CN111011382A

CN111011382A - β application of glucan and succinoglycan in preventing and controlling soil-borne fungal diseases

Info

Publication number: CN111011382A
Application number: CN201911241486.7A
Authority: CN
Inventors: 李晶; 朱庭锐; 张建法
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2020-02-17
Filing date: 2020-02-17
Publication date: 2020-04-17
Anticipated expiration: 2040-02-17
Also published as: CN111011382B

Abstract

The invention can induce the increase of the quantity of streptomyces in soil by adding β -glucan and succinyl glycan mixture into the soil, and simultaneously induce the increase of the activity of hydrolytic enzymes such as β -glucanase, chitinase and cellulase in the soil, and can effectively prevent and control various soil-borne pathogenic fungal diseases through the combined action of the streptomyces and various hydrolytic enzymes.

Description

β application of glucan and succinoglycan in preventing and controlling soil-borne fungal diseases

Technical Field

The invention belongs to the technical field of biological control, and relates to application of β -glucan and succinyl glycan in prevention and control of soil-borne fungal diseases.

Background

The soil-borne diseases refer to diseases that pathogens come from soil and attack plants from roots or stems of the plants, and have the characteristics of wide distribution, serious harm, wide host range, multiple propagation ways, long survival time and the like, and have great harm to the plants. The prevention and control of soil-borne diseases by a biological method is a technology with less environmental risk. Wherein, the biological prevention and control by adding an environment-friendly inducer from an external source mainly depends on the effects of competitive growth, antagonism, heavy parasitism and the like of soil microbial populations, has little damage to the ecological environment and has higher feasibility. Reasonable inducer is selected, the microorganism with antagonistic action on pathogenic fungi in the soil is enriched through the induction action, and simultaneously the activity of hydrolase system capable of degrading the cell wall of the pathogenic fungi is induced to be increased, so that the method has very important significance for reducing soil-borne diseases.

The actinomycetes are the microorganisms which are discovered to have biological control effect at the earliest time, the actinomycetes are various in species and have different metabolic functions, and Streptomyces (Streptomyces) has obvious effect on controlling plant diseases. For example, agricultural streptomycin can be used for preventing and controlling various plant pathogenic bacteria such as crucifer soft rot, cucumber angular leaf spot, phytophthora phaseoli, tomato canker and the like. The actinomycete living preparation Mycostop is mainly used for preventing and treating common soil-borne pathogens such as pythium, fusarium, phytophthora and rhizoctonia. However, the above preparations are produced on a large scale and then added to soil, and have limited adaptability to the soil environment.

β -glucan is a kind of natural synthetic non-starch polysaccharide, its main characteristic is that the polysaccharide backbone contains β -1, 3-glucoside with high proportion, β -glucan is used as important component of cell wall, and is extensively existed in microbial, plant and animal kingdom, at the same time some microbes also have the ability of secreting extracellular β -glucan.

Disclosure of Invention

The inventor firstly discovers that β -glucan and succinyl polysaccharide with different action mechanisms are combined together, and streptomyces in soil can be further enriched by controlling the proportional relation of the glucan and the succinyl polysaccharide, so that the activity of soil hydrolase is improved, and soil-borne pathogenic fungi are prevented and controlled.

The technical scheme for realizing the purpose of the invention is as follows:

β -application of glucan and succinoglycan in preventing and controlling soil-borne fungal diseases, which comprises the following steps:

adding a β -glucan and succinyl polysaccharide mixture into soil according to the mass ratio of β -glucan and succinyl polysaccharide being 1: 0.1-1: 2.0.

In the invention, the β -glucan is β -glucan which is conventionally used in the field, can be yeast glucan, laminarin, lentinan or schizophyllan, and is preferably the yeast glucan or the laminarin.

In the invention, the structural general formula of the succinoglycan is as follows:

wherein n is 10-1000; succ is succinyl; pyr is pyruvoyl.

In the invention, the mass ratio of the β -glucan to the succinoglycan is preferably 1: 0.5-1: 2.0.

In the invention, the addition amount of the β -glucan and succinoglycan mixture is 0.01-0.5% of the mass of the soil, and preferably 0.05-0.25% of the mass of the soil.

In the invention, the β -glucan and succinoglycan mixture can be added directly into solid powder or prepared into mixture solution for spraying.

Compared with the prior biological control technology, the invention has the following advantages:

(1) β -glucan and succinyl glycan mixture has better selective enrichment effect on streptomyces, can induce β -glucanase, chitinase, cellulase and other hydrolase activities in soil to be obviously increased, and the two are synergistically enhanced, and the effect of the mixture on preventing and controlling soil-borne fungal diseases is obviously better than that of the mixture singly added with β -glucan or succinyl glycan.

(2) The effect of the mixture on preventing and controlling soil-borne fungal diseases can be further promoted by regulating the proportional relation of β -glucan and succinyl glycan.

(3) The method has the advantages of low economic cost, high feasibility and environmental friendliness.

Drawings

FIG. 1 is a graph showing the relative abundance of streptomyces in soil in original forestry, Enshi, over time, after different concentrations of mixed polysaccharides were added in example 1.

FIG. 2 is a graph showing the trend of the relative abundance of streptomyces in soil in the rotavatory field of Nanjing over time, after different concentrations of mixed polysaccharides were added in example 1.

FIG. 3 is a graph showing the relative abundance of streptomyces in soil in potato continuous cropping areas in Tengzhou, with time, after different concentrations of mixed polysaccharides were added in example 1.

FIG. 4 is a graph showing the relative abundance of streptomyces in soil in the continuous cropping field of Nicotiana kunming, as a function of time, after different concentrations of mixed polysaccharides were added in example 1.

FIG. 5 is a graph of the time course of β -glucanase activity in soil after the addition of different concentrations of mixed polysaccharides in example 2.

FIG. 6 is a graph of the change in soil cellulase activity over time after the addition of different concentrations of mixed polysaccharides in example 2.

FIG. 7 is a graph of the time course of chitinase activity in soil after the addition of various concentrations of mixed polysaccharides as in example 2.

FIG. 8 is a statistical graph of the number of culturable fungi in soil after the addition of different concentrations of mixed polysaccharides as described in example 3.

FIG. 9 is a graph showing the relative content of Phytophthora infestans in soil after different concentrations of mixed polysaccharides were added in example 3.

FIG. 10 is a graph showing the inhibitory effect of Streptomyces melaleukii on Fusarium oxysporum in example 4.

Detailed Description

The present invention will be described in more detail with reference to the following examples and the accompanying drawings.

The succinoglycan employed in the examples below has the general structural formula:

wherein n is 10-1000; succ is succinyl; pyr is pyruvoyl. Reference to the acquisition of succinoglycan [ Marczak

Mazur Andrzej,KoperPiotr,

Kamil,Skorupska Anna.Synthesis of Rhizobial Exopolysaccharides andTheir Importance for Symbiosis with Legume Plants.[J].Genes,2017,8(12).]。

Example 1

Determination result of relative abundance of streptomyces in typical soil

The method comprises the steps of selecting original forest land of Enshi of Hubei province, alternative farmland of Nanjing city of Jiangsu province, potato continuous cropping land of Teng city of Shandong province and tobacco continuous cropping land of Kunming city of Yunnan province as test areas, adding β -glucan and succinoglycan (laminarin: succinoglycan ═ 1:1w/w) mixture powder to the surface layer of the selected soil, using the amounts of 0%, 0.05% and 0.25% respectively, then sampling once every 15d until 60d is finished, extracting total DNA of the soil by using an Ezup column type soil DNA extraction kit (living creatures (Shanghai)) and sequencing soil macro genome, analyzing the relative abundance of streptomyces sequences in a soil microbial area system according to the sequencing result, analyzing the relative abundance of streptomyces sequences in the soil microbial area system, and adding β -succinoglycan mixture to the soil micro-area system, and obviously increasing the relative abundance of streptomyces in the soil.

Example 2

β -glucanase, cellulase and chitinase activity change in soil

Selecting alternate cultivated land of Nanjing city, Jiangsu province as a test soil sample, adding β -glucan and succinoglycan (yeast glucan: succinoglycan is 1:10w/w) mixture powder to the surface layer of the selected soil, wherein the use amount is 0%, 0.05% and 0.25% respectively, then sampling once every 15d until 60d is finished, taking laminarin, sodium carboxymethylcellulose and chitin colloid as substrates respectively, measuring the hydrolase activity of the soil leachate at pH5.5, wherein the reaction temperature is 35 ℃, the reaction time is 24h, and figures 5-7 show the change trend of the 3 hydrolase activities in the soil along with time.

Example 3

Soil fungus content determination results

Selecting potato continuous cropping land of Teng city, Shandong province as a soil sample, preparing a mixture of β -glucan and succinoglycan (lentinan: succinoglycan: 1:0.2w/w) into a solution with the concentration of 25g/L, spraying the solution onto the surface of the selected soil with the use amount of 0%, 0.05% and 0.25% respectively, sampling every 15d until 60d is finished, measuring the abundance change process of the culturable fungi in the soil by adopting a dilution and coating plate counting method, namely diluting 1g of soil sample into 10mL of sterile phosphate buffer solution, violently shaking for 30min to uniformly disperse the soil into the buffer solution, then carrying out gradient dilution, finally selecting a proper concentration to coat the PDA plate, culturing for 48h at 30 ℃, counting as shown in figure 8, wherein the fungus content in the soil gradually decreases along with the increase of the adding time and the polysaccharide concentration, and the number of the culturable fungi in the soil with the mixed polysaccharide content of 0.25% decreases to 1 × 10 at 60 days⁴CFU/g。

In addition, the analysis on the relative content of the phytophthora infestans in the soil shows that the relative content of the phytophthora infestans is obviously reduced after the mixed polysaccharide is added (figure 9), and the relative amount of the phytophthora infestans in the soil with the addition amount of the mixed polysaccharide of 0.25 percent is reduced from 0.97 to 0.63 compared with that in a control group at 60 days.

Example 4

Inhibition of streptomycete on fusarium oxysporum

The method comprises the steps of selecting a tobacco continuous cropping land of Kunming city in Yunnan province as a test soil sample, preparing a 25g/L solution from a mixture of β -glucan and succinoglycan (schizomycete polysaccharide: succinoglycan ═ 1:10w/w), spraying the solution onto the surface layer of the selected soil, sampling when the use amount is 0.5 percent to 60 days, separating Streptomyces in the soil sample by adopting a Czochralski culture medium to obtain a purified strain, comparing the strain with a 16S rDNA sequence to obtain a Streptomyces pactum (Streptomyces mellea), and then carrying out an antibacterial test on fusarium oxysporum by using the strain, wherein an antibacterial ring on the fusarium oxysporum always exists in a culture dish with a Streptomyces pactum patch after 4 days of co-culture, and the result shows that the Streptomyces enriched by adopting a β -glucan and succinoglycan mixture has an inhibitory effect on the growth of the fusarium oxysporum.

Example 5

The effect of the addition of β -dextran (laminarin) and succinoglycan mixture was compared to the effect of the addition of β -dextran (laminarin) or succinoglycan alone

Soil samples of the alternate tillage land of Nanjing, Jiangsu province, and Nanjing, respectively, described in example 1 were selected and processed according to the method described in example 1. the relative abundance of streptomyces, β -glucanase activity, and the relative abundance of Fusarium oxysporum in the soil were measured at 30d and 60d, respectively, and the results are shown in tables 1 and 2.

TABLE 1 comparison of measurement results when the mixed polysaccharide is added at a concentration of 0.05%

TABLE 2 comparison of measurement results when the mixed polysaccharide is added at a concentration of 0.25%

The comparison shows that when β -glucan and succinoglycan are used together, the key data index is better than the effect when β -glucan or succinoglycan is used alone, and meanwhile, when the ratio of β -glucan to succinoglycan is in the range of 1: 0.5-1: 2, the effect is optimal.

Claims

Use of β -glucans and succinoglycans for the control of soil-borne fungal diseases.
2. The application of claim 1, comprising the following steps:

adding a β -glucan and succinyl polysaccharide mixture into soil according to the mass ratio of β -glucan and succinyl polysaccharide being 1: 0.1-1: 2.0.
3. The use according to claim 1 or 2, wherein said β -glucan is selected from the group consisting of yeast glucan, laminarin, lentinan and schizophyllan.
4. The use according to claim 1 or 2, wherein the succinoglycan has the general structural formula:

wherein n is 10-1000; succ is succinyl; pyr is pyruvoyl.
5. The use according to claim 1 or 2, wherein the mass ratio of β -glucan to succinoglycan is 1:0.5 to 1: 2.0.
6. The use according to claim 1 or 2, wherein the β -glucan and succinoglycan mixture is added in an amount of 0.01-0.5% by mass of the soil.
7. The use according to claim 1 or 2, wherein the β -glucan and succinoglycan mixture is added in an amount of 0.05% to 0.25% by mass of the soil.
8. The use according to claim 1 or 2, wherein the β -glucan and succinoglycan mixture is added directly to the solid powder or prepared as a mixture solution for spraying.