CN113812317B

CN113812317B - Method for improving tolerance of wheat to copper stress by using microbial agent treatment

Info

Publication number: CN113812317B
Application number: CN202111043168.7A
Authority: CN
Inventors: 陈启和; 刘思宇; 芦红云
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2022-06-10
Anticipated expiration: 2041-09-07
Also published as: CN113812317A

Abstract

The invention discloses a method for improving copper stress tolerance of wheat by using microbial agent treatment, and belongs to the technical field of environmental bioremediation. The microbial agent is halophilic rosette bacteria (Kocuria rosea) ZJUQH with the preservation number of CCTCC NO: M2016754, and can improve the tolerance of wheat to copper stress, slow down the toxic action of copper ion pollution in soil or water on wheat germination and seedlings, and improve the root system dry biomass and grain yield of wheat under copper stress by co-culturing with wheat. The method for reducing the toxicity of the copper ion pollution to the wheat by utilizing the microbial agent treatment is simple to operate, low in cost and environment-friendly, and provides an effective measure for relieving the toxicity of the soil to the wheat copper.

Description

Method for improving tolerance of wheat to copper stress by using microbial agent treatment

Technical Field

The invention relates to the technical field of environmental bioremediation, in particular to a method for improving tolerance of wheat to copper stress by utilizing microbial agent treatment.

Background

Copper is an essential trace element of plants, and is used as a cofactor of a plurality of enzymes and is involved in various metabolic physiological processes in the growth and development processes of plants, including photosynthesis, reactive oxygen metabolism, hormone signal transduction and the like. However, over the last decades, with copper mining, sewage irrigation and the widespread use of copper-containing fungicides, more and more copper enters the soil resulting in severe copper contamination. One national survey shows that copper is one of the major pollutants in agricultural land, and 2.1% of the soil in china is contaminated with copper. Excessive copper can have many undesirable effects on plants, including reduced seed germination, poor root and shoot development, lack of mineral nutrients, oxidative stress, and DNA damage, which can lead to reduced crop yields and hamper sustainable agricultural production. Thus, copper contamination has posed a significant threat to crop growth and food safety, and is a widely-focused environmental and agricultural problem.

Magnesium is a free divalent cation with the highest content in plant cells, is an important component of chlorophyll, is an activator of various enzymes in plants, participates in various physiological processes of the plants such as photosynthesis, and has important significance for the growth and development processes of the plants. Plants growing in serpentine soil formed by weathering of ultramafic rock are easily poisoned by excessive magnesium, the soil magnesium pollution condition which is widely generated is not found in the global scope, and the magnesium deficiency is a more widely existed and concerned problem compared with the soil magnesium excess. Research shows that magnesium ions can reduce the toxicity of copper ions to the root of wheat.

Wheat is a staple crop widely grown throughout the world. It is reported that wheat yield is greatly reduced due to excessive copper content in contaminated soil. In order to alleviate or even avoid copper toxicity in plants including wheat, studies have found that some microorganisms, which are called plant growth promoting bacteria, have been used as biologicals to enhance metal tolerance of plants including wheat, which are widely used for soil remediation, can protect plants from biotic and abiotic stresses (including heavy metal contamination) and promote plant growth, and that several studies have shown that rhizosphere microorganisms play an important role in regulating crop growth and stress resistance. It has been shown that Bacillus sp reduces the superoxide radical and H by increasing the activity of SOD, CAT and POD₂O₂To reduce copper toxicity in wheat seeds. Therefore, the application of the special bacteria can provide an effective measure for relieving the copper toxicity of the plants in the soil or the water body.

Disclosure of Invention

The invention aims to provide a method for reducing the toxic action of copper ion pollution in soil or water environment on wheat by using microbial agent treatment.

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

the invention provides application of Kocuria rosea ZJUQH with a preservation number of CCTCC NO: M2016754 in regulating and controlling the tolerance of wheat to copper stress.

Further, the application includes: the salt-philic Cucumaria rosea (Kocuria rosea) ZJUQH is co-cultured with wheat, so that the tolerance capability of the wheat to copper ion stress is improved.

The research of the invention shows that the co-culture of the halophilic Cokuromobacter roseus (Kocuria rosea) ZJUQH and wheat can obviously reduce the copper toxic effect of copper stress on wheat plants, particularly in the germination and seedling stages.

Further, the co-culturing comprises: firstly, fermenting and culturing halophilic rose kucuria (Kocuria rosea) ZJUQH to prepare zymocyte liquid, then adding the zymocyte liquid into wheat culture soil or water polluted by copper ions, and co-culturing with wheat.

Further, the method for fermentation culture of Kocuria rosea (Kocuria rosea) ZJUQH comprises the following steps: firstly, the cells of Kocuria rosea are inoculated into a seed culture medium and are cultured in a rotating way until OD is reached₆₀₀0.8-1.0; then the seed culture of Kocuria rosea is transferred to the fermentation medium and rotary cultured to OD₆₀₀＝2.5-3.0；

The seed culture medium comprises the following components: 5g/L of casein hydrolysate, 10g/L of sodium citrate, 3g/L of yeast extract, 2g/L of KCl, 5g/L of peptone, 10g/L of magnesium sulfate and pH 7.0;

the fermentation medium comprises the following components: 3-7g/L of casein hydrolysate, 5-15g/L of sodium citrate, 1-5g/L of yeast extract, 0-8g/L of potassium chloride, 3-8g/L of peptone, 3-80g/L of magnesium sulfate and 6.8-7.2 of pH.

The rotary culture conditions of the seed culture medium and the fermentation culture medium are as follows: culturing at 20-25 deg.C and 100-150rpm for 36-60 h.

The addition amount of the zymophyte liquid is 30-40L/m³。

Further, pre-germinated wheat seeds were inoculated in a culture medium containing Kocuria rosea (Kocuria rosea) ZJUQH for co-culture.

The wheat seed pre-germination method comprises the following steps: the wheat seeds preserved in a dry mode are disinfected by sodium hypochlorite and inoculated into gauze soaked with MS culture medium (placed in a sterile culture dish) for pre-germination.

The concentration of the sodium hypochlorite is 3 percent; the disinfection time is 5 min; the pre-germination time after the wheat seeds are inoculated is 48 hours.

The co-culture conditions are 20-25 ℃, 48-52% relative humidity, 5800-6200lux illumination intensity and 12h/12h light and shade cycle.

Furthermore, the invention researches the action mechanism of halophilic Culcita rosea (Kocuria rosea) ZJUQH for improving the tolerance of wheat to copper ion stress, and the result shows that, on one hand, halophilic Culcita rosea (Kocuria rosea) ZJUQH resists the toxicity of copper stress by enhancing the absorption of wheat to magnesium ion; on the other hand, Kocuria rosea (Kocuria rosea) ZJUQH promotes wheat oxidation resistance to suppress the toxic action of copper stress, specifically, to lower wheat root H₂O₂Content, increase Glutathione (GSH) content in wheat root, increase Peroxidase (POD) activity in wheat root.

The invention has the following beneficial effects:

the invention discloses the application of halophilic Cokucuria rosea (Kocuria rosea) ZJUQH in improving the tolerance of wheat to copper stress for the first time, and particularly can slow down the toxic action of copper ion pollution in soil or water on wheat germination and seedlings thereof and improve the root system dry biomass and grain yield of wheat under copper stress by co-culturing a microbial agent and the wheat. The invention provides a method for improving the tolerance of wheat to copper stress by using microbial agent treatment, which is simple to operate, low in cost and environment-friendly, and provides an effective measure for relieving the copper toxicity of soil to wheat.

Drawings

FIG. 1 shows different Cu²⁺Growth of wheat seedlings at concentration.

FIG. 2 shows Cu²⁺Length of wheat seedling bud and root at concentration.

FIG. 3 shows Cu²⁺Cell survival of Kocuria rosea at concentration.

FIG. 4 shows Cu at 100. mu. mol/L²⁺Effect of Kocuria rosea on wheat seedling growth, wherein KR-represents co-cultured group without Kocuria rosea added, KR + represents co-cultured group with Kocuria rosea added, the same as below.

FIG. 5 shows Cu at 100. mu. mol/L²⁺Effect of Kocuria rosea on wheat shoot bud and root length.

FIG. 6 is 100. mu. mol/L Cu²⁺Effect of Kocuria rosea on wheat shoot bud and root dry weight.

FIG. 7 shows 100. mu. mol/L Cu²⁺Effect of Kocuria rosea on copper content in wheat seedlings shoots and roots.

FIG. 8 shows 100. mu. mol/L Cu²⁺Effect of Kocuria rosea on magnesium content in wheat seedlings shoots and roots.

FIG. 9 shows 100. mu. mol/L Cu²⁺Lower Kocuria rosea on wheat seedling root H₂O₂Influence of the content.

FIG. 10 shows 100. mu. mol/L Cu²⁺Effect of Kocuria rosea on wheat seedling root GSH content.

FIG. 11 shows 100. mu. mol/L Cu²⁺Effect of Kocuria rosea on wheat seedling root POD activity.

Detailed Description

The present invention is further illustrated by the following specific examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and nature of the invention.

The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Kocuria rosea salina ZJUQH is separated from Qinghai tea Ka salt lake and preserved in the China center for type culture Collection (CCTCC NO: M2016754) at 2016, 12, 15 and 12 months in Wuhan university in Wuhan, China. The chinese patent application No. 201710077208.7 discloses the strain.

Strain culture medium

The initial slant culture medium comprises the following components: 5g/L of casein hydrolysate, 10g/L of sodium citrate, 3g/L of yeast extract, 2g/L of potassium chloride, 5g/L of peptone, 10g/L of magnesium sulfate, 20g/L of agar and pH 7.0.

The seed culture medium comprises the following components: 5g/L of casein hydrolysate, 10g/L of sodium citrate, 3g/L of yeast extract, 2g/L of KCl, 5g/L of peptone, 10g/L of magnesium sulfate and pH 7.0.

The liquid fermentation culture medium comprises the following components: 3-7g/L of casein hydrolysate, 5-15g/L of sodium citrate, 1-5g/L of yeast extract, 0-8g/L of potassium chloride, 3-8g/L of peptone, 3-80g/L of magnesium sulfate and 6.8-7.2 of pH.

The wheat seeds come from the functional agricultural biotechnology laboratory of the institute of agriculture and biotechnology of Zhejiang university. The wheat is cultured in liquid, MS culture medium without agar is used, and the wheat is put into a 480mL tissue culture bottle for inoculation after being autoclaved.

The MS culture medium comprises: 170mg/L potassium dihydrogen phosphate, 370mg/L magnesium sulfate, 440mg/L calcium chloride dihydrate, 1900mg/L potassium nitrate, 1650mg/L ammonium nitrate, 0.83mg/L potassium iodide, 6.2mg/L boric acid, 22.3mg/L manganese sulfate, 8.6mg/L zinc sulfate, 0.25mg/L sodium molybdate, 0.025mg/L copper sulfate, 0.025mg/L cobalt chloride, 37.3mg/L disodium ethylenediaminetetraacetate, 27.8mg/L ferrous sulfate, 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine nicotinate, 0.5mg/L nicotinic acid and 0.5mg/L pyridoxine hydrochloride.

Example 1 tolerance of wheat seedlings to copper ion stress

1. The wheat seed culture conditions are as follows: the seeds preserved in a dry mode are disinfected by 3% sodium hypochlorite for 5min, inoculated into gauze soaked with MS culture medium (placed in a sterile culture dish) for 48h for pre-germination, and then inoculated into a 480mL tissue culture bottle filled with 150mL of MS culture medium after sterilization.

2. Configuring MS culture media containing copper ions with different concentrations in a 480mL tissue culture bottle: 0. 10, 100, 200 and 400 mu mol/L, inoculating the pre-germinated seeds into tissue culture bottles, inoculating ten seeds into each bottle on a punched filter paper sheet, and making 3 seeds in parallel. Culturing in incubator under conditions of 22 + -0.5 deg.C, 50 + -3% relative humidity, 6000lux illumination intensity, 12h/12h light and dark cycle, and changing nutrient solution (MS culture medium) every 3 days for one week. The lengths of the roots and shoots of wheat were measured after one week of culture, and the results are shown in FIGS. 1 and 2.

As can be seen from FIGS. 1 and 2, as the concentration of copper ions increasesAdditionally, the growth of wheat seedlings was significantly inhibited and the exposure to Cu of 100. mu. mol/L and above was compared to the control (no additional copper ions added)²⁺The length of the root and the bud of the wheat seedling is obviously reduced, and the wheat seed can not normally germinate under the concentration of 400 mu mol/L. Since a certain number of seeds did not germinate even under the condition of 200. mu. mol/L, for data reliability, Cu of 100. mu. mol/L²⁺The concentration already had a significant inhibitory effect, so 100. mu. mol/L was chosen as the copper ion stress concentration in the subsequent examples.

Example 2 tolerance of Kocuria rosea to copper ion stress

1. And (3) strain culture conditions: cells from the slant culture of Kocuria rosea were inoculated into 100mL of fresh seed medium in a 250mL conical flask and cultured on a rotary shaker at 130rpm at 25 ℃ for 36-60 h. Then 5% (v/v) of the seed culture was transferred to 100mL of fermentation medium in a 250mL Erlenmeyer flask and incubated at 25 ℃ for 36-60h on a rotary shaker at 130 rpm.

2. 5% (v/v) of the seed culture was inoculated to 0, 100, 400, 1000, 2000, 4000. mu. mol/L CuSO₄The fermentation medium (2) is cultured for 72 hours at 25 ℃, and the concentration of Cu is measured by a CCK-8 kit²⁺Cell survival at 0. mu. mol/L CuSO₄The addition amount group is a control.

As can be seen from FIG. 3, when Cu²⁺Cell survival of Kocuria rosea with Cu at concentrations below 400. mu. mol/L²⁺The concentration is increased and slightly increased; kocuria rosea at 100 and 400. mu. mol/L Cu²⁺The better survival state can be maintained under the concentration. When Cu²⁺The concentration is continuously increased, the survival of Kocuria rosea is obviously inhibited, when Cu²⁺The cell viability of Kocuria rosea decreased to 35.7% of the control group at a concentration of 4000. mu. mol/L.

Example 3 Effect of Kocuria rosea on wheat seedling growth under copper ion stress

1. Water co-culture conditions: selecting proper copper ion stress concentration, adding 5mL of bacterial liquid cultured for 48h into an MS culture medium of a tissue culture bottle of an inoculation group, inoculating an equivalent amount of sterilized fermentation culture medium without strains into a control group, inoculating pre-germinated seeds into the tissue culture bottle, inoculating ten seeds into each bottle on a perforated filter paper sheet, and enabling each group to be parallel by 3. Culturing in incubator under conditions of 22 + -0.5 deg.C, 50 + -3% relative humidity, 6000lux illumination intensity, 12h/12h light and dark cycle, and changing nutrient solution (MS culture medium) every 3 days for one week.

2. Selecting 100mM as copper ion stress concentration, inoculating the bacterial liquid and wheat seeds in the experimental group according to the method, and inoculating the blank fermentation medium with the same amount in the control group. After one week of culture, the length and dry weight of wheat roots and shoots were measured, and the results are shown in FIGS. 4, 5 and 6.

As can be seen from the figure, the co-cultured group added with Kocuria rosea exposed to 100. mu. mol/L of copper stress has a significant improvement in growth state compared with the control group, the lengths of the wheat seedling bud and root are increased by 95.73% and 48.26%, respectively, and the dry weight is increased by 59.69% and 78.41%, respectively, which indicates that Kocuria rosea has a better effect of alleviating copper toxicity to wheat seedlings.

Example 4 Effect of Kocuria rosea on copper and magnesium ion content in wheat seedlings shoots and roots under copper ion stress

The culture was carried out according to the experimental procedure of example 3, 0.2g of each sample of roots and shoots were taken after drying overnight at 80 ℃ with 55mL of HNO₃-H₂O₂The mixture (v/v 8: 3) was digested with a microwave digestor at 180 ℃ for 1h, and then the ion levels of copper and magnesium were measured by a plasma mass spectrometer using the digestate. Calculation of Cu in tissue from standards²⁺And Mg²⁺And expressed in μ g/g · dw, the results are shown in FIGS. 7 and 8.

As can be seen, the co-cultured group to which Kocuria rosea was added was exposed to 100. mu. mol/L copper stress, compared to the control group, shoot and root Cu²⁺The content does not change significantly, and Mg²⁺The content of (A) is obviously increased, which indicates that Kocuria rosea can strengthen wheat seedlings to Mg²⁺Against the toxic effects of copper stress.

Example 5 Kocuria rosea on wheat seedling root H under copper ion stress₂O₂Influence of the amount

The culture was carried out according to the experimental procedure of example 3, using hydrogen peroxide (H)₂O₂) A content detection kit. According to the specification, 0.1g of wheat root tissue is weighed, 1mL of acetone is added for ice bath homogenization, centrifugation is carried out for 10min at 8000rpm and 4 ℃, and all supernatant is collected and placed on ice to be tested. Using a spectrophotometer, the detection wavelength was 415nm and the standard solution was 1. mu. mol/mL H₂O₂。

H₂O₂Content (. mu. mol/g mass) ═ Δ A measurement ÷ Δ A standard ÷ W

Delta a assay-a tube blank, delta a standard-a tube blank

As can be seen from FIG. 9, the concentration of Cu is 100. mu. mol/L²⁺Stress Kocuria rose H in wheat seed roots₂O₂The content is reduced by 30.14 percent, which shows that Kocuria rosea can reduce H in the roots of wheat seedlings₂O₂The content of the compound is beneficial to the wheat to eliminate ROS so as to resist oxidative damage caused by copper stress.

Example 6 Effect of Kocuria rosea on the GSH content of wheat seedlings root under copper ion stress

The culture was performed according to the experimental procedure of example 3, using a Glutathione (GSH) assay kit. According to the instruction, 0.1g of wheat root tissue is weighed, 0.9mL of physiological saline is added for ice bath homogenization, centrifugation is carried out for 10min at 8000rpm and 4 ℃, and all supernatant is collected and placed on ice to be tested. The detection wavelength was 420nm using a spectrophotometer.

As can be seen from FIG. 10, the concentration of Cu is 100. mu. mol/L²⁺Kocuria rosea increased the GSH content in the wheat seed roots by 45.96% under stress, suggesting that inoculation of Kocuria rosea might inhibit Cu by stimulating GSH production in wheat seedlings²⁺Toxic effects of (1).

Example 7 Effect of Kocuria rosea on POD Activity at wheat seedling roots under copper ion stress

The culture was performed according to the experimental procedure of example 3, using a Peroxidase (POD) test kit. According to the instruction, 0.1g of wheat root tissue is weighed, 0.9mL of physiological saline is added for ice bath homogenization, centrifugation is carried out for 10min at 8000rpm and 4 ℃, all supernatant is collected, and the mixture is placed on ice to be tested. The detection wavelength was 420nm using a spectrophotometer.

As can be seen from FIG. 11, the concentration of Cu is 100. mu. mol/L²⁺The Kocuria rosea enhances the POD activity in the wheat seed root by 83.86% under stress, which shows that the Kocuria rosea treatment can enhance the POD activity in the wheat seedling root and is beneficial to the wheat to eliminate ROS so as to resist the oxidative damage caused by copper stress.

Claims

1. An application of Kocuria rosea ZJUQH in regulating and controlling the copper stress tolerance of wheat is disclosed, wherein the preservation number of the Kocuria rosea ZJUQH is CCTCC NO: M2016754.

2. The application of claim 1, wherein the application comprises: the halophilic Cuckoo bacteria (Kocuria rosea) ZJUQH and wheat are co-cultured, so that the tolerance capacity of the wheat to copper ion stress is improved.

3. The use of claim 2, wherein said co-culturing comprises: firstly, fermenting and culturing halophilic rose Cuke bacteria (Kocuria rosea) ZJUQH to obtain zymogen liquid, then adding the zymogen liquid into wheat culture soil or water polluted by copper ions, and co-culturing with wheat.

4. The use as claimed in claim 3, wherein the method of fermentation culture of Kocuria rosea (Kocuria rosea) ZJUQH comprises: firstly, the cells of Kocuria rosea are inoculated into a seed culture medium and are cultured in a rotating way until OD is reached₆₀₀0.8-1.0; then the seed culture of Kocuria rosea is transferred to the fermentation medium and rotary cultured to OD₆₀₀＝2.5-3.0；

the fermentation medium comprises the following components: 3-7g/L of casein hydrolysate, 5-15g/L of sodium citrate, 1-5g/L of yeast extract, 0-8g/L of potassium chloride, 3-8g/L of peptone, 3-80g/L of magnesium sulfate and 6.8-7.2 of pH;

the rotary culture conditions of the strain in the seed culture medium and the fermentation culture medium are as follows: culturing at 20-25 deg.C and 100-150rpm for 36-60 h.

5. The use according to claim 3, wherein the amount of the zymogen liquid added is 30-40L/m³。

6. Use according to claim 3, characterized in that the pre-germinated wheat seeds are co-cultivated by inoculating them in a culture medium containing Kocuria rosea (Kocuria rosea) ZJUQH.

7. The use of claim 6, wherein the co-cultivation conditions are 20-25 ℃, 48-52% relative humidity, 5800-.

8. Use according to claim 1, wherein Kocuria rosea salina (Kocuria rosea) ZJUQH is against the toxic effects of copper stress by enhancing the uptake of magnesium ions by wheat.

9. Use according to claim 1, characterized in that Cucumaria roseophila (Kocuria rosea) ZJUQH promotes the antioxidant action of wheat to inhibit the toxic action of copper stress.

10. The use according to claim 9, wherein the antioxidant effect is a reduction of wheat root H₂O₂Content, increase glutathione content in wheat root, and increase peroxidase activity in wheat root.