Method for relieving salt stress effect of ricinus communis by using arbuscular mycorrhizal fungi and application

Abstract

The invention discloses a method for relieving castor salt stress by arbuscular mycorrhizal fungi and application, and belongs to the technical field of plant protection. The arbuscular root fungus is a mixed strain of glomus mosseae enzyme and rhizopus intraradices according to a spore ratio of 1: 1. Experiments prove that inoculation of arbuscular mycorrhizal fungi can effectively relieve damage of salt stress to castor-oil plants, the net photosynthetic rate, the stomatal conductance, the intercellular CO2 concentration, the transpiration rate and the actual photochemical efficiency are remarkably increased by improving the activity of antioxidant enzyme and promoting the content of proline, soluble sugar and soluble protein, and the salt stress resistance of the castor-oil plants is improved. The arbuscular mycorrhizal fungi are inoculated, the decreasing amplitude of a non-photochemical quenching coefficient is reduced, the photosynthetic structure is protected, and the photosynthetic rate is improved, which indicates that the arbuscular mycorrhizal fungi are inoculated to effectively relieve the damage of salt stress to the castor, improve the salt tolerance of the plant and solve the key growth problem of the castor caused by soil salinization; in addition, no potential safety hazard and risk of mutation of germplasm genes exist in the treatment process, the safety of prevention and control measures is greatly improved, and the method is a green, safe and environment-friendly method for relieving the harm of salt stress to the castor.

Description

Technical Field

[0001] The invention relates to the technical field of plant protection, and in particular to a method and application of alleviating castor oil salt stress by utilizing arbuscular mycorrhizal fungi. Background Art

[0002] Soil salinization has become an increasingly common and serious ecological problem, which not only harms agricultural production and grassland ecosystems, but also inhibits global plant growth and productivity. With the over-exploitation of natural resources by humans, soil salinization has become more and more serious, and soil environmental problems are particularly prominent, so food security is also threatened. my country's saline-alkali land is mainly distributed in 23 provinces, municipalities, and autonomous regions, covering an area of ​​about 6.67 million hectares. Among them, the saline-alkali soil in Northeast my country is characterized by high content of NaHCO3 and Na2CO3. In addition to being rich in high content of Na + In addition, it will also cause a high pH value, which is more harmful to plants, which greatly limits the application of vegetation in the Northeast. At present, how to utilize saline soil has become a difficult problem for environmental protection. The utilization of saline soil mainly includes two aspects: ① Reducing soil salinity to a level that plants can adapt to; ② In order to better adapt to the salt in the soil, improve the salt tolerance of plants. Planting salt-tolerant plants on saline soil can reduce the soil salt concentration to a certain extent, and improve the physical and chemical properties of saline soil by using plant root growth. Similarly, improving plant salt tolerance also has positive significance. Therefore, conducting research on the mechanism of plant salt tolerance and improving plant salt tolerance can provide an important theoretical basis for the rational development and utilization of saline soil.

[0003] Castor (Ricinus communis L.) is an annual or perennial herb of the genus Ricinus in the Euphorbiaceae family. It has strong adaptability, tolerance to barrenness, and tolerance to salinity and alkali. It can grow well in relatively harsh environments or severely polluted conditions, and shows good adaptability and tolerance to polluted environments. With the advancement of science and technology, the comprehensive utilization of castor has been continuously deepened, and the research on castor in ecological management has also developed from simple and low-efficiency to multi-level and high-efficiency, especially the research on its ecological adaptability and stress resistance mechanism under salt stress environment has been continuously deepened.

[0004] Arbuscular mycorrhiza fungi (AMF) has been widely concerned as an important plant biocontrol fungus. Mycorrhiza is a very common and important symbiotic phenomenon in nature. It is widely present in farmland soil and can form symbiotic structures with a variety of plants, accounting for 9%-55% of the total soil microorganisms. The formation of most arbuscular mycorrhizae is beneficial to the plant's nutrient absorption in the soil, promoting plant growth, and improving plant disease resistance. Therefore, it is extremely necessary to develop a method and application of using arbuscular mycorrhizae to alleviate the salt stress of castor oil.

[0005] After searching the literature, no publicly reported literature on the mechanism of symbiosis between castor and arbuscular mycorrhiza to alleviate salt stress has been retrieved. Therefore, using microorganisms to regulate plant growth and improve its stress resistance and nutrient absorption has the characteristics of low cost, simple operation, green and environmental protection, good economic benefits and relatively broad application prospects. The results obtained in this study are conducive to further revealing the salt tolerance mechanism of castor, strengthening the comprehensive development of castor, and making it better used in the utilization and improvement of saline soil, which has important economic, social and ecological benefits. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a method and application of using arbuscular mycorrhizal fungi to alleviate the salt stress of castor. The technical solution provided by the present application is used to promote the growth of castor plants under salt stress and improve the stress resistance.

[0007] In order to solve the above technical problems, the present invention provides a method and application of using arbuscular mycorrhizal fungi to alleviate castor salt stress. The arbuscular mycorrhizal fungi are mixed strains of Glomerella mosseae and Rhizocystis endoradiata in a spore ratio of 1:1.

[0008] Furthermore, the application includes the following steps: the test soil is a mixture of farmland soil and fine sand (soil: sand = 3: 1, v: v). Before the test, the soil and fine sand are sterilized to eliminate fungal spores in the soil.

[0009] Furthermore, each pot was inoculated with 30 g of inoculant or water filtrate of sterilized and non-sterilized inoculants by equal weight at 2 cm below castor seeds to ensure that the soil nutrient status and microbial flora of the two treatment groups were highly consistent.

[0010] The second object of the present invention is to provide a method and application of a mixed strain of arbuscular mycorrhizal fungi in alleviating the salt stress of castor oil plant. The mixed strain of arbuscular mycorrhizal fungi is a mixture of Funneliformis mosseae and Rhizophagus intraradices in a spore ratio of 1:1.

[0011] Compared with the prior art, the present invention has the following advantages and effects:

[0012] The invention inoculates the castor seedling root system with mixed strains of arbuscular mycorrhizal fungi (Glomus mosesi and Rhizocystis endoradicis mixed in a spore ratio of 1:1), and finds that the inoculation of arbuscular mycorrhizal fungi can effectively alleviate the damage of salt stress to castor, improve the salt tolerance of plants, and solve the key growth problem of castor caused by soil salinization; and the mixed strains of arbuscular mycorrhizal fungi, as a highly efficient and safe biological fertilizer, have a significant improvement effect on the growth and development of castor seedlings, and as a renewable and sustainable resource, its ecological economic benefits and environmental sustainable effects are better than other biological fertilizers. Inoculation of arbuscular mycorrhizal fungi shows good application effects in castor cultivation, such as convenient operation, strong operability, small dosage, simple steps, low cost, quick effect, safety and reliability, etc., and has broad prospects in the cultivation and application of castor and other plants. The safety of prevention and control measures is greatly improved, and it is a green, safe and environmentally friendly method for reducing the harm of salt stress to castor. DETAILED DESCRIPTION

[0013] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] In the following examples, the instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels. The methods described are all conventional methods unless otherwise specified, and the raw materials used can be obtained from public commercial channels unless otherwise specified.

[0015] 1. Materials and Methods

[0016] Plant material and treatment

[0017] Experimental materials: The plant material for the test is castor 'Fenbi No. 10'. Select seeds with full grains and complete structure, soak them in 0.5% sodium hypochlorite (NaClO) solution for 10 minutes for disinfection, then rinse the seed surface with sterile distilled water, wipe off the excess water on the seed surface with absorbent tissue after rinsing, and dry them for later use.

[0018] Soil selection: The test soil is a mixture of farmland soil and fine sand (soil: sand = 3:1, v:v). Filter with a 5 mm sieve. Before the test, the soil and fine sand were sterilized with high pressure steam to eliminate fungal spores in the soil.

[0019] Pot test: Select the treated soil and put it into plastic pots of uniform specifications according to the same mass. Inoculate 30g of fungicide or add the water filtrate of sterilized inoculum and non-sterilized inoculum of equal weight in each pot 2cm below the castor seeds to ensure that the soil nutrition status and microbial community of the two groups are highly consistent. The whole test was carried out in a greenhouse with an average temperature of 25℃ and a light time of 12h. In order to avoid mutual influence between the roots of the seedlings and ensure emergence, 3 castor seeds were sown in the middle of each plastic pot and watered with an equal amount of distilled water every day.

[0020] Sixth week after sowing, 150 mM salt stress treatment was started. 300 mL of irrigation was applied daily to keep the soil under 150 mM salt stress. The stress was continued for one week and a control was set up. There were a total of 4 treatments, namely, plants grown under normal soil conditions (NS); plants inoculated with arbuscular mycorrhizal fungi and grown under normal soil conditions (NS+AMF); plants grown under salt stress soil conditions (SS); plants inoculated with arbuscular mycorrhizal fungi and grown under salt stress soil conditions (SS+AMF).

[0021] Castor samples were sampled 24 hours after salt stress treatment. All samples were frozen in liquid nitrogen immediately after measurement and then stored at -80℃ for future use.

[0022] Gas exchange parameters determination:

[0023] Four to five fully expanded leaves were randomly selected from each pot of plants, and the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs) and intercellular carbon dioxide concentration (Ci) of the first fully expanded functional leaf from the top of the plants downward were measured using the Li-6400 (LI-COR, USA) portable photosynthesis measurement system at a light intensity of 1000 μmol·m-2·s-1 and a leaf chamber temperature controlled at 25°C.

[0024] Calculation of water use efficiency:

[0025] The water use efficiency here refers to the amount of CO2 assimilated by plants or leaves for each amount of water transpired, that is, the ratio of photosynthetic rate to transpiration rate, expressed in μmolCO2·mmol -1 H2O means.

[0026] Determination of fluorescence parameters:

[0027] The fluorescence parameters of the first fully expanded functional leaf from the top of the plant were measured using a portable fluorescence instrument FMS2 produced by Hansatech, UK. Four plants were selected for each measurement, and each plant was measured three times. The experimental data were tabulated in average values.

[0028] Osmotic regulating substances determination

[0029] The proline content was determined by ninhydrin colorimetry. Under stress conditions, MDA is a lipid peroxidation product, and its content is often used to explain the degree of peroxidation. The malondialdehyde (MDA) content was determined by thiobarbituric acid method. The soluble sugar content was determined by anthrone colorimetry. The soluble protein content was determined by Coomassie brilliant blue staining.

[0030] Antioxidant enzyme activity assay:

[0031] Superoxide dismutase (SOD) was determined by the nitroblue tetrazolium (NBT) method, peroxidase (POD) was determined by the guaiacol method, catalase (CAT) was determined by the hydrogen peroxide method, and ascorbate peroxidase (APX) was determined by a specific kit.

[0032] Determination of inorganic ion content:

[0033] The samples were measured by inductively coupled plasma optical emission spectrometry (ICP).

[0034] Data analysis

[0035] Excel was used for data processing, SPSS25.0 was used for variance analysis, and p-value less than 0.05 was considered significant. Excel2016, Origin2018 and R-4.0.3 were used for drawing. Variance analysis was used for statistical analysis, followed by Duncan's multivariate comparison test. The superscript lowercase letters of photosynthetic parameters between different samples were significantly different (p<0.05).

[0036] 2. Results and Analysis

[0037] Effects of arbuscular mycorrhizal fungi on the photochemical efficiency of Ricinus communis under salt stress

[0038] The effects of inoculation of arbuscular mycorrhizal fungi on the photosynthetic parameters and chlorophyll fluorescence parameters of castor under salt stress are shown in Table 1. Salt stress can reduce the photosynthesis of castor, while inoculation of arbuscular mycorrhizal fungi can significantly increase the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), actual photochemical efficiency (ФPSII) and photochemical quenching coefficient (qP) of castor, reduce the non-photochemical quenching coefficient (NPQ) of castor, and alleviate the decline in castor photosynthesis caused by salt stress.

[0039] Compared with the control group, inoculation of arbuscular mycorrhizal fungi under salt stress can increase the Pn, Gs, Ci, and Tr of castor, which are increased by 0.40, 0.49, 0.06, and 1.00 times, respectively. Chlorophyll fluorescence parameters can well reflect photosynthetic efficiency, especially ФPSII, and inoculation of arbuscular mycorrhizal fungi can increase the ФPSII of castor, which can significantly increase by 78.26% under normal soil conditions and 59.01% under salt stress, effectively alleviating the negative impact of salt stress. In addition, inoculation of arbuscular mycorrhizal fungi can also increase the quantum yield (Fv / Fm) and qP of the maximum PSII of castor under salt stress, which are increased by 1.64% and 72.22%, respectively. It has a certain promoting effect on the reduction of NPQ, which is reduced by 34.55% under normal soil conditions and 15.83% under salt stress. The above results showed that the photosynthesis and gas exchange rates were weakened under salt stress, but they could be alleviated by inoculating arbuscular mycorrhizal fungi, which stabilized the effects of salt stress on the photosynthetic parameters and chlorophyll fluorescence parameters of castor oil plant.

[0040] Table 1 Effects of arbuscular mycorrhizal fungi on the photochemical efficiency of Ricinus communis under salt stress

[0041]

[0042]

[0043] Note: Different lowercase letters in the same index indicate significant differences at the P < 0.05 level

[0044] Note:Different lowercase letters under the same index indicate significant difference at the P<0.05level

[0045] Effects of Arbuscular Mycorrhizal Fungi on Osmotic Regulation of Ricinus communis Under Salt Stress

[0046] In order to quantify the effect of salt stress on castor biochemical indicators, the content of proline (Pro), malondialdehyde (MDA), soluble sugar (SS), and soluble protein (SP) were further measured and analyzed (Table 2). Compared with the control group, salt stress increased the content of MDA and decreased the content of Pro, SS, and SP, causing secondary oxidative stress and oxidative damage to castor. Arbuscular mycorrhizal fungi alleviated the oxidative damage of castor by significantly increasing the content of Pro, SS, and SP and significantly reducing the content of MDA.

[0047] When plants are subjected to stress, they will produce a large number of superoxide free radicals, which will cause membrane lipid peroxidation and produce MDA. Excessive accumulation of MDA will cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, leading to changes in the structure and function of cell membranes. Therefore, membrane lipid peroxidation is an important sign of plant cell membrane damage. Inoculation with arbuscular mycorrhizal fungi can significantly reduce the MDA content of castor under salt stress, which is 20.75% and 19.95% lower than that without inoculation, respectively, reducing the oxidative damage of castor. The accumulation of osmotic regulating substances is one of the important mechanisms for plants to survive under adversity. Inoculation with arbuscular mycorrhizal fungi can increase the Pro, SS and SP contents of castor, which are significantly increased by 19.01%, 30.67% and 2.12% respectively under normal soil conditions, and by 18.58%, 78.41% and 12.70% respectively under salt stress, effectively alleviating the negative impact of salt stress.

[0048] Table 2 Effects of arbuscular mycorrhizal fungi on osmotic regulating substances in castor oil plant under salt stress

[0049]

[0050]

[0051] Note: Different lowercase letters in the same index indicate significant differences at the P < 0.05 level

[0052] Note:Different lowercase letters under the same index indicate significant difference at the P<0.05level

[0053] Effects of arbuscular mycorrhizal fungi on antioxidant enzyme activities in Ricinus communis under salt stress

[0054] When plants are subjected to abiotic stress, the metabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is changed. In the early stage of stress, low concentrations of ROS and RNS can act as signal molecules to participate in the regulation of plant growth, development, and stress response processes. As the stress continues, high concentrations of ROS and RNS accumulate, disrupting the redox homeostasis in plant cells and breaking the original dynamic balance in the body. Due to the accumulation of a large amount of reactive oxygen species in the cells, oxygen stress is generated, which triggers irreversible oxidative damage to macromolecules such as lipids, proteins, and nucleic acids, destroys the metabolic process, and ultimately leads to plant cell death. Therefore, maintaining intracellular redox homeostasis is crucial to improving plant stress resistance. In order to remove excess reactive oxygen, the antioxidant enzyme system needs to be activated to regulate the content of enzymes and non-enzymes to a balanced state. The activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) were further measured and analyzed (Table 3).

[0055] The effects of arbuscular mycorrhizal fungi on the antioxidant enzyme activities of castor under salt stress are shown in Table 3. Compared with the control group, inoculation with arbuscular mycorrhizal fungi increased the activities of CAT, SOD, POD and APX by 9.53%, 14.95%, 39.15% and 10.31% respectively under normal soil conditions, and by 8.59%, 22.27%, 17.51% and 55.07% respectively under salt stress, reducing the oxidative damage of castor. The results show that inoculation with arbuscular mycorrhizal fungi increased the antioxidant enzyme activities of castor, thereby enhancing its antioxidant capacity to cope with salt stress. Inoculation with arbuscular mycorrhizal fungi can enhance the antioxidant defense system of castor and effectively reduce the degree of oxidative damage caused by salt stress.

[0056] Table 3 Effects of arbuscular mycorrhizal fungi on antioxidant enzyme activities of Ricinus communis under salt stress

[0057]

[0058]

[0059] Note: Different lowercase letters in the same index indicate significant differences at the P < 0.05 level

[0060] Note:Different lowercase letters under the same index indicate significant difference at the P<0.05level

[0061] Effects of inoculation with arbuscular mycorrhizal fungi on inorganic ion content in castor oil plants under salt stress

[0062] Inorganic ion content plays an important role in the salt tolerance mechanism of crop osmotic regulation. For example, potassium ions (K + ) is the most abundant cation in plants. It is not only a macronutrient necessary for plant growth and development, but also plays an important role in regulating ion balance, osmotic regulation, maintaining cell turgor pressure and protein synthesis. 2+ ) is not only important for maintaining the integrity and stability of plant cell membranes, but also has an important relationship with the salt tolerance of plants. Under salt stress, inoculation of arbuscular mycorrhizal fungi can significantly reduce the sodium ion (Na + ) content, increasing K + , phosphorus ion content, reduce ion toxicity, and alleviate the negative impact of salt stress on plant growth.

[0063] The effects of inoculation with arbuscular mycorrhizal fungi on the inorganic ion content of castor beans under salt stress are shown in Table 4. Compared with the control group, inoculation with arbuscular mycorrhizal fungi can significantly reduce the content of Na in castor beans. + The content of castor oil can be reduced by 34.27% under normal soil conditions and by 39.89% under salt stress. In addition, inoculation of arbuscular mycorrhizal fungi significantly increased the K content of castor oil under salt stress. + , and phosphorus ion content increased by 21.85% and 11.78% respectively.

[0064] Table 4 Effects of arbuscular mycorrhizal fungi on inorganic ion content of castor oil plant under salt stress

[0065]

[0066]

[0067] Note: Different lowercase letters in the same index indicate significant differences at the P < 0.05 level

[0068] Note:Different lowercase letters under the same index indicate significant difference at the P<0.05level

[0069] Summarize

[0070] In the present study, inoculation of arbuscular mycorrhizal fungi under salt stress effectively improves the electron cycle transfer rate, fluorescence quantum yield and photosynthetic rate in the photosystem, and helps CO2 accumulation. The salt tolerance of castor is improved by increasing the content of proline, soluble sugar, soluble protein and the activity of antioxidant enzymes (CAT, SOD, POD and APX). The oxidative damage of castor caused by salt stress is alleviated by reducing the MDA content. The osmotic regulation ability of castor is enhanced by adjusting the inorganic ion content, reducing ion toxicity, and alleviating the negative impact of salt stress on plant growth.

[0071] The results of the study showed that inoculation with arbuscular mycorrhizal fungi can improve the salt tolerance of castor, which is helpful to strengthen the comprehensive development of castor and make it better applied in the utilization and improvement of saline soil. As a highly efficient and safe biological fertilizer, the mixed strains of arbuscular mycorrhizal fungi have a significant effect on the growth and development of castor seedlings. As a renewable and sustainable resource, its eco-economic benefits and environmental sustainability are better than other biological fertilizers.

[0072] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method and application of using arbuscular mycorrhizal fungi to alleviate castor oil salt stress, characterized in that The mixed species of arbuscular mycorrhizal fungi include Funneliformis mosseae and Rhizophagus intraradices, which are mixed in a spore ratio of 1:

1.

2. The method and application of using arbuscular mycorrhizal fungi to alleviate castor oil salt stress according to claim 1, characterized in that , which was achieved by the following method: the test soil was a mixture of farmland soil and fine sand (soil: sand = 3:1, v:v). Before the test, the soil and fine sand were sterilized to eliminate fungal spores in the soil.

3. The use of arbuscular mycorrhizal fungi in the treatment of castor oil plant against salt stress according to claim 2, characterized in that : Each pot was inoculated with 30 g of fungicide or water filtrate of sterilized inoculum and non-sterilized inoculum at 2 cm below the castor seeds to ensure that the soil nutritional status and microbial community of the two groups of treatments were highly consistent.

4. The method and application of arbuscular mycorrhizal fungi in alleviating salt stress of castor oil plant according to any one of claims 2 to 3, characterized in that The arbuscular mycorrhizal fungal agent can enhance the photosynthesis capacity of castor-oil plant under salt stress conditions.

5. The method and application of mycorrhizal fungi in alleviating salt stress of castor oil plant according to any one of claims 2 to 3, characterized in that The arbuscular mycorrhizal fungal agent can enhance the antioxidant capacity of castor under salt stress conditions.

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