtrate is directly dried by a spray dryer, and then crushed and sieved to obtain chitosan oligosaccharide. The amount of chitosan in step 1) and the amount of chitosanase and chitinase added in step 2) are in a ratio of 1 g:60 U:75 U, the amount of chitosan in step 1) and the volume of water dilution are 100 g:1 L, the acidic solution is any one of glacial acetic acid, succinic acid or maleic acid, and the pH value is 5.0-6.0; In step 2), the chitosanase is derived from Bacillus mycoides, specifically the chitosanase encoded by the gene sequence shown at positions 11174 to 12541 of GenBank Accession No. QTTY0100044, and the chitinase is derived from Pseudoalteromonas undina, specifically the chitinase encoded by the gene sequence shown at positions 291495 to 293117 of GenBank Accession No. LITK01000002; The chitosanase and chitinase are both obtained by recombinant expression through a common Pichia pastoris expression system, and the chitosanase and chitinase activities obtained by recombinant expression are 4800U / g and 2700U / g, respectively; In step 2), the predetermined temperature is 40° C., 50° C. or 45° C., the reaction time is 80 min, 100 min or 90 min, the volume ratio of the amount of NaOH solution added in step 2) to the volume ratio of the dilution water in step 1) is 1 mL:100 mL, and the mass fraction of the NaOH solution is 2%, in units of g / 100 g.

2. The preparation process according to claim 1, characterized in that: The pH value in step 1) is 5.0, 5.5 or 6.

0.

3. The preparation process according to claim 1, characterized in that: The Pichia pastoris in the recombinant expression of the Pichia pastoris expression system is Pichia pastoris GS115, and the expression vector is pPIC9K.

4. A water-soluble fertilizer, characterized in that: The water-soluble fertilizer is composed of chitosan oligosaccharides, amino acids, alginic acid, humic acid, water-soluble organic carbon, urea, potassium sulfate and water obtained by the preparation process of any one of claims 1 to 3. The content of each component in the water-soluble fertilizer is: chitosan oligosaccharides 5-15 g / L, amino acids 80-100 g / L, alginic acid 2-5 g / L, humic acid 6-10 g / L, water-soluble organic carbon 200-280 g / L, urea 10-15 g / L, potassium sulfate 20-25 g / L, the solvent is water, and the pH of the water-soluble fertilizer is 7.

3.

5. Application of the chitosan oligosaccharide obtained by the preparation process of any one of claims 1 to 3 or the water-soluble fertilizer according to claim 4 in agriculture, wherein the application is to improve the yield, quality and disease resistance of lettuce, tomato and cucumber, and the disease is cucumber mosaic virus disease.

Images