CN114656301A

CN114656301A - Chlorella biofertilizer capable of promoting growth and improving disease resistance

Info

Publication number: CN114656301A
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: 2022-06-24
Anticipated expiration: 2042-04-19
Also published as: CN114656301B

Abstract

The invention discloses a chlorella biofertilizer for promoting growth and improving disease resistance. Belongs to the technical field of biological fertilizers. 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 following beneficial effects: (1) the chlorella extract and the intelligent intelligence in the chlorella biofertilizer provided by the invention have a synergistic effect, so that the effects of promoting plant growth and improving plant disease resistance are obviously higher than those of using a single component, the root activity 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 resistance to diseases is increased; (2) the chlorella biofertilizer disclosed by the invention is few in components, simple and feasible in preparation process, low in production cost, easy to control in production process, beneficial to large-scale industrial production and field application and easy to popularize and apply, and can effectively extract and retain bioactive substances in chlorella.

Description

Chlorella biofertilizer capable of 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 chemical fertilizers make important contribution to preventing and treating diseases and improving crop yield, but with the continuous increase of the usage amount of pesticides and chemical fertilizers, the problems of grain quality safety and environmental safety in China are increasingly prominent. Particularly, along with the excessive use of pesticides and fertilizers in recent years, environmental pollution and ecological deterioration are caused, and a series of serious ecological environmental problems such as pesticide residue, soil structure damage, agricultural product quality reduction and the like appear. The reason for this is that there are problems of scientific use of medicinal fertilizer and quality of pesticide and chemical fertilizer. The quality safety of pesticides and chemical fertilizers seriously restricts the sustainable development of agriculture, and directly or indirectly threatens the physical and psychological health of people. Therefore, starting from the quality safety of pesticides and chemical fertilizers, the green and ecological environment-friendly fertilizer is widely popularized, the plant growth is promoted, the disease resistance of the plants is effectively improved, and the use of pesticides is reduced.

The chlorella is a common unicellular green alga which is widely distributed in the nature, has a large biomass, can be grown and propagated by utilizing light energy autotrophy and organic carbon sources under heterotrophic conditions, and has about 15 types and more than one hundred types of varieties in the world. The content of the crude protein of the chlorella is particularly high and can reach more than 50 percent, particularly, the content of the crude protein of the chlorella pyrenoidosa is higher than that of the crude protein (40 percent) of the soybean, and the quality of the protein is much better. Among the essential amino acids, lysine, arginine, leucine, and valine are high, phenylalanine, isoleucine, and threonine are high, histidine is slightly low, and methionine and tryptophan are low. Also contains abundant physiological active ingredients, such as: growth Factor (CGF), beta-carotene, chlorophyll, vitamins, trace elements, polysaccharide, phycocyanin, unsaturated fatty acid, etc. The chlorella has high nutrient content and health care function, so the chlorella is developed and used in the fields of protein source feed, food, beverage and medical products in sequence.

The intelligent agent is a novel green plant endophyte metabolite mixture. It improves crop yield and quality by improving metabolic efficiency of plants; increasing the resistance and resilience of plants to abiotic stress; improving the quality attributes (sugar content, color, fruit number, etc.) of the agricultural products; improving the water utilization rate, improving the physical and chemical properties of soil, regulating the microbial flora of the soil and the like. The intelligent agent does not have direct bactericidal activity when being used as a plant immunity inducer, and pathogenic bacteria are not easy to generate drug resistance to the agent, so the agent can be mixed with bactericide or fertilizer, and has wide application prospect.

At present, the development of chlorella biological fertilizers mostly focuses on the mixing of chlorella and biological organic matters such as chemical fertilizers, bacillus, livestock excrement, straws and the like, and the components of the mixed fertilizers are complex, the steps are complicated, and the stability of biological activity is questionable. The invention uses the chlorella extract as the main raw material, and avoids the problems of unstable activity and incomplete exertion of effective components caused by directly using the chlorella as the raw material. Therefore, the chlorella is used as a raw material, various bioactive substances such as protein, growth factors, amino acids and the like in the chlorella are extracted, and meanwhile, the intelligent agent with the effects of improving the photosynthesis and the yield of plants is added, so that the chlorella is 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. According to the chlorella biological fertilizer provided by the invention, crude protein in chlorella extract is not less than 45%, chlorophyll is not less than 6.5mg/g, and the chlorella biological fertilizer also contains intelligent agent capable of improving plant disease resistance; meanwhile, the preparation process of the chlorella biofertilizer is simple and easy to implement, can effectively extract and retain bioactive substances in the chlorella, is low in production cost, is easy to control, and is beneficial to large-scale industrial production and field application.

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

a 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 as follows: 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 system activity, promote chlorophyll synthesis, enhance photosynthesis, promote sugar accumulation and conversion, improve plant growth vigor, and further achieve the purpose of improving quality and yield. The intelligent agent is a crude ethanol extract of endophytic fungi Paecilomyces vannayi in wild sea buckthorn plants, and can improve the disease resistance and stress resistance of plants by regulating the metabolism of the plants and inducing the self defense mechanism of the plants. The two are mixed for use, so that the metabolic efficiency of the plant is improved, the yield and the quality of the crop are improved, the resistance of the plant to pathogenic bacteria is increased, and a good synergistic effect is achieved.

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.

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

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

(1) preparing an algae solution:

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

(2) preparing an original algae solution:

sequentially carrying out enzymolysis on the algae liquid by using cellulase, pectinase, alkaline protease and halophyacinosidase to obtain a raw algae liquid;

(3) preparing a chlorella extract:

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

The beneficial effects are as follows: in the process of producing the alga-derived biostimulant by using the alga, on one hand, the alga body is digested to ensure that the cell wall is completely broken as far as possible, and then the content in the cell is released and the micromolecule substance which is degraded by macromolecular substance and is easy to absorb and utilize in the cell is obtained; on the other hand, the plant hormone in the plant extract is well preserved. In the early production process of the seaweed-derived biostimulant, people pay more attention to the extraction of polysaccharide, mineral elements, trace elements and other nutrient substances, and the high-temperature and high-pressure extraction mode is generally adopted, so that the phytohormone contained in the seaweed is damaged, and the growth promotion effect of the produced fertilizer on crops is not obvious.

According to the invention, cellulase, pectinase, protease and fucosidase are sequentially added into chlorella solution for enzymolysis to obtain raw chlorella solution, the raw chlorella solution is filtered, and the 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 retained, the utilization rate of effective ingredients of chlorella is improved, and the method has the advantages of high wall breaking rate, rapidness and simplicity in operation.

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

The beneficial effects are as follows: the minimum concentration is set to ensure sufficient product concentration, and the maximum concentration is set to ensure that the chlorella can be sufficiently degraded after the degrading enzyme is added, and the chlorella cannot be completely degraded due to too high 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 carrying out enzymolysis for 1h at the temperature of 52-60 ℃ and the pH value of 6.0-6.4; adding 0.1-0.15% of pectinase, and carrying out enzymolysis for 1-2 h at the temperature of 48-56 ℃ and the pH value of 3.5-4.5; then adding 0.1% of 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% 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 filtrate to obtain original algae solution.

The chlorella biofertilizer is used in promoting plant growth and raising plant disease resistance.

Furthermore, water is required to be added for dilution during use, and the spraying or root irrigation is adopted.

Furthermore, the water is diluted by 500-1000 times when being sprayed and diluted by 300-800 times when being irrigated to roots.

Further, spraying or irrigating the roots once every 7-10 days, wherein the spraying or irrigating period is 2-3 months.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: (1) the chlorella extract and the intelligent intelligence in the chlorella biofertilizer provided by the invention have a synergistic effect, so that the effects of promoting plant growth and improving plant disease resistance are obviously higher than those of using a single component, the root activity 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 resistance to diseases is increased; (2) the chlorella biofertilizer disclosed by the invention is few in components, simple and feasible in preparation process, low in production cost, easy to control in production process, beneficial to large-scale industrial production and field application and easy to popularize and apply, and can effectively extract and retain bioactive substances in chlorella.

Drawings

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

FIG. 1 is a schematic diagram showing plant height change and root length after spray treatment in example 4 of the present invention;

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

FIG. 3 is a schematic diagram showing the variation of the seedling growth index and growth function after the spraying treatment in example 4 of the present invention;

FIG. 4 is a schematic diagram showing the changes in dry weight and stem thickness of plants after spray treatment in example 4 of the present invention;

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

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

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

FIG. 8 is a schematic diagram showing the changes in dry weight and stem thickness of plants after root irrigation according to example 5 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Intelligent intelligent organic fertilizer (liquid) is purchased from: shandong Pengbo Biotech, Inc.

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

The preparation method of Chlorella comprises (Chlorella is Chlorella pyrenoidosa):

s1 algal species activation: placing chlorella algae seeds in a light incubator for culture and activation;

s2 shake flask culture: placing the activated chlorella strain in a first culture bottle for illumination culture, and then transferring the chlorella strain into a shake flask for dark culture to obtain a seed solution;

s3 seed culture: transferring the seed liquid after the shake flask culture to a seed tank for dark culture;

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

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

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

See patent 202210155408.0 for details.

Example 1

(1) Performing high-density heterotrophic culture on Chlorella (Chlorella concentration is 30 hundred million/ml) to obtain Chlorella solution.

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

(3) And (3) filtering the raw algae liquid obtained in the step (2), and taking the supernatant to obtain a chlorella extract with crude protein content being not less than 45% and chlorophyll content being not less than 6.5mg/g of active ingredients.

(4) And (4) mixing the chlorella extract obtained in the step (3) with an intelligent agent according to the volume ratio of 1000:1 to obtain the chlorella biofertilizer.

Example 2

(2) Adding 0.1% cellulase into the algae solution, and performing enzymolysis for 1h at 60 deg.C and pH of 6.4; 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 deg.C and pH of 9.5; finally adding 0.01% fucosidase, and performing enzymolysis for 0.5h at 55 deg.C and pH of 6.7; obtaining the original algae liquid.

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

(4) And (4) mixing the chlorella extract obtained in the step (3) with an intelligent agent according to the volume ratio of 1000:2 to obtain the chlorella biofertilizer.

Example 3

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

(4) And (4) mixing the chlorella extract obtained in the step (3) with an intelligent agent according to the volume ratio of 1000:3 to obtain the chlorella biofertilizer.

Comparative example 1

Comparative example 2

Intelligence clever.

Example 4

The chlorella biofertilizer prepared in the embodiments 1-3, the chlorella extract prepared in the comparative example 1 and the intelligent spraying method in the comparative example 2 are sprayed, and the specific operations are as follows:

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

Evaluation indexes are as follows: plant height variation, root length, leaf area variation, chlorophyll content, strong seedling index, growth function, plant dry weight, and stem thickness variation. Wherein, the first and the second end of the pipe are connected with each other,

the strong seedling index is the quality of the whole plant stem multiplied by the stem thickness/plant height;

growth function ═ whole plant dry mass/time of seedling.

The growth index change of the pepper after 2 months is shown in figures 1-4, and the control effect of the pepper on pepper virus diseases and epidemic diseases is shown in table 1.

TABLE 1 prevention of pepper virus and epidemic diseases after spray treatment

Example 5

The biofertilizer for the chlorella prepared in the embodiments 1-3, the chlorella extract prepared in the comparative example 1 and the intelligent root-irrigation treatment in the comparative example 2 are carried out, and the specific operations are as follows:

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

Evaluation indexes are as follows: plant height variation, root length, leaf area variation, chlorophyll content, strong seedling index, growth function, plant dry weight and stem thickness variation. Wherein the content of the first and second substances,

growth function ═ whole plant dry mass/time of seedling.

The growth index change of the pepper after 2 months is shown in figures 5-8, and the control effect of the pepper on pepper virus diseases and epidemic diseases is shown in table 2.

TABLE 2 prevention of Pepper Virus and disease after root irrigation

As can be seen from fig. 1 to 8 and tables 1 to 2, after the chlorella biofertilizers prepared in examples 1, 2 and 3 were sprayed or root-irrigated on pepper plants, plant height, root length, leaf area, chlorophyll, seedling growth index, growth function, plant dry weight and stem thickness were all improved, and a good control effect was also achieved against pepper virus diseases and epidemic diseases, indicating that the chlorella biofertilizers significantly promoted the growth of pepper plants and improved the disease resistance of pepper against diseases. Before application, comparative example 1 and comparative example 2 show that only the chlorella extract and the intelligent agent are applied to promote the growth of plants and improve the disease resistance, but the effect is obviously lower than the effect of the chlorella extract and the intelligent agent after the chlorella extract and the intelligent agent are mixed, which shows that the chlorella extract and the intelligent agent provided by the invention can have synergistic effect, and the effect is obviously higher than the effect of using a single component to promote the growth of plants and improve the disease resistance.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred 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 capable of 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.

2. The chlorella biofertilizer for promoting growth and improving disease resistance as claimed in claim 1, which comprises 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 chlorella extract has a crude protein content of 45% or more and a chlorophyll content of 6.5mg/g or more;

the chlorella is Chlorella pyrenoidosa.

4. The chlorella biofertilizer for promoting growth and improving disease resistance as claimed in any one of claims 1 or 2, wherein the chlorella extract is prepared by the following method:

(1) preparing an algae liquid:

(2) preparing an original algae solution:

(3) preparing a chlorella extract:

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

6. The chlorella biofertilizer for promoting growth and improving disease resistance as claimed in claim 4, wherein the specific operation of step (2) is: adding 0.05-0.1% of cellulase into the algae liquid, and carrying out enzymolysis for 1h at the temperature of 52-60 ℃ and the pH value of 6.0-6.4; adding 0.1-0.15% of pectinase, and carrying out enzymolysis for 1-2 h at the temperature of 48-56 ℃ and the pH value of 3.5-4.5; then adding 0.1% of 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; and finally, adding 0.01% fucosidase, and carrying out enzymolysis for 0.5h at the temperature of 48-55 ℃ and the pH value of 5.8-6.7 to obtain a raw algae solution.

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

8. The use of claim 7, wherein the use is carried out by diluting with water and spraying or rooting.

9. The use of claim 8, wherein the dilution is 500-1000 times for spraying and 300-800 times for irrigating roots.

10. The use according to claim 9, wherein the spraying or rooting is performed every 7 to 10 days for a period of 2 to 3 months.