CN114656301B

CN114656301B - Chlorella biofertilizer for promoting growth and improving disease resistance

Info

Publication number: CN114656301B
Application number: CN202210412127.9A
Authority: CN
Inventors: 汪少丽; 王萌; 宫海峰; 吴春峰; 刘保友; 吴建燕; 张占田; 王雪颖; 王洪涛; 张伟; 陈海宁
Original assignee: Yantai Hongyuan Biological Fertilizer Co ltd; Shandong Yantai Academy of Agricultural Sciences
Current assignee: Yantai Hongyuan Biological Fertilizer Co ltd; Shandong Yantai Academy of Agricultural Sciences
Priority date: 2022-04-19
Filing date: 2022-04-19
Publication date: 2023-05-26
Anticipated expiration: 2042-04-19
Also published as: CN114656301A

Abstract

The invention discloses a chlorella biofertilizer for promoting growth and improving disease resistance. Belongs to the technical field of biological fertilizer. The biological fertilizer comprises the following components in parts by volume: 1000 parts of chlorella extract and 1-3 parts of intelligent agent. Compared with the prior art, the invention has the beneficial effects that: (1) The chlorella extract and the intelligent synergistic effect in the chlorella biofertilizer are obviously higher than that of single components, so that the activity of root systems is effectively promoted, the photosynthesis of leaves is obviously improved, the yield of crops is obviously improved, the metabolic efficiency of plants is improved, and the disease resistance is increased; (2) The chlorella biological fertilizer has the advantages of few components, simple and feasible preparation process, effective extraction and retention of bioactive substances in chlorella, low production cost, easy control of the production process, contribution to large-scale industrial production and field application, and easy popularization and application.

Description

Chlorella biofertilizer for promoting growth and improving disease resistance

Technical Field

The invention relates to the technical field of biofertilizers, in particular to a chlorella biofertilizer for promoting growth and improving disease resistance.

Background

Pesticides and fertilizers make an important contribution to disease control and crop yield improvement, but with the continuous increase of the usage amount of pesticides and fertilizers, the grain quality safety and environmental safety problems faced by China are increasingly prominent. Especially, in recent years, with the excessive use of pesticides and fertilizers, environmental pollution and ecological deterioration are caused, and a series of serious ecological environment problems such as pesticide residues, soil structure damage, agricultural product quality reduction and the like are caused. The reasons for the method are not only the problem of scientific medical fertilizers, but also the quality problem of pesticides and fertilizers. The quality safety of pesticides and fertilizers severely restricts the sustainable development of agriculture, and threatens the physical and mental health of people directly or indirectly. Therefore, the quality safety of pesticides and fertilizers is started, the green ecological environment-friendly fertilizer is promoted greatly, the disease resistance of plants is improved effectively while the growth of the plants is promoted, and the use of pesticides is reduced.

Chlorella is a common unicellular green algae, is widely distributed in nature, is mostly in fresh water areas, has large biomass, can utilize light energy to autotrophy, can also utilize organic carbon sources to grow and reproduce under heterotrophic conditions, and has 15 kinds of chlorella in the world at present and hundreds of varieties. The content of the crude protein of the chlorella is particularly high and can reach more than 50%, especially the chlorella pyrenoidosa is the highest, and the content of the crude protein is much higher than that of the crude protein (40%) of the soybean, and the protein quality is much better. Among essential amino acids, lysine, arginine, leucine and valine are high in content, phenylalanine, isoleucine and threonine are high in content, histidine is low in content, and methionine and tryptophan are low in content. Also contains abundant physiologically active ingredients such as: growth Factors (CGF), beta-carotene, chlorophyll, vitamins, trace elements, polysaccharides, phycocyanin, unsaturated fatty acids, etc. Because the chlorella has higher nutrient content and has the health care function, the chlorella is developed for the fields of protein source feed, food, beverage and medical products.

Intelligent intelligence is a novel green plant endophyte metabolite mixture. It improves crop yield and quality by improving metabolic efficiency of plants; increasing the resistance and restoring force of the plant to abiotic stress; improving quality attribute (sugar content, color, fruit seed number, etc.) of agricultural products; improving the water utilization rate, improving the physicochemical property of the soil, regulating the microbial flora of the soil, and the like. The intelligent agent used as a plant immunity inducer has no direct bactericidal activity, is not easy to generate drug resistance to germs, can be mixed with bactericides or fertilizers, and has wide application prospect.

At present, the development of chlorella biological fertilizers is mainly focused on the mixing of chlorella with fertilizers, bacillus, livestock manure, straws and other biological organic matters, the components of the mixed fertilizers are complex, the steps are complicated, and the stability of biological activity is questionable. The invention uses chlorella extract as main raw material, which avoids the problems of unstable activity and incomplete exertion of effective components caused by directly using chlorella as raw material. The invention uses chlorella as raw material to extract various bioactive substances such as protein, growth factor, amino acid and the like, and simultaneously adds intelligent intelligence with the effect of improving photosynthesis and yield of plants, thus being an agricultural green organic fertilizer.

Disclosure of Invention

In view of the above, the invention provides a chlorella biofertilizer for promoting growth and improving disease resistance. The chlorella biofertilizer provided by the invention has the advantages that the crude protein in chlorella extract is not less than 45%, chlorophyll is not less than 6.5mg/g, and the chlorella biofertilizer also has the intelligent intelligence for improving plant disease resistance; meanwhile, the preparation process of the chlorella biological fertilizer is simple and easy to implement, can effectively extract and retain bioactive substances in the chlorella, has low production cost, is easy to control, and is beneficial to large-scale industrial production and field application.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the chlorella biofertilizer for promoting growth and improving disease resistance comprises the following components in parts by volume: 1000 parts of chlorella extract and 1-3 parts of intelligent agent.

The beneficial effects are that: the chlorella extract has high content of high-activity bioactive substances (mainly containing various bioactive substances such as protein, growth factors, amino acids and the like), can effectively improve root activity, promote synthesis of chlorophyll, enhance photosynthesis, promote accumulation and conversion of sugar, improve plant growth vigor, and further achieve the aim of improving quality and yield. As a plant immunity-inducing agent, intelligent smart (intelligent smart is crude ethanol extract of endophytic fungus paecilomyces Paecillomyces vannayi in wild sea buckthorn plants) can improve disease resistance and stress resistance of plants by regulating metabolism of the plants and inducing own defense mechanisms of the plants. The two are mixed for use, so that the metabolic efficiency of the plants is improved, the crop yield and quality are improved, the resistance of the plants to pathogenic bacteria is improved, and the synergistic effect is good.

Further, the chlorella biological fertilizer for promoting growth and improving disease resistance comprises the following components in parts by volume: 1000 parts of chlorella extract and 3 parts of intelligent agent;

the chlorella is Chlorella pyrenoidosa.

Further, the content of crude protein in the chlorella extract is not less than 45%, and the content of chlorophyll is not less than 6.5mg/g.

Further, the preparation method of the chlorella extract comprises the following steps:

(1) Preparing an algae liquid:

performing high-density heterotrophic culture on chlorella to obtain an algae liquid;

(2) Preparing a raw algae liquid:

sequentially carrying out enzymolysis on the algae liquid by cellulase, pectase, alkaline protease and dunaliella salina glycosidase to obtain a raw algae liquid;

(3) Preparing chlorella extract:

filtering the raw algae liquid, and taking supernatant to obtain chlorella extract with crude protein content of not less than 45% and chlorophyll content of not less than 6.5mg/g.

The beneficial effects are that: in the process of producing seaweed-derived biostimulant by using algae, on one hand, algae are digested, so that cell walls are completely broken as much as possible, and then the contents in cells are released, and small molecular substances which are degraded by macromolecular substances in the cells and are easy to be absorbed and utilized are obtained; on the other hand, the phytohormone therein is well reserved. In the early seaweed-derived biostimulant production process, people attach great importance to the extraction of nutrients such as polysaccharide, mineral elements and trace elements, and a high-temperature and high-pressure extraction mode is generally adopted, so that plant hormones contained in seaweed are destroyed, and the produced fertilizer has no obvious effect on the growth promotion of crops.

According to the invention, cellulase, pectase, protease and fucosidase are sequentially added into chlorella algae liquid for enzymolysis to obtain the raw algae liquid, the raw algae liquid is filtered, and supernatant is taken to obtain the chlorella extract containing active ingredients with crude protein content of not less than 45% and chlorophyll content of not less than 6.5mg/g, so that bioactive substances in chlorella can be effectively extracted and reserved, the utilization rate of the chlorella active ingredients is improved, and the wall breaking rate is high, and the method is rapid and simple to operate.

Further, the concentration of chlorella in the step (1) is 30 hundred million/ml.

The beneficial effects are that: the minimum concentration is set to ensure enough product concentration, and the maximum concentration is set to ensure that the chlorella can be fully degraded after the degrading enzyme is added, and the chlorella cannot be fully degraded due to overlarge concentration, so that the product yield is reduced.

Further, the specific operation of the step (2) is as follows: adding 0.05% -0.1% of cellulase into the algae liquid, and performing enzymolysis for 1h under the conditions that the temperature is 52-60 ℃ and the pH value is 6.0-6.4; adding 0.1-0.15% pectase, and performing enzymolysis for 1-2 h at 48-56 ℃ and pH value of 3.5-4.5; then adding 0.1% alkaline protease, and carrying out enzymolysis for 0.5h under the conditions that the temperature is 40-65 ℃ and the pH value is 7.5-9.5; finally adding 0.01% of fucosidase, and carrying out enzymolysis for 0.5h under the conditions that the temperature is 48-55 ℃ and the pH value is 5.8-6.7; filtering to obtain the filtrate, and obtaining the crude algae liquid.

The application of chlorella biofertilizer in promoting plant growth and improving plant disease resistance.

Furthermore, water is added for dilution during use, and a spraying or root irrigation mode is adopted for carrying out.

Further, the water is diluted by 500-1000 times when in spray use, and diluted by 300-800 times when in root irrigation use.

Further, spraying or root irrigation is carried out once every 7-10 days, and the period of spraying or root irrigation is 2-3 months.

Compared with the prior art, the invention has the beneficial effects that: (1) The chlorella extract and the intelligent synergistic effect in the chlorella biofertilizer are obviously higher than that of single components, so that the activity of root systems is effectively promoted, the photosynthesis of leaves is obviously improved, the yield of crops is obviously improved, the metabolic efficiency of plants is improved, and the disease resistance is increased; (2) The chlorella biological fertilizer has the advantages of few components, simple and feasible preparation process, effective extraction and retention of bioactive substances in chlorella, low production cost, easy control of the production process, contribution to large-scale industrial production and field application, and easy popularization and application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing plant height variation and root length after spray treatment according to example 4 of the present invention;

FIG. 2 is a graph showing the change of leaf area and chlorophyll content after spray treatment in example 4 of the present invention;

FIG. 3 is a graph showing the change of the index and the growth function of the strong seedlings after the spraying treatment in the embodiment 4 of the invention;

FIG. 4 is a graph showing the dry weight and stem thickness change of the plant after spray treatment according to example 4 of the present invention;

FIG. 5 is a diagram showing the plant height change and root length after root irrigation treatment according to embodiment 5 of the present invention;

FIG. 6 is a graph showing the leaf area change and chlorophyll content after root irrigation treatment according to example 5 of the present invention;

FIG. 7 is a graph showing the change of the index and the growth function of the strong seedlings after the root irrigation treatment in example 5 of the present invention;

FIG. 8 is a graph showing the dry weight and stem thickness change of the plant after the root irrigation treatment according to example 5 of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Intelligent smart organic fertilizer (liquid) was purchased from: shandong's Boston Biotechnology Co., ltd.

The experimental methods not mentioned in the examples are conventional experimental methods, and are not described in detail herein.

The preparation method of the chlorella comprises the following steps of:

s1, activating algae: placing chlorella species in an illumination incubator for culture and activation;

s2, shake flask culture: placing the activated chlorella algae in a first culture bottle for illumination culture, and transferring the culture bottle to a shake flask for light-shielding culture to obtain seed liquid;

s3, seed culture: transferring the seed liquid after shake flask culture into a seed tank for light-shielding culture;

s4, culturing in a main fermentation tank: placing the seed liquid after seed culture in a main fermentation tank, supplementing 0.1g/l citric acid at 48h, and opening valves of the main fermentation tank and an illumination culture tank to circularly flow and receive illumination; at 60h, supplementing urea with the concentration of 1g/l, closing valves of the main fermentation tank and the illumination culture tank, and fermenting and culturing for 84h;

the main fermentation tank is internally provided with a fermentation medium, and the fermentation medium at least comprises:

the amino acid fermentation broth contains at least 15wt% glutamic acid.

See patent 202210155408.0 for details.

Example 1

(1) The chlorella is subjected to high-density heterotrophic culture (the concentration of the chlorella is 30 hundred million/ml) to obtain an algae liquid.

(2) Adding 0.05% cellulase into the above algae liquid, and performing enzymolysis at 52deg.C and pH of 6.0 for 1 hr; adding 0.1% pectase, and performing enzymolysis at 48deg.C and pH of 3.5 for 1 hr; then adding 0.1% alkaline protease, and performing enzymolysis for 0.5h at 40 ℃ and pH value of 7.5; finally adding 0.01% of fucosidase, and performing enzymolysis for 0.5h at 48 ℃ and pH value of 5.8; obtaining the raw algae liquid.

(3) Filtering the crude alga solution obtained in the step (2), and taking supernatant to obtain chlorella extract with crude protein content of not less than 45% and chlorophyll content of not less than 6.5mg/g active ingredient.

(4) Mixing the chlorella extract obtained in the step (3) with intelligent smart fertilizer according to the volume ratio of 1000:1 to obtain the chlorella biofertilizer.

Example 2

(2) Adding 0.1% cellulase into the above algae liquid, and performing enzymolysis at 60deg.C and pH of 6.4 for 1 hr; adding 0.15% pectase, and performing enzymolysis at 56 deg.C and pH of 4.5 for 2 hr; then adding 0.1% alkaline protease, and performing enzymolysis for 0.5h at 65 ℃ and pH value of 9.5; finally adding 0.01% of fucosidase, and performing enzymolysis for 0.5h at 55deg.C and pH of 6.7; obtaining the raw algae liquid.

(3) Filtering the crude alga solution obtained in the step (2), and taking supernatant to obtain the chlorella extract containing the active ingredients with the crude protein content of not less than 45% and the chlorophyll content of not less than 6.5mg/g.

(4) Mixing the chlorella extract obtained in the step (3) with intelligent smart fertilizer according to the volume ratio of 1000:2 to obtain the chlorella biofertilizer.

Example 3

(2) Adding 0.07% cellulase into the above algae liquid, and performing enzymolysis for 1 hr at 55deg.C and pH of 6.2; adding 0.12% pectase, and performing enzymolysis at 50deg.C and pH of 4.0 for 1.5 hr; then adding 0.1% alkaline protease, and performing enzymolysis for 0.5h at 60 ℃ and pH value of 8.5; finally adding 0.01% of fucosidase, and performing enzymolysis for 0.5h at 50deg.C and pH of 6.2; obtaining the raw algae liquid.

(4) Mixing the chlorella extract obtained in the step (3) with intelligent smart fertilizer according to the volume ratio of 1000:3 to obtain the chlorella biofertilizer.

Comparative example 1

Comparative example 2

Intelligent intelligence.

Example 4

The chlorella biofertilizer prepared in examples 1 to 3, the chlorella extract prepared in comparative example 1 and the intelligent agent prepared in comparative example 2 were sprayed, and the specific operations are as follows:

diluting according to the proportion (volume ratio) of fertilizer to water of 1:500, and spraying after 2 true leaves grow from the pepper seedlings, wherein the spraying period is 2 months after spraying every 7 days.

Evaluation index: plant height variation, root length, leaf area variation, chlorophyll content, strong seedling index, growth function, plant dry weight and stem thickness variation. Wherein, the liquid crystal display device comprises a liquid crystal display device,

strong seedling index = whole plant dry mass x stem thickness/plant height;

growth function = whole plant dry mass/time to grow seedlings.

The change of the growth index of the capsicum after 2 months is shown in fig. 1 to 4, and the control effect of the capsicum on the capsicum virus disease and epidemic disease is shown in table 1.

TABLE 1 control of capsicum virus disease and epidemic disease after spray treatment

Example 5

The biofertilizer of chlorella prepared in examples 1 to 3, the chlorella extract prepared in comparative example 1 and the intelligent agent in comparative example 2 were subjected to root irrigation, and the specific operations are as follows:

diluting according to the ratio (volume ratio) of fertilizer to water of 1:500, and irrigating roots after 2 true leaves of the pepper seedlings grow, wherein root irrigation is performed once every 7 days, and the root irrigation period is 2 months.

strong seedling index = whole plant dry mass x stem thickness/plant height;

growth function = whole plant dry mass/time to grow seedlings.

The change of the growth index of the capsicum after 2 months is shown in fig. 5 to 8, and the control effect of the capsicum on the capsicum virus disease and epidemic disease is shown in table 2.

TABLE 2 control of capsicum virus disease and epidemic disease after root irrigation treatment

As can be seen from fig. 1 to 8 and tables 1 to 2, after the chlorella biofertilizer prepared in example 1, example 2 and example 3 is sprayed or root-irrigated on pepper plants, the plant height, root length, leaf area, chlorophyll, seedling strengthening index, growth function, plant dry weight and stem thickness are all improved, and the chlorella biofertilizer has good control effects on pepper virus diseases and epidemic diseases, which indicates that the chlorella biofertilizer obviously promotes the growth of pepper plants and improves the disease resistance of peppers. From the prior application, comparative example 1 and comparative example 2, only the application of the chlorella extract and the intelligent agent has a certain effect of promoting the growth and improving the disease resistance of plants, but the effect is obviously lower than the effect of the chlorella extract and the intelligent agent after the mixture, which shows that the chlorella extract and the intelligent agent provided by the invention can have a synergistic effect, and the effect is obviously higher than the effect of promoting the growth and improving the disease resistance of plants by using a single component.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The chlorella biofertilizer for promoting growth and improving disease resistance is characterized by comprising the following components in parts by volume: 1000 parts of chlorella extract and 1-3 parts of intelligent agent;

the preparation method of the chlorella extract comprises the following steps:

(1) Preparing an algae liquid:

(2) Preparing a raw algae liquid:

adding 0.05% -0.1% of cellulase into the algae liquid, and performing enzymolysis for 1h under the conditions that the temperature is 52-60 ℃ and the pH value is 6.0-6.4; adding 0.1-0.15% pectase, and performing enzymolysis for 1-2 h at 48-56 ℃ and pH value of 3.5-4.5; then adding 0.1% alkaline protease, and carrying out enzymolysis for 0.5h under the conditions that the temperature is 40-65 ℃ and the pH value is 7.5-9.5; finally, adding 0.01% of fucosidase, and carrying out enzymolysis for 0.5h at 48-55 ℃ and pH value of 5.8-6.7 to obtain a raw algae liquid;

(3) Preparing chlorella extract:

filtering the raw algae liquid, and taking supernatant to obtain chlorella extract with crude protein content of not less than 45% and chlorophyll content of not less than 6.5mg/g;

the disease resistance is anti-capsicum virus disease and epidemic disease.

2. The chlorella biofertilizer for promoting growth and improving disease resistance of claim 1, comprising the following components in parts by volume: 1000 parts of chlorella extract and 3 parts of intelligent agent.

3. The chlorella biofertilizer for promoting growth and improving disease resistance according to any one of claims 1 or 2, wherein the content of crude protein in chlorella extract is equal to or greater than 45%, and the chlorophyll content is equal to or greater than 6.5mg/g;

the chlorella is Chlorella pyrenoidosa.

4. The chlorella biofertilizer for promoting growth and improving disease resistance of claim 1, wherein the concentration of chlorella in step (1) is 30 billion/ml.

5. Use of the chlorella biofertilizer of any one of claims 1 or 2 for promoting plant growth and improving plant disease resistance;

the plant is pepper;

the plant disease resistance is anti-capsicum virus disease and epidemic disease.

6. Use according to claim 5, wherein the use is carried out by diluting with water and spraying or root irrigation.

7. The use according to claim 6, wherein the dilution is 500-1000 times when in spray use and 300-800 times when in root irrigation use.

8. The use according to claim 7, wherein the spraying or rooting is performed once every 7 to 10 days, with a spraying or rooting period of 2 to 3 months.