Application of marine microalgae in improving plant stress resistance and seaweed fertilizer

Abstract

The invention discloses an application of marine microalgae in improving plant stress resistance and seaweed fertilizer. In the application, the marine microalgae is Dunaliella salina, which is applied to the planting environment where the plant is located. The invention improves the stress resistance of the plant and promotes the growth of the plant by applying Dunaliella salina to the planting environment where the plant is located.

Description

Technical Field

[0001] The invention relates to the field of marine microalgae, and in particular to an application of marine microalgae in improving plant stress resistance and a seaweed fertilizer. Background Art

[0002] Plants often encounter adverse stress during their growth, such as high temperature, drought, and salinity. Long-term stress can cause a reduction in the yield or even death of plants such as crops, causing damage to agricultural production. Saline-alkali land lacks organic matter, has low indigenous microbial diversity, and artificial exogenous microorganisms are also easily lost. As the degree of salinity in the soil increases, the soil water potential decreases, and plants have difficulty absorbing water, causing physiological drought in plants and hindering their growth and development. In addition, saline-alkali stress can interfere with or inhibit the normal physiological activities of plant cells, change the structure and function of antioxidant enzymes, break the balance (production and removal) of ROS (Reactive oxygenspecies) in plants, cause oxidative and antioxidant stress, accumulate a large amount of reactive oxygen, and cause oxidative damage. Traditional saline-alkali soil improvement measures mainly include water conservancy and chemical improvement. Water conservancy improvement requires the establishment of a large drainage and irrigation system, which is costly; although chemical methods are more effective, they are prone to secondary pollution.

[0003] In recent years, the introduction of biological methods has injected new vitality into the improvement of saline-alkali land, such as the application of algae resources in agricultural production. However, there are no reports on the effect of marine microalgae, such as Dunaliella salina, on the improvement of saline-alkali land and the promotion of plant growth in saline-alkali land. Summary of the invention

[0004] In order to solve the problem of plant growth in a saline-alkali environment, the present invention proposes an application of marine microalgae in improving plant stress resistance and seaweed fertilizer.

[0005] The technical problem of the present invention is solved by the following technical solutions:

[0006] In a first aspect, the present invention provides a use of marine microalgae in improving plant stress resistance, wherein the marine microalgae is Dunaliella salina, which is applied to a planting environment where the plant is located.

[0007] In a second aspect, the present invention further provides a seaweed fertilizer for improving plant stress resistance, wherein the seaweed fertilizer contains Dunaliella halogenica.

[0008] The beneficial effects of the present invention compared with the prior art include:

[0009] The present invention proposes an application of marine microalgae in improving plant stress resistance and seaweed fertilizer, which can improve plant stress resistance and promote plant growth by applying Dunaliella salina to the planting environment where the plant is located. The present invention can provide technical support for crop planting, especially rapeseed planting, in saline-alkali environments.

[0010] Other beneficial effects of the embodiments of the present invention will be further described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a schematic diagram of rapeseed seed germination rate-days in the saline-alkali solution environment experiment in Example 1 of the present invention;

[0012] Figure 2 Schematic diagram of rapeseed seed emergence rate-days in saline-alkali solution environment experiment in Example 1 of the present invention;

[0013] Figure 3 It is a schematic diagram of the germination or emergence of rapeseed seeds on the seventh day in the saline-alkali solution environment experiment in Example 1 of the present invention;

[0014] Figure 4 Schematic diagram of rapeseed seed emergence rate-days in saline-alkali soil environment experiment in Example 1 of the present invention;

[0015] Figure 5a is a schematic diagram of the malondialdehyde content in rapeseed after culturing in a saline-alkali soil environment for 28 days in Example 2 of the present invention;

[0016] Figure 5b is a schematic diagram of the proline content in rapeseed after culturing in a saline-alkali soil environment for 28 days in Example 2 of the present invention;

[0017] Figure 5c is a schematic diagram of the superoxide dismutase content in rapeseed after culturing in a saline-alkali soil environment for 28 days in Example 2 of the present invention;

[0018] Figure 5d It is a schematic diagram of the peroxidase content in rapeseed after culturing in a saline-alkali soil environment for 28 days in Example 2 of the present invention. DETAILED DESCRIPTION

[0019] The present invention is further described below with reference to the accompanying drawings and in combination with preferred embodiments. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other without conflict.

[0020] An embodiment of the present invention provides an application of marine microalgae in improving plant stress resistance. The marine microalgae is Dunaliella salina, which is applied to a planting environment where the plant is located.

[0021] In some embodiments, the planting environment is a saline solution, and the Dunaliella salina is applied to the saline solution in the form of at least one of an active algae suspension, dry algae powder, and dry algae powder algae suspension, wherein each liter of the saline solution contains 10 8 ~10 9 Dunaliella salina algae cells.

[0022] In some embodiments, the planting environment is saline-alkali soil, and the Dunaliella salina is applied to the saline-alkali soil in the form of an active algae suspension and / or a dry algae powder suspension, wherein each gram of the saline-alkali soil contains 10 6 ~10 7 Dunaliella salina algae cells.

[0023] In some embodiments, the plant is rapeseed.

[0024] In some embodiments, the stress resistance refers to salt and alkali resistance.

[0025] In some embodiments, the improving plant stress resistance is achieved by activating the antioxidant system in the plant.

[0026] In some embodiments, the method for activating the antioxidant system in the plant is to increase the activity of antioxidant enzymes in the plant, and the antioxidant enzyme is at least one of peroxidase and superoxide dismutase.

[0027] In some embodiments, the active algae suspension is prepared by inoculating Dunaliella salina into artificial seawater with a salinity of 35‰, culturing for 20 to 30 days, centrifuging at 4000 to 6000r, washing with purified water 3 to 4 times, and then adding a predetermined amount of purified water; wherein the salinity of the artificial seawater is provided by coral sea salt. 10 ~10 11 Dunaliella salina algae cells.

[0028] In some embodiments, the dry algae powder is prepared by inoculating Dunaliella salina into artificial seawater with a salinity of 35‰, culturing for 20 to 30 days, centrifuging at 4000 to 6000 r / min, washing with pure water for 3 to 4 times, and drying in an oven at 30 to 40°C; the salinity of the artificial seawater is provided by coral sea salt.

[0029] In some embodiments, the dry algae powder algae suspension is prepared by suspending the dry algae powder in purified water. 9 ~10 10 Dunaliella salina algae cells.

[0030] In some embodiments, the Dunaliella salina is cultured in f / 2-Si culture medium under the following culture conditions: light intensity of 4000±500 lux, and a ratio of light cycle to light-dark cycle of 14h:10h.

[0031] In some embodiments, the f / 2-Si medium comprises 8.82×10 -4 M NaNO 3 ,3.62×10-5 M NaH 2 PO 4 ·H 2 O, trace metal element mother solution and vitamin mother solution, wherein the trace metal element mother solution includes 1.17×10 -5 MFeCl 3 6H 2 O, 1.17×10 -5 M's Na 2 EDTA·2H 2 O, 3.93×10 -8 MCuSO 4 ·5H 2 O, 2.60×10 -8 M's Na 2 MoO 4 ·2H 2 O, 7.65×10 -8 M ZnSO 4 7H 2 O, 4.20×10 -8 M CoCl 2 6H 2 O and 9.10×10 -7 MnCl 2 ·4H 2 O, the vitamin mother solution includes 2.96×10 -7 Vitamin B 1 , 2.05×10 -9 M of vitamin H and 3.69×10 -10 Vitamin B 12 .

[0032] The embodiment of the present invention further provides a seaweed fertilizer for improving plant stress resistance, wherein the seaweed fertilizer contains Dunaliella halogenica.

[0033] In some embodiments, the Dunaliella salina is present in the seaweed fertilizer in the form of at least one of an active algae suspension, dry algae powder, and a dry algae powder algae suspension.

[0034] The present invention is further described below through specific examples.

[0035] (1) Cultivation of Dunaliella salina:

[0036] The algae used in the experiment of the present invention is Dunaliella sp., which was purchased from Xiamen University.

[0037] Before the experiment, Dunaliella salina was first domesticated and expanded in f / 2-Si medium to adapt it to laboratory culture conditions. The growth cycle of Dunaliella salina is about 12 days. After the algal cells enter the stable period, 1 / 10 of the volume of the algal liquid is taken for re-inoculation to achieve semi-continuous culture.

[0038] The seawater used in f / 2-Si culture medium is artificial seawater (35g coral sea salt is added to 1L pure water, the same below), sterilized in an autoclave with a salinity of 35‰. Dunaliella salina is cultured in an artificial climate chamber with a light intensity of 4000lux and a light cycle to light-dark cycle ratio of 14h:10h.

[0039] In this experiment, 1L of Dunaliella salina algae solution was cultured in a 3L conical flask. The position of the culture flask was randomly changed twice a day and shaken to ensure uniform illumination of the algae solution in the conical flask.

[0040] In the embodiment of the present invention, f / 2-Si medium without silicon addition (i.e., reduced silicon or -Si) is used to culture Dunaliella salina. The formula of the f / 2-Si medium is shown in Table 1, where "---" represents blank. Prepare 950 mL of artificial seawater, add the components and volumes shown in Table 1 to 950 mL of seawater, and then dilute to 1 L with artificial seawater.

[0041] Table 1

[0042]

[0043] Preparation of trace metal element mother solution: prepare 950 mL of ultrapure water, add the trace metal element mother solution formula ingredients in Table 2 to the ultrapure water and make up to 1 L, filter and sterilize twice and store in a 4°C refrigerator for use. "---" in the table indicates blank.

[0044] Table 2

[0045]

[0046]

[0047] Prepare vitamin mother solution: prepare 950 mL of ultrapure water, add the vitamin mother solution formula ingredients in Table 3 to the ultrapure water and make up to 1 L, filter and sterilize twice and freeze in a -20℃ refrigerator for later use. "---" in the table indicates blank.

[0048] Table 3

[0049]

[0050] (2) Preparation of active algae suspension:

[0051] The Dunaliella salina obtained in (1) was inoculated into 1 L of artificial seawater. After culturing for 30 days, the mixture was centrifuged at 4000-6000 r. After washing with purified water for 3-4 times, purified water was added and shaken to prepare an active algae suspension. The algae cell density was 6.88*10 10 cells / L (Each liter of active algae suspension contains 6.88*10 10 Dunaliella salina algae cells).

[0052] (3) Preparation of dried algae powder:

[0053] The Dunaliella salina obtained by culture in (1) was inoculated into 1 L of artificial seawater, and after culture for 30 days, the Dunaliella salina was centrifuged at 4000-6000 r, and washed with purified water for 3-4 times, and then dried in an oven at 30-40° C. for 48 hours to prepare dry algae powder.

[0054] (4) Preparation of dry algae powder and algae suspension:

[0055] The dry algae powder obtained in (3) was suspended in pure water to prepare a dry algae powder algae suspension. The algae cell density was 6.88*10 10 cells / L (Each liter of active algae suspension contains 6.88*10 10 Dunaliella salina algae cells).

[0056] Taking rapeseed as an example, the experimental process and test parameters are as follows: Experimental Example 1: Experiment on improving the germination of rapeseed seeds in a saline-alkali environment by Dunaliella salina.

[0057] 1. Material preparation:

[0058] Experimental subjects: rapeseed seeds.

[0059] Drug configuration:

[0060] (1) Saline solution: Prepare 20 mmol saline solution (NaCl: Na 2 SO 4 :NaHCO 3 :Na 2 CO 3 =1:9:9:1 (molar ratio)).

[0061] (2) Saline-alkali soil: 225 g simulated substrate (nutrient soil and vermiculite volume ratio 1:1), watered with saline-alkali solution three times (interval: wait until the saline-alkali solution is completely penetrated before watering again), 100 ml each time.

[0062] (3) Ordinary soil: 225 g simulated matrix (nutrient soil and vermiculite volume ratio 1:1), watered with pure water three times (interval: wait until the saline solution is completely penetrated before watering again), 100 ml each time.

[0063] 2. Experimental process:

[0064] After the rapeseed seeds were sterilized with 20% (volume) hydrogen peroxide for 5 minutes, they were washed with purified water 5 times to remove the hydrogen peroxide solution, and the following experiment was conducted:

[0065] (1) Saline and alkali liquid environment experiment:

[0066] Active algae suspension-salt-alkali solution (named TA group): Add active algae suspension to 500ml of saline-alkali solution to make each liter of saline-alkali solution contain 6.88*10 8 Place gauze in the germination device, add 500ml of "active algae suspension-salt-alkali solution", select 10 small rapeseed seeds of the same size and place them in the germination device for hydroponics for 7 days.

[0067] Dry algae powder-salt-alkali solution (named BA group): add dry algae powder to 500ml of salt-alkali solution, and each liter of salt-alkali solution contains 6.88*10 8 Place gauze in the germination device, add 500ml of "dry algae powder-salt-alkali solution", select 10 small rapeseed seeds of the same size and place them in the germination device for hydroponics for 7 days.

[0068] Saline-alkali solution (named CKY group): gauze was placed in the germination device, 500 ml of saline-alkali solution was added, 10 small rapeseed seeds of the same size were selected and placed in the germination device, and hydroponically cultivated for 7 days.

[0069] Purified water (named as CK group): gauze was placed in the germination device, 500 ml of purified water was added, 10 small rapeseed seeds of the same size were selected and placed in the germination device for hydroponics for 7 days.

[0070] The volumes used in the above groups were equal, and 3 parallel experiments were set up for each group.

[0071] Measuring indicators:

[0072] The seed germination rate and emergence rate were recorded every day. The germination rate of rapeseed seeds was recorded every day. Figure 1 As shown, rapeseed seed germination rate-days Figure 2 As shown in Table 4, the data are all averages.

[0073] Table 4

[0074]

[0075]

[0076] Schematic diagram of rapeseed germination or emergence on the 7th day in three parallel experiments of CK, CKY, BA and TA groups in the saline-alkali environment experiment Figure 3 shown.

[0077] from Figure 1 , Figure 2 , Figure 3 It can be concluded from the data in Table 4 that the germination of rapeseed seeds was inhibited in the simulated saline-alkali environment, but the addition of active algae suspension and dry algae powder promoted the germination and emergence of rapeseed seeds.

[0078] (2) Saline-alkali soil environment experiment:

[0079] Active algae suspension-saline-alkali soil (named TA group): In saline-alkali soil, select 10 small rapeseed seeds of the same size and place them in a pot, and evenly apply 100 ml of active algae suspension diluted 20 times, so that each gram of soil contains an average of 1.53*10 6 Dunaliella salina cells were cultured for 7 days.

[0080] Dry algae powder suspension-saline-alkali soil (named BA group): In saline-alkali soil, 10 small rapeseed seeds of the same size were selected and placed in a pot, and 100 ml of dry algae powder suspension diluted 20 times was evenly applied, so that each gram of soil contained an average of 1.53*10 6 Dunaliella salina cells were cultured for 7 days.

[0081] Saline-alkali soil (named CKY group): In saline-alkali soil, 10 small rapeseed seeds of the same size were selected and placed in a pot, 100 mL of pure water was evenly applied, and cultured for 7 days.

[0082] Ordinary soil (named as CK group): In ordinary soil, 10 small rapeseed seeds of the same size were selected and placed in a pot, 100 mL of pure water was evenly applied, and cultured for 7 days.

[0083] The volumes used in the above groups were equal, and 3 parallel experiments were set up for each group.

[0084] Measuring indicators:

[0085] The seed germination rate was recorded every day. Figure 4 As shown in Table 5, all data are averages.

[0086] Table 5

[0087]

[0088] from Figure 4It can be concluded from the data in Table 5 that the emergence of rapeseed seeds is inhibited in the saline-alkali soil environment. The addition of active algae suspension and dry algae powder suspension can significantly promote the emergence speed of rapeseed seeds.

[0089] Experimental Example 2: Experiment on improving the stress resistance of rapeseed in saline-alkali soil environment by using Dunaliella salina.

[0090] 1. Material preparation:

[0091] Experimental object: rapeseed seeds. The rapeseed seeds were sterilized with 20% hydrogen peroxide for 5 minutes, and then washed with pure water for 5 times to wash away the hydrogen peroxide solution.

[0092] Drug configuration:

[0093] (1) Saline solution: Prepare 20 mmol saline solution (NaCl: Na 2 SO 4 :NaHCO 3 :Na 2 CO 3 =1:9:9:1 (molar ratio)).

[0094] (2) Saline-alkali soil: 225 g simulated substrate (nutrient soil and vermiculite volume ratio 1:1), watered with saline-alkali solution three times (interval: wait until the saline-alkali solution is completely penetrated before watering again), 100 ml each time.

[0095] (3) Ordinary soil: 225g simulated matrix (nutrient soil and vermiculite volume ratio 1:1), watered with pure water three times (interval: wait until the saline solution is completely penetrated before watering again), 100ml each time

[0096] 2. Experimental process

[0097] Saline-alkali soil environment:

[0098] Active algae suspension-saline-alkali soil (named TA group): In saline-alkali soil, 10 small rapeseed seeds of the same size were selected and placed in a pot, and 100 ml of active algae suspension diluted 20 times was evenly applied. On average, each gram of soil contained 1.53*10 6 Dunaliella salina cells were cultured for 28 days.

[0099] Dry algae powder suspension-saline-alkali soil (named TA group): In saline-alkali soil, 10 small rapeseed seeds of the same size were selected and placed in a pot, and 100 ml of dry algae powder suspension diluted 20 times was evenly applied. On average, each gram of soil contained 1.53*10 6 Dunaliella salina cells were cultured for 28 days.

[0100] Saline-alkali soil (named CKY group): In saline-alkali soil, 10 small rapeseed seeds of the same size were selected and placed in pots, 100 ml of pure water was evenly applied, and cultured for 28 days.

[0101] Ordinary soil (named as CK group): In ordinary soil, 10 small rapeseed seeds of the same size were selected and placed in a pot, 100 ml of pure water was evenly applied, and cultured for 28 days.

[0102] The volumes used in the above groups were equal, and 3 parallel experiments were set up for each group.

[0103] 3. Measurement indicators:

[0104] After culturing in saline-alkali soil for 28 days, the malondialdehyde (MDA), proline (Pro), superoxide dismutase (SOD), and peroxidase (POD) of rapeseed were measured. Figure 5a As shown in Figure 2, the proline (Pro) content of each group of rapeseed is as follows: Figure 5b As shown in Figure 2, the superoxide dismutase (SOD) content of each group of rapeseed is as follows: Figure 5c As shown in Figure 2, the peroxidase (POD) content of each group of rapeseed is as follows: Figure 5d The contents of malondialdehyde (MDA), proline (Pro), superoxide dismutase (SOD) and peroxidase (POD) in each group of rapeseed are shown in Table 6, where all data are averages.

[0105] Table 6

[0106]

[0107] from Figure 5a-5d From the data in Table 6, it can be concluded that in the saline-alkali soil environment, the proline (Pro) content of rapeseed treated with active algae suspension (TA group) and dry algae powder suspension (BA group) decreased, and the plant salt-alkali stress was weakened. At the same time, the malondialdehyde (MDA) content of rapeseed decreased, the degree of membrane lipid peroxidation decreased, and the overall degree of oxidative stress decreased. This shows that the application of active algae suspension and dry algae powder suspension can increase the content of plant protective enzymes (POD, SOD), which is beneficial for rapeseed to resist oxidative stress.

[0108] The embodiment of the present invention also provides a seaweed fertilizer for improving plant stress resistance, wherein the seaweed fertilizer contains Dunaliella salina. In specific implementation, the Dunaliella salina is present in the seaweed fertilizer in the form of active algae suspension, dry algae powder or dry algae powder algae suspension.

[0109] Beneficial effects of the embodiments of the present invention: The embodiments of the present invention studied the role of Dunaliella salina in improving plant stress resistance and its growth-promoting effect on plants. Dunaliella salina can be used as a potential seaweed fertilizer, which can improve plant stress resistance and promote plant growth in saline-alkali environments. Two different production types (active algae suspension and dry algae powder) of Dunaliella salina can improve the seed germination rate, emergence speed, and stress resistance of rapeseed. However, compared with the active algae suspension, the cost of dry algae powder is relatively high due to the additional operation step of "drying". The research of the present invention provides technical support for crop planting, especially rapeseed planting, in saline-alkali environments.

[0110] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art of the present invention, several equivalent substitutions or obvious variations can be made without departing from the concept of the present invention, and the performance or use is the same, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. An application of marine microalgae in improving plant stress resistance, characterized in that: The plant is rapeseed, the stress resistance refers to salt-alkali resistance, and the marine microalgae is Dunaliella salina, which is applied to the planting environment where the plant is located to improve the seed germination rate and emergence speed of rapeseed and the salt-alkali resistance of rapeseed, wherein when the planting environment is saline-alkali solution, the Dunaliella salina is applied to the saline-alkali solution in the form of at least one of active algae suspension, dry algae powder and dry algae powder algae suspension; when the planting environment is saline-alkali soil, the Dunaliella salina is applied to the saline-alkali soil in the form of active algae suspension and / or dry algae powder algae suspension; The active algae suspension is prepared by inoculating Dunaliella salina into artificial seawater with a salinity of 35‰, culturing for 20 to 30 days, centrifuging at 4000 to 6000 r, washing with purified water for 3 to 4 times, and then adding a predetermined amount of purified water; The dry algae powder is prepared by inoculating Dunaliella salina into artificial seawater with a salinity of 35‰, culturing for 20 to 30 days, centrifuging at 4000 to 6000 r, washing with pure water for 3 to 4 times, and drying in an oven at 30 to 40°C. The dry algae powder algae suspension is prepared by suspending the dry algae powder in pure water; The salinity of the artificial seawater is provided by coral sea salt; The improvement of plant stress resistance is achieved by increasing the activity of antioxidant enzymes in plants to activate the antioxidant system in the plants.

2. The use according to claim 1, characterized in that The planting environment is saline-alkali solution, each liter of which contains 10 8 ~10 9 Dunaliella salina algae cells.

3. The use according to claim 1, characterized in that The planting environment is saline-alkali soil, and each gram of saline-alkali soil contains 10 6 ~10 7 Dunaliella salina algae cells.

4. The use according to claim 2 or 3, characterized in that: The Dunaliella salina is cultured in f / 2-Si culture medium under the following culture conditions: light intensity of 4000±500 lux, and a light cycle to light-dark cycle ratio of 14h:10h.

5. The use according to claim 4, characterized in that: The f / 2-Si culture medium includes 8.82×10 -4 M NaNO3, 3.62×10 -5 M of NaH2PO4·H2O, trace metal element mother solution and vitamin mother solution, wherein the trace metal element mother solution includes 1.17×10 -5 M of FeCl3·6H2O, 1.17×10 -5 M Na2EDTA·2H2O, 3.93×10 -8 M CuSO4·5H2O, 2.60×10 -8 M Na2MoO4·2H2O, 7.65×10 -8 M ZnSO4·7H2O, 4.20×10 -8 M CoCl2·6H2O and 9.10×10 -7 M of MnCl2·4H2O, the vitamin mother solution includes 2.96×10 -7 Vitamin B1, 2.05×10 -9 M of vitamin H and 3.69×10 -10 Vitamin B 12 .

6. The use according to claim 1, characterized in that: The antioxidant enzyme is at least one of peroxidase and superoxide dismutase.

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