CN114868544A - Method for relieving double stress poison of waterlogging and salt of plant seedlings - Google Patents

Abstract

The application relates to the technical field of biology, in particular to a method for relieving double stress poison of waterlogging and salt of plant seedlings; the method comprises the following steps: obtaining salt stress experimental crops; obtaining waterlogging stress experimental crops; carrying out double stress treatment on the experimental crops to obtain double stress experimental crops; respectively spraying exogenous brassinolide on the leaf surfaces of each stress experimental crop to obtain a treated experimental crop group; carrying out leaf detection, and respectively carrying out continuous cultivation to obtain a plurality of groups of detection data; judging whether the exogenous brassinolide has influence on the experimental crops subjected to the double stress treatment according to a plurality of groups of detection data; if so, spraying exogenous brassinolide to the crop plants to be treated to obtain the crop plants with the double stresses of waterlogging and salt relieved; the leaf detection comprises leaf length and width value detection, leaf pigment content detection, soluble sugar content detection, relative conductivity detection and photosynthetic characteristic detection; the dual stress crop can be relieved.

Description

Method for relieving double stress poison of waterlogging and salt of plant seedlings

Technical Field

The application relates to the technical field of biology, in particular to a method for relieving double stress poison of waterlogging and salt of plant seedlings.

Background

According to statistics, about 7% of the land area in the world is affected by salinization, and about 10% of the land area is affected by waterlogging disasters, and with the increasing severity of the soil salinization phenomenon, the area of the salinization land is about 9.91 multiplied by 107hm ² The soil area along the river in the coastal region which is easily affected by waterlogging disasters is the largest and accounts for more than 75 percent of the total disaster area; in agricultural production, unreasonable fertilization and irrigation measures aggravate the process of secondary salinization of soil, cause waterlogging to crops, greatly reduce the yield of the crops due to high salinity and waterlogging, even cause failure, cause the salinization of the soil and the waterlogging disaster to become huge environmental pressure for restricting agricultural development, and cause the global crop yield loss of up to 20 percent and billions of agricultural economic losses each year.

The waterlogging disaster can cause the increase of anaerobic respiration of plants, harmful substances are accumulated in soil, and the normal growth and development of roots are inhibited, so that the overground part is influenced, the problems that the chlorophyll synthesis of leaves is less, the leaves are aged quickly, the accumulation amount of photosynthetic products is reduced, membrane lipid peroxidation is caused, and the like easily occur to crops, and finally the yield of the crops is reduced; salt stress also has similar effects, but in addition, salt stress causes a reduction in the osmotic potential in plants, thereby causing physiological drought and affecting the water-absorbing capacity of plants.

Brassinolide (BR) is an important steroid hormone in plants, can regulate and control gene expression and physiological metabolism of biological activity pathways such as plant seed germination, photosynthesis, reproductive growth, stress response and the like, and Exogenous Brassinolide (EBR), namely 2, 4-epibrassinolide commonly used in agricultural production at present is an artificially synthesized high-activity brassinolide analogue; although the exogenous brassinolide also plays a positive role in improving the resistance and tolerance of plants to adverse environmental stresses (such as low temperature, salt damage, drought, HMs stress and the like), the research focuses on the stress aspect of single factor at present, but an effective relieving method is still lacked aiming at the toxicity of double waterlogging and salt stresses suffered by crops in partial areas, so that how to relieve the toxicity of double waterlogging and salt stresses by using the exogenous brassinolide is a technical problem which needs to be solved at present.

Disclosure of Invention

The application provides a method for relieving double stress poison of waterlogging and salt of plant seedlings, and aims to solve the technical problem that the double stress poison of waterlogging and salt of crops is difficult to relieve in the prior art.

In a first aspect, the present application provides a method for alleviating double stress poisoning of water logging and salt of young plants, the method comprising:

carrying out salt stress treatment on the experimental crops to obtain salt stress experimental crops;

carrying out waterlogging stress treatment on the experimental crops to obtain waterlogging stress experimental crops;

carrying out double stress treatment on the experimental crops by salt stress treatment and waterlogging stress treatment to obtain double stress experimental crops;

respectively spraying exogenous brassinolide on the leaf surfaces of the salt stress experimental crops, the waterlogging stress experimental crops and the double stress experimental crops to obtain treated experimental crop groups;

respectively carrying out leaf detection on the treated experimental crop groups, and respectively carrying out continuous cultivation to obtain a plurality of groups of detection data;

judging whether the exogenous brassinolide has influence on the experimental crops subjected to the double stress treatment according to a plurality of groups of detection data;

if so, spraying exogenous brassinolide to the crop plants to be treated to obtain the crop plants with the double stresses of waterlogging and salt relieved;

the leaf detection comprises leaf length and width value detection, leaf pigment content detection, soluble sugar content detection, relative conductivity detection and photosynthetic characteristic detection.

Optionally, the spraying treatment of the exogenous brassinolide comprises:

continuously spraying exogenous brassinolide of 0.09-0.11 mu mol/L for 3 days.

Optionally, the salt stress treatment comprises:

and adding 45-55 mmol/day of salt solution into the soil of the experimental crop until the concentration of the salt solution in the soil is more than or equal to 100 mmol.

Optionally, the waterlogging stress treatment includes: carrying out waterlogging stress treatment in the tendril pulling period and the pod bearing period of the experimental crops, wherein the tendril pulling period is that 7-8 pieces of compound leaves appear in the experimental crops or 25-30 days after the experimental crops are sowed,

the pod bearing period is that the pod bearing rate of the experimental crop is more than or equal to 50% or the experimental crop is sowed for 40 d-50 d.

Optionally, the time for the salt stress treatment, the water logging stress treatment and the double stress treatment is 1-2 weeks.

Optionally, the leaf pigment content detection includes:

taking the leaves of the treated experimental crop group to obtain a leaf group;

shearing the leaves of the leaf group, and extracting the mixed solution in the dark to obtain an extracting solution;

performing spectrophotometric detection on the extracting solution to obtain a plurality of groups of OD values;

and calculating a plurality of groups of OD values to respectively obtain the chlorophyll a content, the chlorophyll b content, the total chlorophyll content and the carotenoid content of the leaf groups.

Optionally, the calculation formula of the chlorophyll a content is as follows:

chlorophyll a content ═ V/(1000 × W) (12.7 × OD663-2.69 × OD645),

the calculation formula of the chlorophyll b content is as follows:

chlorophyll b content ═ V/(1000 × W) (22.9 × OD645-4.68 × OD663),

the calculation formula of the total content of chlorophyll is as follows:

total chlorophyll content ═ V/(1000 × W) (20.21 × OD645+8.02 × OD663),

the formula for calculating the carotenoid content is:

carotenoid content ═ 4.7 × OD440-0.27 ═ 20.21 × OD645+8.02 × OD663) ] × V/(1000 × W);

wherein V is the volume of the extracting solution, W is the fresh weight of the leaves of the leaf group, OD645 is the absorbance value of the extracting solution on a 645nm spectrophotometer, and OD663 is the absorbance value of the extracting solution on a 663nm spectrophotometer.

Optionally, the mixed solution comprises absolute ethyl alcohol, acetone and water; the volume ratio of the absolute ethyl alcohol to the acetone to the water is 4-5: 1.

Optionally, the photosynthetic characteristic detection comprises leaf net photosynthetic rate and intercellular CO of leaves of the treated experimental crop group ₂ And (4) detecting the concentration, the porosity conductance and the transpiration rate.

Optionally, the relative conductivity detection comprises:

pretreating the leaves of the treated experimental crop group to obtain a leaching liquor;

determining a first conductivity R1 of the leachate;

heating the leaching solution, cooling and shaking up, and then measuring the conductivity to obtain a second conductivity R2;

calculating relative conductivity according to the first conductivity R1 and the second conductivity R2;

wherein the relative conductivity is R1/R2 100%.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

the method for relieving the water logging of the plant seedlings and the toxic hazard of double salt stress is characterized in that the experimental crops subjected to double stress are respectively treated with the experimental crops subjected to independent stress of salt stress treatment and water logging stress treatment, the leaf pigment content, the soluble sugar content, the relative conductivity and the photosynthetic characteristic are detected, the internal conditions of the experimental crops can be comprehensively reflected, the photosynthetic products of photosynthesis of the experimental crops can be accurately determined, the photosynthetic products under different stress conditions are analyzed, the effect of relieving the double stress by the exogenous brassinolide can be accurately and effectively determined, and further the experimental crops subjected to double stress can be relieved by the exogenous brassinolide.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

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, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.

FIG. 1 is a schematic flow diagram of a process provided in an embodiment of the present application;

FIG. 2 is a schematic flow chart of leaf pigment content detection provided in the embodiments of the present application;

FIG. 3 is a schematic flow chart of relative conductivity detection provided by an embodiment of the present application;

FIG. 4 is a graph showing the effect of exogenous EBR on the conductivity of kidney bean seedling leaves under the dual stress of waterlogging and salt provided by the examples of the present application;

FIG. 5 is a graph showing the effect of exogenous EBR on soluble sugar content of kidney bean seedling leaves under dual stress of waterlogging and salt, provided in the examples of the present application;

FIG. 6 is a graph of the effect of exogenous EBR on net photosynthetic rate of kidney bean seedling leaves under dual stress of waterlogging and salt as provided in the examples herein;

FIG. 7 is a graph showing the effect of exogenous EBR on intercellular carbon dioxide of leaves of kidney bean seedlings under the dual stress of waterlogging and salt provided in the examples of the present application;

FIG. 8 is a graph showing the effect of exogenous EBR on stomatal conductance of kidney bean seedling leaves under double stress of waterlogging and salt, provided by the examples of the present application;

FIG. 9 is a graph showing the effect of exogenous EBR on transpiration rate of kidney bean seedling leaves under dual stress of waterlogging and salt, provided in the examples of the present application;

FIG. 10 is a graph showing the effect of exogenous EBR on actual photosynthetic quantum yield of kidney bean seedling leaves under dual stress of waterlogging and salt, provided by the examples of the present application;

FIG. 11 is a graph showing the effect of exogenous EBR on maximum photochemical efficiency of kidney bean seedling leaves under dual stress of waterlogging and salt as provided in the examples of the present application;

FIG. 12 is a graph showing the effect of exogenous EBR on photochemical reflectance index of kidney bean seedling leaves under double stress of waterlogging and salt provided in the examples of the present application;

FIG. 13 is a graph showing the effect of exogenous EBR on the vegetation decay index of kidney bean seedling leaves under the double stress of waterlogging and salt.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all 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 application.

The inventive thinking of the application is that: actual photosynthetic efficiency Y (II), Y (II) represents the actual original photochemical efficiency of the PS II reaction center under the condition of partial closing, and Y (II) can reflect the share of energy used for electron transfer in illumination of plant leaves in absorbed light energy and is closely related to the intensity of carbon assimilation reaction;

the photochemical quenching coefficient qP is the proportion of light energy absorbed by an antenna pigment in a PS II reaction center for chemical electron transfer, is directly related to processes such as electron transfer, photosynthetic oxidation and the like, and reduces the proportion of reaction centers opened in the PS II low-reaction by qP and electrons participating in carbon dioxide fixation; the maximum photochemical quantum yield Fv/Fm is different under the non-stress environment according to different plant species, but the reduction degree of the maximum photochemical quantum yield Fv/Fm is an important index for reflecting the degree of damage of a plant photosynthetic mechanism caused by environmental stress;

the transpiration rate is an important way for dissipating moisture in the plant body, can promote the conduction of the moisture in the plant body and accelerate the transportation of mineral substances, and carbon dioxide molecules enter the plant body from pores during transpiration so as to influence the photosynthesis rate;

the leaf air hole conductivity is the door for the water vapor and carbon dioxide to enter and exit, and controls the photosynthesis and transpiration of plants at the same time;

since the chlorophyll content of the plant leaves directly determines the photosynthesis intensity when the external conditions are fixed, the pigment content of the plant leaves also needs to be analyzed.

The chlorophyll a content, the chloroplast number per unit leaf area and the chlorophyll content per unit weight determine the utilization rate of the plant on light energy; the pigment is an important component of a thylakoid membrane and is a receptor of light energy, wherein chlorophyll a is favorable for absorbing long-wave light, chlorophyll b is favorable for absorbing short-wave light, and the carotenoid is not only a photosynthetic pigment, but also an endogenous antioxidant, and can absorb residual energy in cells and quench active oxygen besides a certain function in photosynthesis, thereby preventing membrane lipid peroxidation. The ratio of carotene to chlorophyll is related to the capability of the plant to endure the stress, and the change of the ratio of chlorophyll a to b can reflect the intensity of the photosynthetic activity of the leaves.

In one embodiment of the present application, as shown in fig. 1, there is provided a method for alleviating double stress poisoning of water logging and salt of young plants, the method comprising:

s1, carrying out salt stress treatment on the experimental crops to obtain salt stress experimental crops;

s2, carrying out waterlogging stress treatment on the experimental crops to obtain waterlogging stress experimental crops;

s3, carrying out double stress treatment on the experimental crops through salt stress treatment and waterlogging stress treatment to obtain double stress experimental crops;

s4, respectively spraying exogenous brassinolide on leaf surfaces of the salt stress experimental crops, the waterlogging stress experimental crops and the double stress experimental crops to obtain treated experimental crop groups;

s5, respectively carrying out leaf detection on the treated experimental crop groups, and respectively carrying out continuous cultivation to obtain a plurality of groups of detection data;

s6, judging whether the exogenous brassinolide has influence on the experimental crops subjected to the double stress treatment or not according to a plurality of groups of detection data;

if so, spraying exogenous brassinolide to the crop plants to be treated to obtain the crop plants with the double stresses of waterlogging and salt relieved;

the leaf detection comprises leaf length and width value detection, leaf pigment content detection, soluble sugar content detection, relative conductivity detection and photosynthetic characteristic detection.

In some alternative embodiments, the spray application treatment of the exogenous brassinolide comprises:

continuously spraying exogenous brassinolide of 0.09-0.11 mu mol/L for 3 days.

In the embodiment of the application, the active effect of continuously spraying the 0.09-0.11 mu mol/L exogenous brassinolide for 3d is that the exogenous brassinolide can be ensured to effectively act on the leaf surfaces of the experimental crops under the condition of the concentration, so that the improvement of the exogenous brassinolide on the interiors of the crops is ensured, the experimental crops suffering from double stress toxicity of waterlogging and salt can be relieved, and the accuracy of subsequent experiments is ensured; when the concentration is greater than or less than the end point of the range, the effect of relieving is not obvious, and the accuracy of subsequent experiments is poor.

In some alternative embodiments, the salt stress treatment comprises:

and adding 45-55 mmol/day of salt solution into the soil of the experimental crop until the concentration of the salt solution in the soil is more than or equal to 100 mmol.

In the embodiment of the application, the salt solution concentration and the salt solution amount added into the soil are limited, so that the salt stress on the experimental crops can be effectively realized under the conditions of the concentration and the salt solution amount.

In some alternative embodiments, the water logging stress treatment comprises: carrying out waterlogging stress treatment in the tendril pulling period and the pod bearing period of the experimental crops, wherein the tendril pulling period is that 7-8 pieces of compound leaves appear in the experimental crops or 25-30 days after the experimental crops are sowed,

the pod bearing period is that the pod bearing rate of the experimental crop is more than or equal to 50% or the experimental crop is sowed for 40 d-50 d.

In this application embodiment, through prescribing a limit to the time of waterlogging stress processing respectively, can guarantee that experimental crop is in photosynthesis's active period this moment to it is obvious to the influence of photosynthesis to guarantee waterlogging treatment, and then guarantees the accurate judgement to follow-up experiment.

In some alternative embodiments, the salt stress treatment, the water logging stress treatment and the double stress treatment are all performed for a period of 1 week to 2 weeks.

In the embodiment of the application, the stress treatment time is limited, so that the stress treatment stages can be effectively and fully treated, the accuracy is provided for subsequent experiments, and whether exogenous brassinolide has influence on experimental crops subjected to double stress treatment is accurately judged.

In some alternative embodiments, as shown in fig. 2, the leaf pigment content detection comprises:

s101, taking the leaves of the treated experimental crop group to obtain a leaf group;

s102, shearing the leaves of the leaf group, and extracting the mixed solution under the dark condition to obtain an extracting solution;

s103, carrying out spectrophotometric detection on the extracting solution to obtain a plurality of groups of OD values;

and S104, calculating the plurality of groups of OD values to respectively obtain the chlorophyll a content, the chlorophyll b content, the total chlorophyll content and the carotenoid content of the leaf groups.

In some alternative embodiments, the chlorophyll-a content is calculated by the formula:

chlorophyll a content ═ V/(1000 × W) (12.7 × OD663-2.69 × OD645),

the calculation formula of the chlorophyll b content is as follows:

chlorophyll b content ═ V/(1000 × W) (22.9 × OD645-4.68 × OD663),

the calculation formula of the total content of chlorophyll is as follows:

total chlorophyll content ═ V/(1000 × W) (20.21 × OD645+8.02 × OD663),

the formula for calculating the carotenoid content is:

carotenoid content ═ 4.7 × OD440-0.27 ═ 20.21 × OD645+8.02 × OD663) ] × V/(1000 × W);

wherein V is the volume of the extracting solution, W is the fresh weight of the leaves of the leaf group, OD645 is the absorbance value of the extracting solution on a 645nm spectrophotometer, and OD663 is the absorbance value of the extracting solution on a 663nm spectrophotometer.

In the embodiment of the application, by limiting the calculation modes of the chlorophyll a content, the chlorophyll b content, the chlorophyll total amount and the carotenoid content, the data such as the actual photosynthetic efficiency, the maximum photochemical quantum yield, the photochemical reflection index and the vegetation attenuation index can be respectively calculated through the content data, so that the photosynthesis state of the experimental crop can be more comprehensively reflected, and the influence degree of the exogenous brassinolide on the experimental crop subjected to double stress treatment can be accurately judged.

In some alternative embodiments, the mixed liquor comprises absolute ethanol, acetone, and water; the volume ratio of the absolute ethyl alcohol to the acetone to the water is 4-5: 1.

In the embodiment of the application, the volume ratio of absolute ethyl alcohol, acetone and water is limited within the range, so that the pigments of the leaves such as chlorophyll and carotenoid in the leaves of the treated experimental crop group can be effectively and sufficiently extracted, the sufficient extraction of the pigment content of the leaves is ensured, and the influence degree of exogenous brassinolide on the experimental crops subjected to double stress treatment can be accurately judged.

In some alternative embodiments, the photosynthetic feature detection comprises leaf net photosynthetic rate, intercellular CO, etc. of the leaves of the treated set of test crops ₂ And (4) detecting the concentration, the porosity conductance and the transpiration rate.

In the embodiment of the application, the net photosynthetic rate of the leaves and intercellular CO are carried out on the leaves of the treated experimental crop group ₂ The influence of exogenous brassinolide on the photosynthesis of the leaves can be intuitively obtained by detecting the concentration, the air hole conductivity and the transpiration rate, so that the accuracy of the experiment is ensured.

In some alternative embodiments, as shown in fig. 3, the relative conductivity detection comprises:

s201, preprocessing the leaves of the treated experimental crop group to obtain a leaching liquor;

s202, determining a first conductivity R1 of the leaching solution;

s203, heating the leaching liquor, cooling, shaking up, and then measuring the conductivity to obtain a second conductivity R2;

s204, calculating to obtain relative conductivity according to the first conductivity R1 and the second conductivity R2;

wherein the relative conductivity is R1/R2 100%.

In the embodiment of the application, the relative conductivity of the treatment experimental group can be accurately obtained by limiting the specific test mode of the relative conductivity, so that the internal state of the crop can be effectively reflected, and accurate guarantee is provided for judging the influence degree of the exogenous brassinolide on the experimental crop subjected to the double stress treatment

Example 1

The first, material and method:

1. experimental materials:

the method takes the vining kidney beans as test objects, seeds are provided by bean research institute of Jianghan university, and the test site is located in a bean research and research base of Hubei province.

2. The experimental method comprises the following steps:

salt stress treatment: and (3) cultivating the seeds of the sprawl beans, and adding 50mmol of salt solution into the soil every day after the seedling stage of the sprawl beans grows out of the first compound leaf until the concentration of NaCl salt solution in the soil reaches 100 mmol.

Water logging stress treatment: the treatment time of waterlogging stress treatment is 2 periods, namely a tendril pulling period (7-8 pieces of compound leaves appear in the experimental crops, which is about 25-30 days after the experimental crops are sown) and a pod bearing period (the pod bearing rate is more than or equal to 50 percent, and about 40-50 days after the experimental crops are sown); the waterlogging stress treatment adopts a random block design, and the total number of the treatments is 3: the method comprises the following steps of normally watering, water logging stress and waterlogging stress, wherein the water logging stress and the waterlogging stress are carried out by sleeving a plastic pot outside a cultivation plastic pot of each experimental crop and laying plastic cloth between the plastic pots to ensure that water does not leak, the normally watered soil keeps the relative water content of 70-80%, the waterlogging stress treatment keeps the water surface in the pot higher than the soil surface by 2cm and is in a water accumulation state, the water logging stress treatment keeps the water surface flush with the soil surface, water is not accumulated, the treatment time is divided into 3 days, and after the waterlogging stress treatment is finished, water is permeated to the normal soil water content.

The test materials were divided into 2 groups, one group was sprayed with EBR solution in the morning and evening for 3 days continuously, and the other group was sprayed with distilled water as a control. Spraying distilled water on the leaf surface until the leaf surface is wet completely and drops, spraying EBR with corresponding concentration in other treatments until the drops drop, wherein the spraying time is 18:00 every day, the EBR is diluted by the same mother solution, the EBR is used immediately after preparation, and the concentration after preparation is 0.1 mu mol/L.

The test treatment groups were specifically: no EBR + no salt + normal watering (CK group), no EBR + no salt + waterlogging (S1 group), no EBR +100mM salt + normal watering (S2 group), no EBR +100mM salt + waterlogging (S3 group), EBR + no salt + normal watering (S4 group), EBR + no salt + waterlogging (S5 group), EBR +100mM salt + normal watering (S6 group) and EBR +100mM salt + waterlogging (S7 group).

3. The determination method comprises the following steps:

3.1 pigment content of seedling leaves:

the leaves were cut into pieces, 0.1g of the pieces were put into 10mL of a mixed extract (ethanol: acetone: water: 4.5:1), extracted in the dark until the leaves were completely white to obtain an extract, and the mixed extract was used as a control to measure OD440, OD645 and OD663 in a spectrophotometer, respectively.

The calculation formula is as follows:

chlorophyll a content ═ V/(1000 × W) (12.7 × OD663-2.69 × OD645),

the calculation formula of the chlorophyll b content is as follows:

chlorophyll b content ═ V/(1000 × W) (22.9 × OD645-4.68 × OD663),

the calculation formula of the total content of chlorophyll is as follows:

total chlorophyll content ═ V/(1000 × W) (20.21 × OD645+8.02 × OD663),

the formula for calculating the carotenoid content is:

carotenoid content ═ 4.7 × OD440-0.27 ═ 20.21 × OD645+8.02 × OD663) ] × V/(1000 × W);

wherein V is the volume of the extracting solution, W is the fresh weight of the leaves of the leaf group, OD645 is the absorbance value of the extracting solution on a 645nm spectrophotometer, and OD663 is the absorbance value of the extracting solution on a 663nm spectrophotometer.

3.2 determination of conductivity and soluble sugar content:

1. determination of the relative conductivity: taking plant leaves with the same size (ensuring the integrity of the leaves as much as possible and containing less stem nodes), washing with tap water, washing with distilled water for 3 times, sucking surface water with filter paper, cutting the leaves into long strips with proper length (needing to avoid main veins), quickly weighing 3 fresh samples, each 0.1g of the fresh samples, respectively placing the fresh samples into 10mL deionized water scale test tubes, covering the test tubes with glass plugs, soaking at room temperature for 12h, measuring the first conductivity of the leaching solution Rl. with a conductivity meter, heating in a boiling water bath for 30min, cooling to room temperature, shaking uniformly, measuring the second conductivity of the leaching solution R2 again, wherein the relative conductivity is RI/R2 × 100%.

Soluble sugar content: and (3) measuring the content of the soluble sugar by using an ultraviolet-visible spectrophotometer: wiping off surface dirt on the fresh kidney bean leaves to be detected, shearing, uniformly mixing, weighing 0.10-0.30 g and 3 parts in total, respectively putting the weighed fresh kidney bean leaves into 3 graduated test tubes, adding 5-10 mL of distilled water, sealing by a plastic film, extracting in boiling water for 30min (each extraction is carried out for 2 times), filtering an extracting solution into a 25mL volumetric flask, repeatedly washing the test tube and residues, and fixing the volume to the scales. Color development measurement 0.5mL of sample extract is absorbed into a 20mL graduated test tube (repeated for 2 times), 1.5mL of distilled water is added, then 0.5mL of anthrone ethyl acetate reagent and 5mL of concentrated sulfuric acid are sequentially added into the test tube, the test tube is fully oscillated, the test tube is immediately placed into a boiling water bath, the temperature is accurately kept for 1min tube by tube, the test tube is naturally cooled to room temperature after being taken out, a blank is used as a reference, and the optical density of the test tube is measured at the wavelength of 630 nm.

3.3 photosynthetic characteristics of the seedling leaves:

photosynthetic parameters such as net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs) and transpiration rate (Tr) of vegetable soybean leaf were measured using LI-6400 portable photosynthetic analyzer. The measurement time was 9 a.m.: 00-11: 30, keeping the indoor air circulation, randomly measuring 3 seedlings with consistent growth in each group, and taking an average value.

4. And (3) data analysis:

the detection of all test data is repeated for 3 times, statistical data arrangement is carried out, data analysis is carried out by IBMSPSSStatics 20, single-factor analysis of variance (ANOVA) is carried out on the test data by a Duncan new complex range method, the difference significance (P <0.05) between all treatment groups is checked, the significance is recorded and marked by letters, the average value of all the treatment groups and the control group is subjected to chart making, the standard error is calculated, the obtained standard error is added into a statistical chart by an error line of a positive-negative deviation strip end mode, and the letters are marked.

Secondly, result and analysis:

the effect of EBR on leaf aspect ratio and yield of kidney bean seedlings under dual stress of waterlogging and salt:

the effect of EBR on leaf aspect ratio and yield of kidney bean seedlings under double stress of water logging and salt for 2 weeks is shown in table 1:

TABLE 1

As can be seen from table 1: as the length-width ratio of the leaves is one of important physiological indexes of plants, when the plants are stressed by different types and degrees of environments, one of the important indications of stress is the change of the length-width ratio. After the kidney beans are stressed for two weeks, the group with the largest leaf length-width ratio value is EBR + salt-free + normal watering, and the ratio is obviously improved by 30.77% compared with the group without EBR + salt-free + waterlogging with the lowest leaf length-width ratio. And the length-width ratios of the four treatment groups sprayed with the EBR are slightly higher than those of the corresponding treatment groups not sprayed with the EBR, which shows that the length-width ratio of the leaves can be improved by applying the EBR, and the influence of stress on the length-width ratio of the plants is relieved. The yield of the EBR + salt-free + normal watering group was highest and 54.7% higher than that of the EBR + salt-free + waterlogging group.

The effect of EBR on chlorophyll and carotenoid content of kidney bean seedling leaves under double stress of waterlogging and salt is shown in table 2:

TABLE 2

Note that different lower case letters indicate significant differences between treatments (P < 0.05).

The chlorophyll a/b ratio is an important index for measuring the negative tolerance of plants. Researches show that the low ratio of chlorophyll to a/b is beneficial to improving the absorption of far-red light by plants, is beneficial to the plants to absorb more light under the condition of low light intensity so as to improve the photosynthetic efficiency, and is a physiological adaptation formed by the plants under the condition of low light intensity.

From table 3, it can be seen that: in the treatment without spraying the EBR, compared with the CK group, the treatment of waterlogging stress (S1 group) and single salt stress (S2 group) has the advantages that the content of photosynthetic pigments in the leaves of the kidney bean seedlings and the content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid in the S1 group are respectively reduced by 16.94%, 25%, 18.78% and 16.71% compared with the CK group, and the difference is obvious (P is less than 0.05); the photosynthetic pigment content of S2 is 0.51, 0.46 and 0.72 times of that of CK group, EBR is applied to waterlogging stress (S5 group and S6 group), the content of each photosynthetic pigment basically has no obvious change, and chlorophyll a/b is increased under single salt stress, but the difference is not obvious.

In the treatment without salt and waterlogging stress, after EBR is sprayed, compared with the treatment without EBR, the content of chlorophyll a is increased by 16.6%, the content of chlorophyll b is reduced by 26.5%, the content of total chlorophyll is reduced by 18.8%, and the difference is obvious, but the content changes of chlorophyll a/b and carotenoid are not obvious. Under the double stress of salt and waterlogging, after the EBR is sprayed, compared with the condition that the EBR is not sprayed, the content of chlorophyll a is reduced by 57.4%, the content of chlorophyll b is reduced by 68%, the total content of chlorophyll is reduced by 59.8%, the content of carotenoid is reduced by 58.0%, the difference is obvious, and the content of chlorophyll a/b is increased by 34.4%, and the difference is obvious.

The effects of EBR on leaf conductivity and soluble sugar content of kidney bean seedlings under double stress of waterlogging and salt are shown in fig. 4 and 5.

As shown in FIG. 4, the single salt stress, the single waterlogging stress or the double stresses of the two all damage the cell membranes of the kidney bean seedlings, and the conductivity of the leaves is increased. When EBR is not sprayed, single salt stress (S2 group) is increased by 122.49% compared with CK, and the difference is obvious; the single waterlogging stress (S1 group) and the double stress (S3 group) are respectively increased by 35.93 percent and 17.51 percent compared with the CK group, and the difference is not significant. Under EBR remission, most of the treated groups had reduced cell membrane damage, as indicated by a decrease in their relative conductivity. EBR (S4 group) was applied to normally grown kidney beans and had a leaf relative conductivity of 3.765, which was the lowest value in the treatment group, and was 4.32% lower than the CK group, with no significant difference. EBR (group S6) conductivity significantly decreased by 49.47% compared to S2 (P <0.05) applied under monosalt stress; however, when EBR was applied under waterlogging stress alone (group S5), the conductivity was significantly increased by 54.77% compared to S1; EBR (S7 group) was applied under dual salt and waterlogging stress with a significant increase in conductivity of 53.09%. Therefore, EBR can alleviate the increase in conductivity caused by salt stress, and cannot alleviate the increase in conductivity caused by waterlogging stress.

As can be seen from FIG. 5, since soluble sugar is one of the main substances for regulating the osmotic pressure and energy storage of cells, the osmotic pressure of the plant can be increased or decreased to various degrees in order to maintain the normal osmotic pressure of the cells under external stress, and EBR can slow down the change of EBR within a certain range. When water logging stress is exhibited, soluble sugar content increases; upon salt stress, the soluble sugar content decreases. Waterlogging stress (S1 group) was significantly increased by 1.72-fold (P <0.05) compared to CK, and monosalt stress (S2 group) was significantly decreased by 44.73% compared to CK group; whereas the change in soluble sugar content was much slowed down after EBR application (S5 and S6 groups). EBR was applied in the normal-growing kidney bean-treated group (S4 group), whose soluble sugar content was significantly increased 77.26% compared to the control group; the content of the S7 group sprayed with EBR is increased by 2.99 percent compared with the S3, and the difference is not significant.

The effect of EBR on photosynthesis indicators of kidney bean seedlings under double stress of waterlogging and salt is shown in fig. 6 to 9:

as shown in fig. 6 to 9, the net photosynthetic rate of the kidney bean seedlings was affected regardless of the water-logging stress, the single salt stress or the double stress of the water-logging stress and the salt, and showed various degrees of decrease depending on the stress; and for other indices: stomatal conductance, intercellular carbon dioxide concentration, transpiration rate and stressed plants are all increased compared with CK group. The net photosynthetic rates of the S1-S7 groups were reduced compared to the CK group of the control group, and the treated groups except the S3 group were significantly lower than CK (P <0.05), and the intercellular carbon dioxide concentrations of the seven treated groups were significantly higher than the CK group.

Under the double stress of waterlogging and salt, the net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration and transpiration rate of the fertilizer are respectively 0.9052, 1.71, 0.585 and 1.614 times of those of the CK group; bean seedlings that were EBR-stressed under double stress (group S7) showed overall performance of various photosynthetic indicators compared to group S3.

The effect of EBR on the fluorescence parameters of kidney bean seedling leaves under double stress of waterlogging and salt is shown in fig. 10 and 11:

the actual photosynthetic quantum yield Y (II) represents the actual photosynthetic efficiency, the range is 0.229-0.276, and the actual photosynthetic efficiency is higher within the altitude of 2900 m-3100 m; the actual photosynthetic quantum yield Y (II) is in negative correlation with the total content of chlorophyll, the content of chlorophyll a and chlorophyll b and the ratio of chlorophyll a/b.

As can be seen from fig. 10, for the treatment group without EBR spraying, the value of y (ii) was lowest in the CK group, the value of y (ii) was 296% higher in the no-salt + waterlogging treatment group, the difference was significant (P <0.05), the value of y (ii) in the no-salt + waterlogging treatment group was 129% higher than the value of y (ii) in the no-salt + waterlogging treatment group, the difference was significant (P <0.05), the value of y (ii) in the salt + normal watering treatment group was 137% higher, 73.2% higher than the value of y (ii) in the no-salt + normal watering group, but no difference was found (P < 0.05); for the EBR-sprayed treatment group, the Y (II) values were the lowest for the salt + normal-watering treatment group, and the Y (II) values were 16.8%, 46.5%, and 82.3% higher for the salt + waterlogged treatment group, the salt-free + normal-watering treatment group, and the salt-free + waterlogged treatment group than for the salt + normal-watering treatment group, but were not different (P < 0.05).

Because Fv/Fm refers to the maximum photochemical efficiency of PS II, the primary light energy conversion efficiency in a PS II reaction center is reflected, and Fv/Fo reflects the potential activity of PS II and is 2 important parameters for indicating the photochemical reaction condition.

As can be seen from FIG. 11, for the treatment groups without spraying EBR, the Fv/Fm values of the salt-free and normal watering groups are the lowest, the Fv/Fm values of the salt-free and waterlogging treatment groups and the salt-free and waterlogging treatment groups are 102% and 121% higher than those of the salt-waterlogging treatment groups, and the difference is significant (P is less than 0.05), and the Fv/Fm values of the salt-normal watering treatment groups are 70.0% higher than those of the salt-normal watering treatment groups but are not different (P is less than 0.05); the Fv/Fm under the stress condition is higher than the control without any stress, the Fv/Fm value of the salt-free waterlogging treatment group is the highest in the treatment group sprayed with the EBR, the Fv/Fm value of the salt-free waterlogging treatment group is higher than that of the salt-normal watering treatment group, the salt-waterlogging treatment group and the salt-free waterlogging treatment group by 109%, 52.1% and 57.0%, the difference is obvious (P is less than 0.05), and the difference among the 3 treatments of the salt-normal watering, the salt-waterlogging treatment group and the salt-free waterlogging treatment group is not obvious, so that the EBR can relieve the damage caused by the salt stress to a certain extent and cannot relieve the damage caused by the waterlogging stress.

The effect of EBR on photochemical reflectance index of kidney bean seedlings under double stress of waterlogging and salt is shown in fig. 12 and 13:

because the change of the carotenoid content in the plant is one of the important indexes of the plant responding to the environmental stress, and the Photochemical Reflectance Index (PRI) is related to the photosynthetic utilization rate (LUE) and is very sensitive to the change of the carotenoid (especially yellow pigment) of the living plant, the research and analysis of the carotenoid in the leaf blade by utilizing the index has very important significance for researching the luminous energy utilization rate of the plant in the photosynthesis under the stress environment. As can be seen from fig. 12 and the significance comparison, there is no significant difference between the treatment group to which EBR is applied and the corresponding group to which EBR is not applied, and the effect of increasing or decreasing PRI is not the same, which indicates that EBR has no significant effect on PRI value in the same stress treatment. The PRI value of the CK group in the control group is stabilized between 0.02 and 0.03 before and after, and no significant difference exists along with the change of the growth time; under the stress of monosalt, the PRI before and after the plant is higher than that of the plant with normal growth, and respectively increases 35.71% (EBR + monosalt spraying after 1-week treatment), 30.76% (EBR + monosalt not spraying after 1-week treatment), 104.35% (EBR + monosalt spraying after 2-week treatment) and 12.5% (EBR + monosalt not spraying after 2-week treatment); under single waterlogging stress, the PRI of the plant and the PRI of the plant without stress have no significant difference, the PRI value of the plant under double stress and the PRI value of the plant without stress have the maximum difference, and are all significantly higher than those of the plant without stress, and are respectively 1.46 times (EBR + double stress sprayed for 1 week), 1.65 times (EBR + double stress not sprayed for 1 week), 2.48 times (EBR + double stress sprayed for 2 weeks) and 2.375 times (EBR + double stress not sprayed for 2 weeks), so that the PRI value of the plant can be obviously increased under double or single salt stress, especially under double stress.

The PSRI is a vegetation decay index, is used for improving the sensitivity of the ratio of the carotenoid to the chlorophyll to the maximum extent, the increase of the PSRI indicates the increase of the canopy stress and the beginning of vegetation senescence, and has important application to vegetation health monitoring, plant physiological stress detection and crop production and yield analysis. Similar to the PRI detection results, as can be seen from fig. 13 and the significant differences, the PSRI values of the kidney bean seedlings under the double stress of salt and water-logging exceed those of other treatment groups in the same period regardless of the presence or absence of EBR, which indicates that the stress of the plants under the double stress exceeds that of the plants under single stress, up to 0.051 is 1.37 times that of the control group, and the stress resistance of the plants increases and the PSRI value decreases with the increase of the stress time.

3. Conclusion and discussion:

(1) after the double stress treatment of waterlogging and salt for two weeks, the length and width of the leaves are obviously reduced, and the trend of reducing the length and width of the leaves is relieved to a certain extent by adding the EBR.

(2) EBR has no effect of improving the chlorophyll content and soluble sugar under the double stress of waterlogging and salt, but shows a remarkable reduction trend in partial results, but obviously improves the chlorophyll a/b value.

(3) Waterlogging, single salt and double stress all affect the net photosynthetic rate, intercellular carbon dioxide concentration, stomatal conductance and transpiration rate of the kidney bean seedlings, and the influence is correspondingly increased gradually: the net photosynthetic rate is reduced, the intercellular carbon dioxide concentration, the stomatal conductance and the transpiration rate are increased. The EBR has different effects on each index in the stressed plants, but the EBR ensures that each photosynthetic data of the stressed plants are close to the data of the bean seedlings which are added with the EBR and are not stressed, so that the EBR has no obvious difference.

From the above conclusions, it can be clearly concluded that EBR has an obvious alleviating effect on kidney beans under double stress of salt and water logging, and thus EBR can be used as one of alleviating means for kidney beans under double stress.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

(1) according to the method provided by the embodiment of the application, the experimental crops subjected to double stress are respectively and simultaneously subjected to salt stress treatment and waterlogging stress treatment and subjected to independent stress, and the effects of relieving the double stress of the exogenous brassinolide are accurately and effectively determined by detecting the length and width values of the leaves, the pigment content of the leaves, the soluble sugar content, the relative conductivity and the photosynthetic characteristic, so that the experimental crops subjected to the double stress can be relieved by utilizing the exogenous brassinolide.

(2) According to the method provided by the embodiment of the application, after the EBR is sprayed, the adverse effects of leaf area reduction, chlorophyll content reduction, net photosynthetic rate reduction, relative conductivity increase and Y (II) increase caused by waterlogging stress, salt stress and double stresses of waterlogging stress and salt can be relieved.

(3) According to the method provided by the embodiment of the application, after the EBR is sprayed, the adverse effects of the reduction of the soluble sugar content, the increase of the PRI value, the transpiration rate, the intercellular carbon dioxide concentration and the improvement of the porosity caused by double stress of waterlogging and salt can be relieved.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice 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 (10)

1. A method for relieving double stress toxicity of waterlogging and salt of plant seedlings, which comprises the following steps:

carrying out salt stress treatment on the experimental crops to obtain salt stress experimental crops;

carrying out waterlogging stress treatment on the experimental crops to obtain waterlogging stress experimental crops;

carrying out double stress treatment on the experimental crops by salt stress treatment and waterlogging stress treatment to obtain double stress experimental crops;

respectively spraying exogenous brassinolide on the leaf surfaces of the salt stress experimental crops, the waterlogging stress experimental crops and the double stress experimental crops to obtain treated experimental crop groups;

respectively carrying out leaf detection on the treated experimental crop groups, and respectively carrying out continuous cultivation to obtain a plurality of groups of detection data;

judging whether the exogenous brassinolide has influence on the experimental crops subjected to the double stress treatment according to a plurality of groups of detection data;

if so, spraying exogenous brassinolide to the crop plants to be treated to obtain the crop plants with the double stresses of waterlogging and salt relieved;

the leaf detection comprises leaf length and width value detection, leaf pigment content detection, soluble sugar content detection, relative conductivity detection and photosynthetic characteristic detection.

2. The method of claim 1, wherein the spray application of exogenous brassinolide comprises:

continuously spraying exogenous brassinolide of 0.09-0.11 mu mol/L for 3 days.

3. The method of claim 1, wherein the salt stress treatment comprises:

and adding 45-55 mmol/day of salt solution into the soil of the experimental crop until the concentration of the salt solution in the soil is more than or equal to 100 mmol.

4. The method of claim 1, wherein the water logging stress treatment comprises: carrying out waterlogging stress treatment in the tendril pulling period and the pod bearing period of the experimental crops, wherein the tendril pulling period is that 7-8 pieces of compound leaves appear in the experimental crops or 25-30 days after the experimental crops are sowed,

the pod bearing period is that the pod bearing rate of the experimental crop is more than or equal to 50% or the experimental crop is sowed for 40 d-50 d.

5. The method according to claim 1, wherein the time for the salt stress treatment, the water-logging stress treatment and the double stress treatment is 1 to 2 weeks.

6. The method of claim 1, wherein the leaf pigment content detection comprises:

taking the leaves of the treated experimental crop group to obtain a leaf group;

shearing the leaves of the leaf group, and extracting the mixed solution in the dark to obtain an extracting solution;

performing spectrophotometric detection on the extracting solution to obtain a plurality of groups of OD values;

and calculating a plurality of groups of OD values to respectively obtain the chlorophyll a content, the chlorophyll b content, the total chlorophyll content and the carotenoid content of the leaf groups.

7. The method according to claim 6, wherein the chlorophyll-a content is calculated by the formula:

chlorophyll a content ═ V/(1000 × W) (12.7 × OD663-2.69 × OD645),

the calculation formula of the chlorophyll b content is as follows:

chlorophyll b content ═ V/(1000 × W) (22.9 × OD645-4.68 × OD663),

the calculation formula of the total content of chlorophyll is as follows:

total chlorophyll content ═ V/(1000 × W) (20.21 × OD645+8.02 × OD663),

the formula for calculating the carotenoid content is:

carotenoid content ═ 4.7 × OD440-0.27 ═ 20.21 × OD645+8.02 × OD663) ] × V/(1000 × W);

wherein V is the volume of the extracting solution, W is the fresh weight of the leaves of the leaf group, OD645 is the absorbance value of the extracting solution on a 645nm spectrophotometer, and OD663 is the absorbance value of the extracting solution on a 663nm spectrophotometer.

8. The method of claim 6, wherein the mixed solution comprises absolute ethanol, acetone, and water; the volume ratio of the absolute ethyl alcohol to the acetone to the water is 4-5: 1.

9. The method of claim 8, wherein said photosynthetic feature detection comprises leaf net photosynthetic rate, intercellular CO, etc. of leaves of said treated set of test crops ₂ And (4) detecting the concentration, the porosity conductance and the transpiration rate.

10. The method of claim 1, wherein the relative conductivity detection comprises:

pretreating the leaves of the treated experimental crop group to obtain a leaching liquor;

determining a first conductivity R1 of the leachate;

heating the leaching solution, cooling and shaking uniformly, and then measuring the conductivity to obtain a second conductivity R2;

calculating relative conductivity according to the first conductivity R1 and the second conductivity R2;

wherein the relative conductivity is R1/R2 100%.

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