Rhizosphere polysaccharides, biostimulants and their applications

Abstract

The present invention relates to the field of agricultural technology. The present invention provides a rhizosphere polysaccharide, a biostimulant and applications thereof. In particular, a rhizosphere polysaccharide having a structure of formula (I) is provided. The biostimulant prepared from the rhizosphere polysaccharide provided by the present invention can stimulate the secretion of endogenous hormones in crops, improve the resistance of crops to biological and abiotic stresses, promote crop germination and root growth, and increase crop yield; formula (I).

Description

Technical Field

[0001] The invention relates to the technical field of agriculture, and in particular to rhizosphere polysaccharide, biostimulant and application thereof. Background Art

[0002] Biostimulants are a type of product that stimulates the process of plant absorption and utilization of nutrients. Their purpose is to improve one or more aspects of the characteristics of plants or their root systems: 1. Nutrient utilization efficiency; 2. Resistance to abiotic stress; 3. Quality traits; 4. Availability of limited nutrients in the soil or rhizosphere. Biostimulants are neither fertilizers nor pesticides, but they can play a special role and value in plant growth. They enhance the innate immunity and induce resistance of plants, thereby reducing the occurrence of diseases and pests and enhancing the ability of plants to resist adverse climatic environments, thereby reducing the application of pesticides and fertilizers, ensuring healthy plant growth, and promoting high-quality and sustainable agricultural development.

[0003] The prior art discloses that CN114111856 discloses propyl dihydrojasmonate (PDJ) and DCPTA as biostimulants, both of which have the function of regulating plant growth. However, according to market feedback, the application of colorless or light yellow biostimulants has a more obvious effect, while the application of brown or black biostimulants has no obvious effect or even no effect, which affects the promotion of the product.

[0004] Therefore, it is very necessary to provide a biostimulant with good effect and good safety. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a rhizosphere polysaccharide. The biostimulant prepared from the rhizosphere polysaccharide provided by the present invention can stimulate the secretion of endogenous hormones in crops, improve the resistance of crops to biotic and abiotic stresses, promote crop germination and root growth, and increase crop yield.

[0006] The present invention provides a rhizosphere polysaccharide having a structure of formula (I),

[0007] Formula (I);

[0008] Here, n is the degree of polymerization, which ranges from 5 to 6.

[0009] The molar ratio of fructose, mannose, xylose and glucose in the rhizosphere polysaccharide of the present invention is 1: (0.16-0.18): (0.16-0.18): (0.01-0.02).

[0010] In some specific embodiments, the molar ratio of fructose, mannose, xylose and glucose in the rhizosphere polysaccharide of the present invention is 1: 0.17: 0.17: 0.02. In some specific embodiments, the molar ratio of fructose, mannose, xylose and glucose in the rhizosphere polysaccharide of the present invention is 1: 0.17: 0.17: 0.015.

[0011] The weight average molecular weight of the rhizosphere polysaccharide of the present invention is 15.43 kDa.

[0012] The method for extracting rhizosphere polysaccharides provided by the present invention comprises:

[0013] A) inoculating rhizosphere bacteria in sugarcane root exudates into the reaction solution to obtain a biological reaction solution;

[0014] B) mixing the bioreactor solution and water, centrifuging to obtain a supernatant; adding ethanol to the supernatant for precipitation to obtain a filter residue;

[0015] C) washing the filter residue with water, and then removing protein with Sevage reagent to obtain an upper layer solution;

[0016] D) After concentrating the upper layer solution, dialyze to obtain the dialysate in the dialysis bag;

[0017] E) The dialyzate is precipitated with ethanol, chromatographically separated and eluted to obtain rhizosphere polysaccharides.

[0018] The method for extracting rhizosphere polysaccharides provided by the present invention first inoculates rhizosphere bacteria in sugarcane root secretions into a biological reaction liquid, and performs biological reaction to obtain the biological reaction liquid.

[0019] The preferred method is as follows: adding rhizosphere bacteria in sugarcane root exudates into a bioreactor containing a bioreactor solution, and reacting at 30° C. for 3 days to obtain a bioreactor solution containing rhizosphere polysaccharides.

[0020] The reaction of the present invention is preferably carried out in a bioreactor, which is not limited in the present invention, and any bioreactor well known to those skilled in the art can be used.

[0021] The bioreactor liquid of the present invention comprises: sugarcane fruit powder, sugarcane peel powder, sugarcane rhizome powder and plant protein powder; the mass ratio of the sugarcane fruit powder, sugarcane peel powder, sugarcane rhizome powder and plant protein powder is 50:5:5:10; the pH value of the bioreactor liquid is adjusted to 7.0.

[0022] In some specific embodiments, the bioreactor solution includes: 50 g / L sugarcane fruit powder, 5 g / L sugarcane peel powder, 5 g / L sugarcane rhizome powder, 10 g / L plant protein powder, and pH=7.0.

[0023] The biological reaction temperature is 30° C. and the reaction time is 2 to 4 days, preferably 3 days.

[0024] The bioreactor liquid is mixed with water and centrifuged to obtain a supernatant. The water is preferably deionized water; the volume ratio of the bioreactor liquid to water is 1:(2-3); the centrifugal speed is 5000-8000 rpm, specifically 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm; or a range between any two of the above; the time is 2-5 min; specifically 2 min, 3 min, 4 min or 5 min; or a range between any two of the above;

[0025] The supernatant is added with ethanol for precipitation to obtain a filter residue. Preferably, 95% ethanol is added to each supernatant, stirred in a reaction kettle at room temperature, allowed to stand, filtered, the filtrate is discarded, and the filter residue is retained.

[0026] Wherein, the ethanol is 95% ethanol; the volume ratio of the supernatant to the ethanol is 1:(2-3); the stirring time at room temperature is 0.5h; the standing specifically includes: standing at 5-10°C for 4-8 hours; the filtration preferably includes: filtering with a 50-200 mesh filter; specifically, it can be 50 mesh, 80 mesh, 100 mesh, 120 mesh, 150 mesh, 180 mesh or 200 mesh. Or a range between any two of the above;

[0027] The filter residue is washed with water; preferably, the process includes: mixing the filter residue and water, stirring at room temperature, filtering, discarding the filter residue, and retaining the filtrate. The mass ratio of the filter residue to water is 1:(2~3). The stirring time at room temperature is 0.5h; the filter screen is 50~200 mesh. Specifically, it can be 50 mesh, 80 mesh, 100 mesh, 120 mesh, 150 mesh, 180 mesh or 200 mesh. Or a range value between any two of the above;

[0028] The filtrate and the Sevage reagent were mixed, stirred at room temperature, and the upper solution was separated; the mass ratio of the filtrate and the Sevage reagent was 1:1; and the stirring time at room temperature was 0.5 h.

[0029] The upper layer solution is concentrated; the concentration is preferably reduced pressure concentration; the parameters of the reduced pressure concentration include:

[0030] 60~70℃, -0.08Mpa.

[0031] After concentration, dialyzation is performed to obtain a dialysate in a dialysis bag; the molecular cutoff of the dialysis bag is 1000 Daltons; the dialysis bag is placed in ultrapure water and stirred for 12 hours, and then the dialysate in the dialysis bag is taken out.

[0032] The dialysate is precipitated with ethanol; ethanol is added to the dialysate, stirred, centrifuged, the supernatant is discarded, the precipitate is collected, and the precipitate is frozen or dried at room temperature to obtain white crystals.

[0033] The ethanol is 95% ethanol; the volume ratio of the dialysate to the ethanol is 1:(2-3); the stirring time at room temperature is 1-2 minutes; the centrifugal speed is 6000 rpm for 1 minute.

[0034] The product is chromatographed and eluted using a chromatography column to obtain rhizosphere polysaccharide.

[0035] The chromatography column of the present invention is diethylaminoethyl cellulose.

[0036] First, pre-treat the chromatography column: add diethylaminoethyl cellulose to ultrapure water, stir well and let it stand at room temperature for 6 to 12 hours to swell. Use wet column packing (20×400 mm), and then use acid water-ultrapure water-alkaline water-deionized water to flush the column in sequence until the eluent is neutral, and the pre-treatment of the chromatography column can be completed. The present invention does not limit the specific operation of flushing the column with acid water-ultrapure water-alkaline water-deionized water, which can be well known to those skilled in the art.

[0037] The above product (white crystals) was added with water to form an aqueous solution, and the sample was chromatographed; the eluent was a NaCl solution; the concentration of the NaCl solution was 0.1 M. That is: 0 M, 0.1 M, 0.2 M, and 0.3 M NaCl solutions were used for elution in sequence, and they were numbered in the order of collection, and the absorbance of each eluate at a wavelength of 490 nm was detected by the phenol-sulfuric acid method; it was shown that elution with 0.1 M NaCl solution could obtain a rhizosphere polysaccharide component with an absorbance of 98.1%.

[0038] The method also includes adding 95% ethanol to precipitate the rhizosphere polysaccharide component and drying it to obtain rhizosphere polysaccharide.

[0039] The ethanol is 95% ethanol; the volume ratio of the rhizosphere polysaccharide solution to ethanol is 1:(2~3); the stirring time at room temperature is 1~2 minutes; then centrifugation is performed at 6000 rpm at room temperature for 1 minute, the supernatant is discarded, the precipitate is collected, and the rhizosphere polysaccharide is obtained after the precipitate is frozen or dried at room temperature.

[0040] The present invention provides a biostimulant, comprising the rhizosphere polysaccharide described in any one of the above technical solutions or the rhizosphere polysaccharide extracted by the extraction method described in any one of the above technical solutions.

[0041] In the present invention, the weight percentage of rhizosphere polysaccharide in the biostimulant is 0.0025% to 100%.

[0042] According to the present invention, the dosage form of the biostimulant includes granules, powders, liquids or seed coatings.

[0043] Specifically,

[0044] The biostimulant granules include 60% to 100% rhizosphere polysaccharide and a granule carrier; specifically, it can be 60%, 70%, 80%, 90% or 100%; the granule carrier is selected from one or more of lignin sulfonate, urea, and potassium phosphate;

[0045] The biostimulant powder comprises 60% to 98% rhizosphere polysaccharide and a powder carrier; specifically, it may be 60%, 70%, 80%, 90% or 98%; the powder carrier is selected from one or more of rice husk powder, kaolin, diatomaceous earth, plant ash and peat soil;

[0046] The liquid biostimulant comprises 0.0025% to 50% rhizosphere polysaccharide and a liquid carrier; specifically, it may be 0.0025%, 0.1%, 1%, 10% or 50%; the liquid carrier is selected from one or more of brassinolide, coronatine, urea, amino acid, phosphate potassium salt, and deionized water;

[0047] The biostimulant seed coating agent includes 0.02%~0.12% rhizosphere polysaccharide and a seed coating agent carrier; specifically, it can be 0.02%, 0.05%, 0.08%, 0.1% or 0.12%; the seed coating agent carrier is selected from one or more of trehalose (alginate), xanthan gum, hydroxymethyl cellulose, coronatine, fludioxonil, and rose red pigment.

[0048] The present invention provides the use of the above-mentioned biostimulant in improving plant stress resistance; the stress resistance includes resistance to salinity, resistance to low temperature, resistance to drought and / or resistance to diseases and insect pests.

[0049] The improvement of stress resistance of the present invention includes promoting the transcription level of the corresponding genes of endogenous salicylic acid and jasmonic acid, and producing a synergistic effect with probiotics. The synergistic effect is an effect in pest resistance. For example, the rhizosphere polysaccharide of the present invention can induce Bacillus thuringiensis to wrap the cucumber root system and kill the root-knot nematodes near the root system.

[0050] The probiotics include but are not limited to Bacillus thuringiensis.

[0051] The method of improving stress resistance of the present invention includes improving the germination rate of seeds under stress of salinity, low temperature, drought and other adverse conditions.

[0052] The salicylic acid and jasmonic acid described in the present invention are salicylic acid and jasmonic acid in protoplast mesophyll cells.

[0053] According to the present invention, the pests include pests caused by nematodes and / or diseases caused by Pseudomonas. The nematodes include root-knot nematodes.

[0054] The Pseudomonas include but are not limited to Pseudomonas olivetica, Pseudomonas syringae, Pseudomonas savastanoi, Pectobacterium carotovorum, and Pseudomonas syringae.

[0055] In summary, the biostimulant prepared from the rhizosphere polysaccharide of the present invention can be used for seed dressing to improve the germination rate of plants under adverse stresses such as salinity, low temperature, and drought; can be used for induction to stimulate the transcription of salicylic acid / jasmonic acid endogenous hormone response genes in plant protoplast mesophyll cells; can be used for leaf spraying to improve the resistance of plants to diseases such as Pseudomonas olive cancer; and can be used for root irrigation to improve the control effect of Bacillus thuringiensis in the rhizosphere of plants on root-knot nematodes and other pests.

[0056] The present invention also provides a method for improving plant stress resistance, which comprises applying the biostimulant described in the above technical solution.

[0057] The present invention has clearly described the above biostimulants, which will not be repeated here, as they are well known to those skilled in the art.

[0058] The application methods of the present invention include but are not limited to root irrigation, seed dressing, and foliar spraying.

[0059] The plant described in the present invention is a monocotyledon and / or a dicotyledon. The monocotyledon is a Gramineae plant, and the dicotyledon is a Leguminosae plant, a Cucurbitaceae plant and / or a Solanaceae plant.

[0060] The grass plants include but are not limited to rice, corn and / or wheat, oats, quinoa, and highland barley.

[0061] The cucurbitaceae plants include but are not limited to watermelon, melon, cucumber, sponge gourd, pumpkin, bitter gourd, sponge gourd, wax gourd,

[0062] The Solanaceae plants include but are not limited to eggplant, tomato, and tobacco.

[0063] The legumes include but are not limited to soybeans, mung beans, peas, broad beans, peanuts (groundnuts), cowpeas, lentils, green beans, soybeans, and red beans (adzuki beans).

[0064] The dosage of the biostimulant for foliar spraying of the present invention is 10-100 mL / plant.

[0065] The dosage of the biostimulant for root irrigation of the present invention is 10-100 mL / plant.

[0066] The dosage of the biostimulant for seed dressing according to the present invention is 0.05-0.2 g / kg of seeds.

[0067] The inventors found that the use of 0.03% to 0.12% rhizosphere polysaccharide for seed coating can effectively improve the salt-alkali resistance germination rate and growth index of the corn seeds; when the ratio of rhizosphere polysaccharide to seeds is 50 mg / kg, the use effect is best.

[0068] The inventors found that the use of 0.02% to 0.08% rhizosphere polysaccharide for seed dressing and coating of soybean seeds can effectively improve the low temperature resistance germination rate and growth index of soybean seeds; when the ratio of rhizosphere polysaccharide to seeds is 100 mg / kg, the use effect is best. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the rhizosphere polysaccharide curve obtained by ion column elution;

[0070] Figure 2 It is the gas chromatography of rhizosphere polysaccharides;

[0071] Figure 3 This is the GPC spectrum of rhizosphere polysaccharides;

[0072] Figure 4 This is the infrared spectrum of rhizosphere polysaccharides;

[0073] Figure 5 Rhizosphere polysaccharide 1 H and 13 C NMR spectrum;

[0074] Figure 6 is the structural formula of rhizosphere polysaccharide; n is the degree of polymerization, which ranges from 5 to 6;

[0075] Figure 7 It is that rhizosphere polysaccharides promote the transcription level of salicylic acid / jasmonic acid responsive genes;

[0076] Figure 8 It is the rhizosphere polysaccharides that improve the resistance of leaves to Pseudomonas carcinomatosa;

[0077] Fig. 9 It is the rhizosphere polysaccharides that promote Bacillus thuringiensis to encapsulate the root system;

[0078] Fig.10 It is rhizosphere polysaccharide seed dressing that promotes the germination and growth of corn with salt and alkali resistance;

[0079] Fig.11 It is a rhizosphere polysaccharide seed dressing that promotes soybean germination and growth against low temperatures. DETAILED DESCRIPTION

[0080] The present invention provides a rhizosphere polysaccharide, a biostimulant and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0081] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0082] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0083] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0084] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0085] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0086] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0087] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0088] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0089] In order to further illustrate the present invention, a rhizosphere polysaccharide, a biostimulant and applications thereof provided by the present invention are described in detail below in conjunction with examples.

[0090] Example 1: Isolation and extraction of rhizosphere polysaccharides

[0091] 1) Add the rhizosphere bacteria in the sugarcane root exudates into a bioreactor filled with a reaction solution (50 g / L of sugarcane fruit powder, 5 g / L of sugarcane peel powder, 5 g / L of sugarcane rhizome powder, 10 g / L of plant protein powder, pH=7.0), and react at 30°C for 3 days to obtain a bioreactor containing rhizosphere polysaccharides.

[0092] 2) Take 10 liters of bioreactor solution as 1 portion, add 2 volumes of deionized water to each portion of bioreactor solution, centrifuge at 5000-8000 rpm for 5 minutes; discard the precipitate and retain the supernatant.

[0093] 3) Take 10 liters of supernatant as 1 portion, add 2 volumes of 95% ethanol to each portion of supernatant, stir at room temperature for 0.5 hour in the reactor, let stand at 5-10°C for 4-8 hours, then filter with a 50-200 mesh filter, discard the filtrate, and retain the filter residue.

[0094] 4) Take 100 g of the filter residue as 1 portion, add 2 times the mass of deionized water to each portion of the filter residue, stir at room temperature in the reactor for 0.5 hour, filter with a 50-mesh filter, discard the filter residue, and retain the filtrate.

[0095] 5) Take 100 ml of the filtrate as 1 portion, add an equal volume of Sevage reagent to each portion of the filtrate, stir at room temperature in the reactor for 0.5 hour, drain the lower and middle layer solutions from the separatory funnel at the bottom of the reactor, and keep the upper layer solution.

[0096] 6) Take 100 ml of the upper layer solution as 1 portion, and evaporate each portion of the upper layer solution at 60°C and -0.08 MPa under reduced pressure to 10 ml of concentrated solution.

[0097] 7) Pour 10 ml of the concentrated solution into a dialysis bag with a molecular cutoff of 1000 Daltons. Place the dialysis bag in 1 liter of ultrapure water and stir for 12 hours, then take out the dialysate in the dialysis bag.

[0098] 8) Take 10 ml of dialysate as 1 portion, add 2 volumes of 95% ethanol to each portion of dialysate, stir at room temperature for 2 minutes, centrifuge at 6000 rpm for 1 minute at room temperature, discard the supernatant, collect the precipitate, and freeze or dry the precipitate at room temperature to obtain white crystals.

[0099] 9) Add 50 g of diethylaminoethyl cellulose to 0.3 liters of ultrapure water, stir well and let stand at room temperature for 12 hours to allow it to swell. Use wet column packing (20×400 mm), and then flush the column with acid water-ultrapure water-alkaline water-deionized water in sequence until the eluent is neutral, and the pretreatment of the chromatography column is completed.

[0100] 10) The white crystals obtained in step 9) were prepared into a 5 g / L aqueous solution, and 5 ml of sample chromatography was evenly added along the edge of the pretreated chromatography column, and 0 M, 0.1 M, 0.2 M, and 0.3 M NaCl solutions were used for elution, and 20 portions were collected for each elution concentration, each of which was 5 ml, and numbered in order of collection. The absorbance of each eluate at a wavelength of 490 nm was detected by the phenol-sulfuric acid method; it was shown that elution with 0.1 M NaCl solution could obtain rhizosphere polysaccharide component a ( Figure 1 ).

[0101] Example 2: Structural Identification of Rhizosphere Polysaccharides

[0102] 1) Take 100 ml of component a solution as 1 portion, add 2 volumes of 95% ethanol to each portion of component a solution, stir at room temperature for 2 minutes, centrifuge at 6000 rpm for 1 minute at room temperature, discard the supernatant, collect the precipitate, and freeze or dry the precipitate at room temperature to obtain rhizosphere polysaccharide.

[0103] 2) The rhizosphere polysaccharide and standard monosaccharides (arabinose, rhamnose, fructose, xylose, mannose, galactose, glucose) were subjected to acid hydrolysis and trimethylsilyl ether derivatization, and the monosaccharide composition of the rhizosphere polysaccharide was determined by gas chromatography. The peak areas of the obtained chromatograms were integrated and then inserted into the standard curve to calculate that the rhizosphere polysaccharide was a heteropolysaccharide composed of fructose, mannose, xylose, and glucose, and the molar ratio was 1:0.17:0.17:0.015 ( Figure 2 ).

[0104] 3) 20 μL of sample was injected, and the weight-average molecular weight of rhizosphere polysaccharide was determined by high-performance gel filtration chromatography. The standard curve was calculated based on the logarithm and retention time of 1 g / L standard dextran with different molecular weights. The results showed that the peak shape of the pure rhizosphere polysaccharide was symmetrical, and the molecular weight of the rhizosphere polysaccharide was 15.43 kDa ( Figure 3 ).

[0105] 4) The pure rhizosphere polysaccharide was mixed with potassium bromide and pressed into tablets, and detected by Fourier transform infrared spectroscopy with a detection range of 4000-400 wavenumbers. The results showed that: 3415 cm -1 The OH bond stretching vibration peak is at 2960 cm -1 2937 cm -1 The peak at 1600cm is the stretching vibration peak of the CH bond, indicating that it is a sugar, and the double peak suggests that it contains fructose; -1 The peak at 1407 cm is the stretching vibration peak of the C=O bond. -1 1390 cm -1 The peak at 1200-1000 cm -1 The peak at 920 cm is the bending vibration peak of COH or COC bond; -1 The characteristic peak of furanose is near 850 cm -1 The characteristic peak of pyranose is near 893cm -1 The characteristic peak of β configuration is near 809 cm -1 The characteristic peak near is the α configuration ( Figure 4 ).

[0106] 5) Dissolve 10 mg of rhizosphere polysaccharide in 1 mL of heavy water for three times and perform nuclear magnetic resonance spectroscopy (NMR) on the sample. 1 H NMR, 13 CNMR determination; by comparing with the displacement values ​​of known polysaccharides, the nuclear magnetic resonance spectrum of rhizosphere polysaccharides was analyzed ( Figure 5 ), and analyze the carbon and hydrogen attribution; based on the experimental results, it can be known that the structure of rhizosphere polysaccharides is Figure 6 shown.

[0107] Example 3: Rhizosphere polysaccharide solution stimulates the transcription of endogenous hormone response genes in protoplast mesophyll cells

[0108] 1) Use ultrapure water to dilute the rhizosphere polysaccharide into 100 µL of protoplast mesophyll cell solution carrying the luciferase encoding gene, so that the final concentration of rhizosphere polysaccharide reaches 0.005%, 0.01% and 0.02%, respectively. 1 µM, 10 µM and 100 µM methyl salicylate and methyl jasmonate solutions are used as positive controls. Ultrapure water is used as solvent control.

[0109] 2) Add luciferin to the substrate in each system for incubation, and use an ELISA reader to test the fluorescence intensity levels of endogenous salicylic acid and jasmonic acid response genes in the cells for 5 consecutive minutes.

[0110] 3) Summarize the obtained test fluorescence signal intensity data, calculate the difference before and after changes, and draw a graph ( Figure 7 ), it was found that 0.005%-0.02% rhizosphere polysaccharide solution can stimulate the transcription of endogenous salicylic acid and jasmonic acid response genes in cells; and the effect is best when its concentration is ~0.01%.

[0111] Example 4: Leaf spraying of rhizosphere polysaccharide solution improves tobacco resistance to Pseudomonas olive cancer

[0112] 1) Treatment Group 1: 0.25 mg of rhizosphere polysaccharide and 0.1 g of potassium sorbate were added to 10 ml of deionized water to prepare a 0.0025% rhizosphere polysaccharide solution; the solution was sprayed on tobacco leaves, 3 leaves per group, and 2 ml per leaf.

[0113] 2) Treatment Group 2: 0.5 mg of rhizosphere polysaccharide and 0.1 g of potassium sorbate were added to 10 ml of deionized water to prepare a 0.005% rhizosphere polysaccharide solution; the solution was sprayed on tobacco leaves, 3 leaves per group, and 2 ml per leaf.

[0114] 3) Control group: 0.5 mg of chitosan and 0.1 g of potassium sorbate were added to 10 ml of deionized water in sequence; the solution was sprayed on tobacco leaves, 3 leaves per group, and 2 ml was sprayed on each leaf.

[0115] 4) One day after leaf spraying, OD 600 = 0.05 of Pseudomonas oliveri suspension was injected into the tobacco leaves of each group.

[0116] 4) Seven days after inoculation, take photos to record the lesions on the leaves of each group ( Figure 8 ), it can be found that the lesion area on the tobacco leaves in the rhizosphere polysaccharide treatment group was significantly smaller than that in the water control group; this shows that leaf spraying of 0.0025% and 0.005% rhizosphere polysaccharide solution can improve the disease resistance of tobacco to Pseudomonas olive cancer.

[0117] Example 5: Root irrigation with rhizosphere polysaccharide solution improves the control effect of Bacillus thuringiensis on cucumber root-knot nematodes

[0118] 1) Genetically transform the plasmid carrying the green fluorescent protein coding gene into the cytoplasm of Bacillus thuringiensis, then amplify and culture the Bacillus thuringiensis, and dilute the bacterial solution with ultrapure water to make its OD 600 When the concentration is about 0.5, you can get the Bacillus thuringiensis suspension.

[0119] 2) Sow the surface-sterilized cucumber seeds into 18 ml of tissue culture solution and culture them at 25-28°C in a light incubator with a light / dark time ratio of 15 / 9 until they reach the one true leaf stage.

[0120] 3) Treatment group: Add 0.5 g of rhizosphere polysaccharide, 10 mg of urea, 1 mg of complex amino acid, and potassium phosphate buffer to 1 ml of sterile deionized water to prepare 50% rhizosphere polysaccharide solution; drip all the solution to the rhizosphere of cucumber seedlings; at the same time, evenly drip 1 ml of Bacillus thuringiensis suspension into the tissue culture solution; then continue tissue culture for 3 days.

[0121] 4) Control group: Add 0.01 μg of coronatine, 10 mg of urea, 1 mg of complex amino acids, and potassium phosphate buffer to 1 ml of sterile deionized water to adjust the pH to 6.0; drip the solution onto the root zone of cucumber seedlings; at the same time, evenly drip 1 ml of Bacillus thuringiensis suspension into the tissue culture solution; then continue tissue culture for 3 days.

[0122] 5) The rhizosphere hydroponic solution of the cucumber seedlings in the treatment group and the control group was aspirated, and the sections were prepared and observed under a fluorescence confocal microscope at a wavelength of GFP488. At the same time, the roots of some cucumber seedlings were taken out, and the root hair area, secondary roots, and crown roots were sliced ​​manually, and the sections were prepared and observed under a fluorescence confocal microscope at a wavelength of GFP488.

[0123] 6) Overlap the observed bright field and dark field images ( Fig. 9 ), it can be found that compared with the control group, the treatment group drip-irrigated with 50% rhizosphere polysaccharide solution had more Bacillus thuringiensis (green) attached to the cucumber rhizosphere.

[0124] 7) The remaining cucumber seedlings of the treatment group and the control group were transplanted into potted plants in a 28°C greenhouse; 5 days after transplanting, the soil around the roots of each cucumber plant was inoculated with ~500 second-instar root-knot nematodes.

[0125] 8) On the 50th day, the root-knot nematode disease and control efficacy of each group of cucumbers were investigated and plotted as Table 1. It can be found that the control efficacy of cucumbers treated with 50% rhizosphere polysaccharide solution against root-knot nematodes reached 54.93%, indicating that rhizosphere polysaccharides can induce Bacillus thuringiensis to wrap the cucumber roots and kill the root-knot nematodes near the roots.

[0126] Table 1

[0127] Grouping disease finger Prevention effect Control group 71.0 — Treatment Group 32.0 54.93%

[0128] Example 6: Rhizosphere polysaccharide seed coating agent promotes corn germination with salt and alkali resistance

[0129] 1) Treatment Group 1: Add 2.5 mg of Rose Bengal, 2.0 mg of Fludioxonil and 1.5 mg of rhizosphere polysaccharide to 5 ml of ultrapure water in sequence to prepare a 0.03% rhizosphere polysaccharide seed coating agent; take 5 ml of 0.03% rhizosphere polysaccharide seed coating agent and 30 g of corn seeds (~100 seeds), stir and mix, and then dry.

[0130] 2) Treatment Group 2: Add 2.5 mg of Rose Bengal, 2.0 mg of Fludioxonil and 3.0 mg of Rhizosphere Polysaccharide to 5 ml of ultrapure water in sequence to prepare a 0.06% Rhizosphere Polysaccharide Seed Dressing Agent; take 5 ml of 0.06% Rhizosphere Polysaccharide Seed Dressing Agent and 30 g of corn seeds (~100 seeds), stir and mix well, and then dry.

[0131] 3) Treatment Group 3: Add 2.5 mg of Rose Bengal, 2.0 mg of Fludioxonil and 6.0 mg of Rhizosphere Polysaccharide to 5 ml of ultrapure water in sequence to prepare a 0.12% Rhizosphere Polysaccharide Seed Dressing Agent; take 5 ml of 0.12% Rhizosphere Polysaccharide Seed Dressing Agent and 30 g of corn seeds (~100 seeds), stir and mix, and then dry.

[0132] 4) Control group: Add 2.5 mg of Rose Bengal, 2.0 mg of Fludioxonil and 3 mg of Trehalose to 5 ml of ultrapure water, take 5 ml of the solution and stir with 25 g of corn seeds (~100 seeds), and then dry.

[0133] 5) Sow the corn seeds that have been fully coated with the seed dressing in potting soil with pH = 10.5 and NaCl = 1.5%, and cultivate them in a greenhouse at 25°C for 6-9 days. There are 3 pots in each group, and 20 seeds are sown in each pot.

[0134] 6) Observe and record the germination of corn 6 days after sowing; 9 days after sowing, pull out the corn seedlings with their roots, clean the rhizosphere soil, and take photos to record the growth of corn ( Fig.10 ).

[0135] 7) On the 6th day, the germination rate of corn seeds was counted, and on the 9th day, the plant height, root length, plant weight and root weight of corn seedlings were counted. It can be found in Table 2 that the use of 0.03%-0.12% rhizosphere polysaccharide for seed coating can effectively improve the salt-alkali resistance germination rate and growth index of corn seeds; when the ratio of rhizosphere polysaccharide to seeds is 50 mg / kg, the best effect is achieved.

[0136] Table 2

[0137] Grouping Usage ratio Germination rate Plant height (cm) Root length (cm) Plant weight (g) Root weight (g) Control group 0 mg / kg 85% 17.97 10.99 1.60 0.38 Treatment Group 1 50 mg / kg 98.33% 19.22 12.07 2.44 0.50 Treatment Group 2 100 mg / kg 90.00% 18.79 11.31 1.98 0.41 Treatment Group 3 200 mg / kg 93.33% 18.51 11.58 1.87 0.42

[0138] Example 7: Rhizosphere polysaccharide seed coating agent promotes soybean germination against low temperature

[0139] 1) Treatment Group 1: Add 2.5 mg of Rose Bengal and 1 mg of rhizosphere polysaccharide to 5 ml of ultrapure water in sequence to prepare a 0.02% rhizosphere polysaccharide seed coating agent; take 5 ml of 0.02% rhizosphere polysaccharide seed coating agent and 20 g of soybean seeds (~100 seeds), stir and mix, and then dry.

[0140] 2) Treatment Group 2: Add 2.5 mg of Rose Bengal and 2 mg of rhizosphere polysaccharide to 5 ml of ultrapure water in sequence to prepare a 0.04% rhizosphere polysaccharide seed coating agent; take 5 ml of 0.04% rhizosphere polysaccharide seed coating agent and 20 g of soybean seeds (~100 seeds), stir and mix, and then dry.

[0141] 3) Treatment Group 3: Add 2.5 mg of Rose Bengal and 4 mg of rhizosphere polysaccharide to 5 ml of ultrapure water in sequence to prepare a 0.08% rhizosphere polysaccharide seed coating agent; take 5 ml of 0.08% rhizosphere polysaccharide seed coating agent and 20 g of soybean seeds (~100 seeds), stir and mix, and then dry.

[0142] 4) Control group: Add 2.5 mg of Rose Bengal and 2.5 mg of Trehalose to 5 ml of ultrapure water in sequence, take 5 ml of the solution and stir with 20 g of soybean seeds (~100 seeds), then dry.

[0143] 5) Store the coated soybean seeds at ~3.5℃ for 24 hours, and then transfer them to a 25℃ greenhouse for sowing. Each group has 3 pots, and each pot has 20 seeds.

[0144] 6) Observe and record the germination of soybeans 7 days after sowing; 9 days after sowing, pull out the soybean seedlings with their roots, clean the rhizosphere soil, and take photos to record the growth of soybeans ( Fig.11 ).

[0145] 7) The germination rate of soybean seeds was counted on the 7th day, and the plant height, root length, plant weight and root weight of soybean seedlings were counted on the 9th day. The plots are shown in Table 3. It can be found that the low temperature resistance germination rate and growth index of soybean seeds can be effectively improved after the soybean seeds are coated with rhizosphere polysaccharides at a content of 0.02%-0.08%. When the ratio of rhizosphere polysaccharides to seeds is 100 mg / kg, the best effect is achieved.

[0146] Table 3

[0147] Grouping Usage ratio Germination rate Plant height (cm) Root length (cm) Plant weight (g) Root weight (g) Control group 0 mg / kg 71.67% 19.14 10.09 1.88 0.28 Treatment Group 1 50 mg / kg 86.67% 19.89 9.61 2.01 0.26 Treatment Group 2 100 mg / kg 93.33% 22.10 12.02 2.28 0.41 Treatment Group 3 200 mg / kg 90.00% 20.32 11.70 2.16 0.38

[0148] Example 8. Preparation of rhizosphere polysaccharide granules and powders

[0149] 1) Mix the raw materials according to the following percentages: 20% urea, 5% potassium dihydrogen phosphate, 5% dipotassium hydrogen phosphate, 10% lignin sulfonate, and the rest is made up to 100% with rhizosphere polysaccharide.

[0150] 2) The above raw materials are subjected to the processes of coarse grinding, stirring and mixing, air flow grinding, remixing, kneading, granulation, screening and drying to obtain rhizosphere polysaccharide granules with a content of 60%.

[0151] 3) Mix the above raw materials according to the following percentages: 10% rice husk powder, 5% kaolin, 5% diatomaceous earth, 10% wood ash, 10% peat soil, and the rest is made up to 100% with rhizosphere polysaccharide.

[0152] 4) After coarse crushing, stirring and mixing, air flow crushing and re-mixing, a rhizosphere polysaccharide powder with a content of 60% can be obtained.

[0153] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting rhizosphere polysaccharides, characterized in that: include: A) inoculating rhizosphere bacteria in sugarcane root secretions into a reaction solution for reaction to obtain a bioreaction solution; the reaction solution comprises: sugarcane fruit powder, sugarcane peel powder, sugarcane rhizome powder and plant protein powder; the mass ratio of the sugarcane fruit powder, sugarcane peel powder, sugarcane rhizome powder and plant protein powder is 50:5:5:10; the pH value of the reaction solution is adjusted to 6.8-7.2, the temperature of the bioreaction is 30°C, and the time is 2-4 days; B) mixing the bioreactor solution and water, centrifuging, and obtaining a supernatant; adding ethanol to the supernatant for precipitation, and obtaining a filter residue; C) washing the filter residue with water, and then removing protein with Sevage reagent to obtain an upper layer solution; D) After concentrating the upper layer solution, dialyze to obtain the dialysate in the dialysis bag; E) The dialyzate is precipitated with ethanol, and then chromatographed and eluted to obtain rhizosphere polysaccharide; the chromatography column is diethylaminoethyl cellulose; the eluent is NaCl solution; the concentration of the NaCl solution is 0.1M.

2. The extraction method according to claim 1, characterized in that Step A) Step B) the centrifugal speed is 5000-8000 rpm, the time is 2-5 min; the ethanol is 95% ethanol; the volume ratio of the supernatant to the ethanol is 1:(2-3); Step D) The dialysis is performed using a dialysis bag with a cut-off volume of 1000 Daltons.

3. A rhizosphere polysaccharide, characterized in that The extract is prepared by the extraction method according to claim 1 or 2.

4. The rhizosphere polysaccharide according to claim 3, characterized in that The molar ratio of fructose, mannose, xylose and glucose in the rhizosphere polysaccharide is 1: (0.16-0.18): (0.16-0.18): (0.01-0.02), and the weight average molecular weight of the rhizosphere polysaccharide is 15.43 kDa.

5. A biostimulant, characterized in that: The invention comprises the rhizosphere polysaccharide extracted by the extraction method according to any one of claims 1 to 2 or the rhizosphere polysaccharide according to any one of claims 3 to 4.

6. The biostimulant according to claim 5, characterized in that: The weight percentage of rhizosphere polysaccharide in the biostimulant is 0.0025% to 100%.

7. The biostimulant according to claim 5, characterized in that The dosage form of the biostimulant includes granules, powders, liquids or seed coatings.

8. The biostimulant according to claim 7, characterized in that: The biostimulant granules include 60% to 100% rhizosphere polysaccharide and a granule carrier; the granule carrier is selected from one or more of lignin sulfonate, urea, and potassium phosphate; The biostimulant powder comprises 60% to 98% rhizosphere polysaccharide and a powder carrier; the powder carrier is selected from One or more of rice husk powder, kaolin, diatomaceous earth, wood ash, and peat soil; The biostimulant liquid comprises 0.0025% to 50% rhizosphere polysaccharide and a liquid carrier; the liquid carrier is selected from one or more of brassinolide, coronatine, urea, amino acids, phosphate potassium salts, and deionized water; The biostimulant seed coating agent comprises 0.02% to 0.12% rhizosphere polysaccharide and a seed coating agent carrier; the seed coating agent carrier is selected from one or more of trehalose, xanthan gum, hydroxymethyl cellulose, coronatine, fludioxonil and rose red pigment.

9. Use of the biostimulant according to any one of claims 5 to 8 in improving plant stress resistance; the stress resistance includes resistance to salinity, resistance to low temperature, resistance to drought and / or resistance to pests and diseases.

10. A method for improving plant stress resistance, comprising applying the biostimulant according to any one of claims 5 to 8.

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