Application of modified enteromorpha polysaccharide to improvement of seed vigor and / or improvement of drought resistance of seeds

Abstract

The invention provides application of modified enteromorpha polysaccharide to improvement of seed vigor and / or improvement of drought resistance of seeds, and belongs to the technical field of plant planting. The invention provides application of modified enteromorpha polysaccharide in improving seed vigor and / or improving drought resistance of seeds. The modified enteromorpha polysaccharide is prepared by performing carboxymethylation modification on enteromorpha polysaccharide. The modified enteromorpha polysaccharide contains more carboxyl groups, so that the chelation capability of the enteromorpha polysaccharide on trace elements can be enhanced. When the modified enteromorpha polysaccharide is used for dressing seeds, the germination of corn seeds under drought conditions can be remarkably promoted, the plant height of seedlings and the total length of root systems are increased, and the drought resistance of corn is improved. The invention further establishes a method for treating the seeds by combining ultrasound, compound bacteria and modified enteromorpha polysaccharide, so that the seed vitality can be better improved, and the drought resistance of the seedlings is enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant cultivation, and particularly relates to application of modified Enteromorpha polysaccharide in improving seed vigor and / or improving drought resistance of seeds. BACKGROUND

[0002] Seed vigor is the sum of seed germination and emergence rate, seedling growth potential, plant stress resistance and production potential, and is an important indicator of seed quality. High-vigor seeds can emerge rapidly and uniformly after being sown in the field, which helps to achieve full, uniform and strong seedlings, and lays a good foundation for crop yield increase. High-vigor seeds have strong resistance to field stress, can escape and resist pests and diseases, and have the ability to compete with weeds due to the strong and vigorous growth of seedlings. Improving seed vigor can improve resistance to early spring low temperature conditions, allow for early sowing, and increase yield under early sowing conditions.

[0003] Physical methods for improving seed vigor include plasma treatment and ultrasonic treatment. Wang Decheng et al. of China Agricultural University established a mathematical model of plasma treatment power and treatment time and comprehensive germination test index of alfalfa seeds, and sought an optimized treatment condition scheme. Tang Xiangru of South China Agricultural University studied the regulation mechanism of ultrasonic seed treatment on rice seed germination. Guangzhou Jin Dang Agricultural Technology Co., Ltd. developed several ultrasonic seed treatment machines. Lu Guohua of the Institute of Agricultural Environment and Sustainable Development, Chinese Academy of Agricultural Sciences studied the effect of ultrasonic seed treatment on seed germination of corn and other crops, and the results showed that the activities of seed amylase and protein-decomposing enzyme were enhanced, and the seed germination potential was improved.

[0004] Chemical components include amino acids, humic acid, inorganic salts, chitin and its derivatives, microorganisms and their metabolites, and plant extracts, etc. with biological stimulation function, which can help to improve seed germination rate. Humic acid and the like can stimulate the synthesis of endogenous hormones such as plant cell division and growth hormones, and directly accelerate the seed germination process. Biological stimulants can improve the tolerance of seeds to abiotic stresses such as drought and high temperature, and the components such as SOD and POD can scavenge active oxygen free radicals, protect cell membrane structure, and reduce the influence of adverse environment on germination.

[0005] Microbial seed coating is to uniformly coat the functional preparation containing active microorganisms (such as nitrogen-fixing bacteria, phosphorus-dissolving bacteria, and biocontrol bacteria) on the surface of seeds. The core principle is to use the physiological characteristics of microorganisms to provide multiple stress regulation and nutrient support for crop growth, thereby improving seed vigor.

[0006] However, the effect of the current methods for improving seed vigor is often limited, and some methods can promote seed growth but have no significant improvement effect on improving seed stress resistance. SUMMARY

[0007] In view of the defects in the prior art, the purpose of the present application is to provide the application of modified enteromorpha polysaccharide in improving seed vigor. The modified enteromorpha polysaccharide can not only promote seed growth, but also improve the stress resistance and drought resistance of seeds.

[0008] The purpose of the present application is achieved by the following technical solutions: The present application provides the application of modified enteromorpha polysaccharide in improving seed vigor and / or improving the drought resistance of seeds, wherein the modified enteromorpha polysaccharide comprises: modified enteromorpha polysaccharide prepared by carboxymethylation modification of enteromorpha polysaccharide.

[0009] Preferably, the preparation method of the modified enteromorpha polysaccharide comprises: Mixing an enteromorpha polysaccharide aqueous solution with a NaOH aqueous solution to activate the hydroxyl group of the polysaccharide and obtain activated enteromorpha polysaccharide; Mixing the activated enteromorpha polysaccharide with a chloroacetic acid solution to perform a carboxymethylation reaction and obtain carboxymethylated enteromorpha polysaccharide, i.e. a reaction system containing modified enteromorpha polysaccharide.

[0010] Preferably, the mass percentage of enteromorpha polysaccharide in the enteromorpha polysaccharide aqueous solution is 10-20 wt%; the mass percentage of NaOH in the NaOH aqueous solution is 10 wt%-30 wt%; the mass ratio of the enteromorpha polysaccharide aqueous solution to the NaOH aqueous solution is 1:(1-2); the temperature for activating the hydroxyl group of the polysaccharide is 20-40℃; and the time for activating the hydroxyl group of the polysaccharide is 2-3h.

[0011] Preferably, the mass percentage of chloroacetic acid in the chloroacetic acid solution is 20%-40%; the mass ratio of the enteromorpha polysaccharide aqueous solution to the chloroacetic acid solution is 10:(1-2); the temperature for the carboxymethylation reaction is 50-80℃; and the time for the carboxymethylation reaction is 1-2h.

[0012] Preferably, the preparation method further comprises separation and purification of the modified enteromorpha polysaccharide, wherein the separation and purification method of the modified enteromorpha polysaccharide comprises: mixing the reaction system containing the modified enteromorpha polysaccharide with ethanol to precipitate the modified enteromorpha polysaccharide.

[0013] The present application provides a method for improving seed vigor, comprising: The modified enteromorpha polysaccharide in the application is mixed with seeds for seed dressing.

[0014] Preferably, the seed dressing method comprises: mixing an enteromorpha polysaccharide aqueous solution with seeds and then drying; the mass concentration of the modified enteromorpha polysaccharide in the enteromorpha polysaccharide aqueous solution is 20-50mg / L; and the mass ratio of the enteromorpha polysaccharide aqueous solution to the seeds is (50-100)g:1kg.

[0015] Preferably, before seed dressing, the process also includes: Seeds are subjected to ultrasonic treatment to obtain ultrasonically treated seeds; The seeds treated with ultrasound were coated with a compound microbial coating agent.

[0016] Preferably, the ultrasonic treatment includes a mixed ultrasonic treatment; the frequency of the first ultrasonic wave in the mixed ultrasonic treatment is 20~40 kHz, and the frequency of the second ultrasonic wave is 60~90 kHz; the duration of the mixed ultrasonic treatment is 30~40 s. The compound bacterial coating agent includes Bacillus subtilis and Bacillus megaterium.

[0017] This invention provides the application of the method described in the above technical solution in improving the drought resistance of plants.

[0018] The beneficial effects of this invention are: This invention provides the application of modified *Ulva prolifera* polysaccharide in improving seed vigor and / or seed drought resistance. The modified *Ulva prolifera* polysaccharide is prepared by carboxymethylation of *Ulva prolifera* polysaccharide. This modified *Ulva prolifera* polysaccharide contains a higher number of carboxyl groups, which enhances its chelating ability for trace elements. Seed treatment with this modified *Ulva prolifera* polysaccharide significantly promotes germination of maize seeds under drought conditions, increases seedling height, and improves the drought resistance of maize seeds. This invention further establishes a method for combined seed treatment using ultrasound, compound bacteria, and modified *Ulva prolifera* polysaccharide, which can better enhance seed vigor and improve the drought resistance of seedlings. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Figure 1 shows the effect of different treatment times with mixed-frequency ultrasound on the germination of hydroponic seeds. Figure 2 Figure 1 shows the germination rate of hydroponic corn seeds after different treatment times with mixed-frequency ultrasound. Figure 3 Figure 1 shows the effect of different treatment times with mixed-frequency ultrasound on the root length of hydroponic corn seeds. Figure 4 This is a schematic diagram of carboxymethylation of Ulva prolifera polysaccharides; Figure 5 The image shows the 1H NMR spectrum of *Ulva prolifera* polysaccharide. Figure 6 The 1H NMR spectrum of the modified *Ulva prolifera* polysaccharide is shown. Figure 7 Figure 1 shows the effect of the modified *Ulva prolifera* polysaccharide treatment group on the seedling height of maize. Figure 8 Figure 1 shows the effect of Bacillus subtilis and Bacillus megater compound inoculant on seed germination rate of maize on days 3 and 7 after sowing under drought stress. Figure 9 Example 6 illustrates the effects of different treatment groups on maize seed germination under drought conditions. Detailed Implementation

[0021] This invention provides the application of modified Ulva prolifera polysaccharide in improving seed vigor and / or improving seed drought resistance, wherein the modified Ulva prolifera polysaccharide comprises: modified Ulva prolifera polysaccharide prepared by carboxymethylation modification of Ulva prolifera polysaccharide.

[0022] The present invention does not have any particular limitation on the carboxymethylation modification method of the *Ulva prolifera* polysaccharide; any conventional modification method in the art can be used.

[0023] As an optional embodiment of the present invention, the preparation method of the modified *Ulva prolifera* polysaccharide includes: The polysaccharide solution of Ulva prolifera was mixed with the NaOH aqueous solution to activate the hydroxyl groups of the polysaccharide, thus obtaining activated Ulva prolifera polysaccharide. The activated Ulva polysaccharide was mixed with a chloroacetic acid solution and subjected to a carboxymethylation reaction to obtain carboxymethylated Ulva polysaccharide, which is a reaction system containing modified Ulva polysaccharide.

[0024] This invention involves mixing an aqueous solution of *Ulva prolifera* polysaccharide with an aqueous solution of NaOH to activate the hydroxyl groups of the polysaccharide, thereby obtaining activated *Ulva prolifera* polysaccharide. As an optional embodiment of this invention, the mass percentage of *Ulva prolifera* polysaccharide in the aqueous solution can be 10-20 wt%, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 wt%. The mass percentage of NaOH in the aqueous solution can be 10-30 wt%, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 wt%. The mass ratio of the *Ulva prolifera* polysaccharide aqueous solution to the NaOH aqueous solution can be 1:(1-2), or 1:1, 1:1.5, or 1:2. This invention does not specifically limit the mixing method; any conventional mixing method in the art can be used. As an optional embodiment of the present invention, the temperature for activating the polysaccharide hydroxyl groups can be 20~40℃, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40℃; the activation time for the polysaccharide hydroxyl groups can be 2~3 hours, or 2, 2.5, or 3 hours. The present invention preferably involves stirring during the polysaccharide hydroxyl group activation process; the stirring speed can be 300~500 r / min, or 400 r / min.

[0025] After obtaining the activated *Ulva prolifera* polysaccharide, the present invention mixes the activated *Ulva prolifera* polysaccharide with a chloroacetic acid solution for a carboxymethylation reaction to obtain carboxymethylated *Ulva prolifera* polysaccharide, i.e., a reaction system containing modified *Ulva prolifera* polysaccharide. As an optional embodiment of the present invention, the mass percentage of chloroacetic acid in the chloroacetic acid solution can be 20-40%, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40%; the solvent of the chloroacetic acid solution can be composed of 50% water and 50% ethanol by volume. As an optional embodiment of the present invention, the amount of chloroacetic acid solution added is based on the *Ulva prolifera* polysaccharide aqueous solution; the mass ratio of the *Ulva prolifera* polysaccharide aqueous solution to the chloroacetic acid solution can be 10:(1-2), or 10:1, 10:1.5, or 10:2. As an optional embodiment of the present invention, the temperature of the carboxymethylation reaction can be 50~80℃ or 70℃; the time of the carboxymethylation reaction can be 1~2h, or 1, 1.5 or 2h. After the carboxymethylation reaction is completed, the present invention obtains carboxymethylated Ulva prolifera polysaccharide, that is, a reaction system containing modified Ulva prolifera polysaccharide.

[0026] After obtaining the reaction system containing modified *Ulva prolifera* polysaccharides, the present invention preferably further includes separating and purifying the modified *Ulva prolifera* polysaccharides in the reaction system. The present invention does not specifically limit the method of separation and purification; any conventional separation and purification method in the art can be used. As an optional embodiment of the present invention, before separating and purifying the modified *Ulva prolifera* polysaccharides in the reaction system, the present invention preferably further includes neutralizing the reaction system. The present invention preferably uses hydrochloric acid aqueous solution for the neutralization reaction. After the neutralization reaction is completed, the present invention separates and purifies the modified *Ulva prolifera* polysaccharides. As an optional embodiment of the present invention, the separation and purification method includes: mixing the neutralized reaction system with ethanol to precipitate the modified *Ulva prolifera* polysaccharides. The present invention does not specifically limit the amount of ethanol used; any conventional amount in the art can be used. As an optional embodiment of the present invention, based on the mass of the *Ulva prolifera* polysaccharide aqueous solution, the mass-to-volume ratio of the *Ulva prolifera* polysaccharide aqueous solution to ethanol can be 1 g: (3~6) mL. After obtaining the precipitated modified *Ulva prolifera* polysaccharides, the present invention preferably further includes washing and drying the modified *Ulva prolifera* polysaccharides. This invention does not specifically limit the washing and drying methods; conventional washing and drying methods in the art can be used. As an optional embodiment of this invention, the washing can be performed with ethanol, preferably multiple times, 2-6 times, or 2, 3, 4, 5, or 6 times; the drying temperature can be 70°C. After drying, this invention obtains a pure modified *Ulva prolifera* polysaccharide. The modified *Ulva prolifera* polysaccharide prepared by the method provided by this invention exhibits a significantly altered molecular structure compared to *Ulva prolifera* polysaccharide, containing a higher concentration of carboxyl groups.

[0027] The present invention uses the modified seaweed polysaccharide described in the above technical solution to treat seeds, which can significantly promote seed germination and seedling growth, and better promote seed growth under drought conditions.

[0028] This invention provides a method for improving seed vigor, comprising: The modified *Ulva prolifera* polysaccharide used in the above-described technical solution is mixed with seeds for seed dressing.

[0029] As an optional embodiment of the present invention, before coating the seeds with modified *Ulva prolifera* polysaccharide, the method preferably further includes: ultrasonic treatment of the seeds to obtain ultrasonically treated seeds; and coating the ultrasonically treated seeds with a compound microbial coating agent. As an optional embodiment of the present invention, the ultrasonic treatment includes mixed ultrasonic treatment; the frequency of the first ultrasonic wave in the mixed ultrasonic treatment can be 20-40 kHz or 25 kHz, and the frequency of the second ultrasonic wave can be 60-90 kHz or 75 kHz. The terms "first" and "second" are not meaningful in this invention and are merely descriptive distinctions for ultrasonic waves. As an optional embodiment of the present invention, the duration of the mixed ultrasonic treatment can be 30-40 seconds, or 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 seconds.

[0030] After obtaining the ultrasonically treated seeds, the present invention preferably coats the obtained seeds with a compound microbial seed coating agent. As an optional embodiment of the present invention, the compound microbial seed coating agent includes Bacillus subtilis and Bacillus megaterium. The present invention does not specifically limit the preparation method of the compound microbial seed agent; any conventional preparation method in the art can be used. As an optional embodiment of the present invention, the preparation method of the compound microbial seed coating agent includes: mixing a Bacillus subtilis suspension and a Bacillus megaterium suspension to obtain the compound microbial seed coating agent. As an optional embodiment of the present invention, the viable count of the Bacillus subtilis suspension can be ≥1×10⁻⁶. 9 CFU / mL, or 1×10 9 ~1×10 10 CFU / mL; the viable count of the Bacillus megaterium suspension can be ≥5×10⁻⁶. 8 CFU / mL, or 5×10 8 ~1×10 9CFU / mL. This invention does not specifically limit the preparation methods of the *Bacillus subtilis* and *Bacillus megaterium* bacterial suspensions; conventional preparation methods in the art can be used. As an optional embodiment of this invention, the preparation method of the *Bacillus subtilis* bacterial suspension includes: culturing *Bacillus subtilis* in a culture medium to obtain a *Bacillus subtilis* bacterial suspension. In this invention, the culture medium can be TSA medium; the culture temperature can be 30°C; the culture time can be 24~36 h, or 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 h; the culture process preferably involves shaking; the shaking speed can be 180 rpm. As an optional embodiment of this invention, the preparation method of the *Bacillus megaterium* bacterial suspension includes: culturing *Bacillus megaterium* in a culture medium to obtain a *Bacillus megaterium* bacterial suspension. In this invention, the culture medium can be a nutrient broth medium; the culture temperature can be 30℃; the culture time can be 36~48h, or 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48h. The culture process is preferably accompanied by shaking; the shaking speed can be 150 rpm. As an optional embodiment of this invention, the volume ratio of the Bacillus subtilis suspension and the Bacillus megaterium suspension can be 1:1. After mixing the Bacillus subtilis suspension and the Bacillus megaterium suspension, the mixture is preferably stirred; the stirring speed can be 300~500 r / min, or 400 r / min; the stirring time can be 10~15 min, or 10, 11, 12, 13, 14 or 15 min. After stirring, glycerol is preferably added to the bacterial solution mixture. As an optional embodiment of this invention, the amount of glycerol added can be 0.5%. The addition of glycerol to the bacterial culture mixture in this invention is mainly to use glycerol as a protective agent to improve the stability of the bacterial cells.

[0031] After obtaining the compound microbial inoculant, the present invention uses the compound microbial inoculant to coat the seeds. The present invention does not specifically limit the coating method; any conventional seed coating method in the art can be used. As an optional embodiment of the present invention, the coating method can be: mixing the seeds with the compound microbial inoculant for coating. When mixing, the mass ratio of the compound microbial inoculant to the seeds can be (50~100) g:(1~2) kg, or it can be 50 g:1 kg, 100 g:1 kg, or 50 g:2 kg. As an optional embodiment of the present invention, the coating temperature can be 10~40℃, or it can be 30℃; the coating time can be 20~60 min, or it can be 30 min.

[0032] As an optional embodiment of the present invention, after the composite microbial coating agent is applied, the resulting seeds can be treated with modified *Ulva prolifera* polysaccharide. As an optional embodiment of the present invention, the seed treatment method can be: mixing the modified *Ulva prolifera* polysaccharide aqueous solution with the seeds and then drying. In the present invention, the mass concentration of the modified *Ulva prolifera* polysaccharide in the aqueous solution can be 20-50 mg / L, or 20, 30, 40, or 50 mg / L; the mass ratio of the modified *Ulva prolifera* polysaccharide aqueous solution to the seeds can be (50-100) g:1 kg, or 50 g:1 kg, 60 g:1 kg, 70 g:1 kg, 80 g:1 kg, 90 g:1 kg, or 100 g:1 kg. The present invention does not specifically limit the drying method; any conventional drying method in the art can be used. As an optional embodiment of the present invention, the drying method can be natural air drying.

[0033] This invention provides the application of the method described in the above technical solution in improving the drought resistance of plants. The results of the embodiments of this invention show that seed dressing with the modified *Ulva prolifera* polysaccharide can significantly promote seed growth under drought conditions, increase seedling height, and improve plant drought resistance. Furthermore, after sequentially subjecting seeds to ultrasonic treatment, coating with a compound microbial agent, and seed dressing with modified *Ulva prolifera* polysaccharide, this invention can more significantly improve seed vigor, more significantly promote seed growth, increase seedling height, and thus improve the drought resistance of plants.

[0034] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Methods for treating seeds using mixed-frequency ultrasonic waves: Seed treatment was performed using mixed-frequency ultrasound at frequencies of 25 kHz and 75 kHz. The ultrasonic seed treatment machine used was a tunnel-type seed treatment device (ZL202111648547.9). The frequencies of the first and second ultrasonic waves were set to 25kHz and 75kHz, respectively. Seeds were transported from one side of the tunnel entrance to the other via a conveyor belt. Different seed treatment times were set by adjusting the conveyor belt speed: 10s, 20s, 30s, 40s, 50s, and 60s. Seeds that did not undergo mixed-frequency ultrasonic treatment served as a control (CK). Each group consisted of 200 seeds for a seed germination experiment, conducted according to GB / T3543.4.

[0036] The results of seed germination after 3 and 7 days of treatment in each experimental group are as follows:Figures 1-3 As shown. Figure 1 The graph shows the effect of different treatment times with mixed-frequency ultrasound on seed germination. Figure 2 This graph shows the germination rate of maize seeds after different treatment times with mixed-frequency ultrasound. Figure 2 The left figure shows the seed germination rate of each experimental group on day 3; the right figure shows the seed germination rate of each experimental group on day 7. Figure 3 This figure shows the effect of different treatment times with mixed-frequency ultrasound on the root length of maize seeds. Figure 3 The left figure shows the root length of each experimental group on day 3; the right figure shows the root length of each experimental group on day 7.

[0037] like Figures 1-3 As shown, the germination rate of corn treated with 40s on day 3 was significantly higher than that of other treatments. On day 7, the germination rates of 30s and 40s treatments were significantly higher than those of the control treatment. The germination rates of 50s and 60s treatments showed a decrease. Therefore, the optimal treatment time for mixed frequency ultrasound is 30-40s. Ultrasonic seed treatment can promote root growth, with 40s treatment being the best. The germination rates of 50s and 60s treatments also showed a decreasing trend, indicating that excessively long treatment times do not significantly promote germination.

[0038] Example 2 A method for carboxymethylation modification of *Ulva prolifera* polysaccharides, comprising the following steps: Add 150g of a pre-prepared aqueous solution containing 20 wt% NaOH to 100g of an aqueous solution containing 10-20 wt% Ulva prolifera polysaccharide. Stir at 30℃ and 400 rpm for 2 h. Then add 20g of a chloroacetic acid solution (composed of 50% water and 50% ethanol) containing 25% chloroacetic acid, and continue the reaction at 70℃ for 1 h. After the reaction is complete, neutralize the reaction solution with a pre-prepared solution containing 10 wt% HCl. Pour the neutralized reaction solution into 500mL of ethanol, and the modified Ulva prolifera polysaccharide precipitates out. Filter, wash the modified Ulva prolifera polysaccharide several times with ethanol, and dry at 70℃ to obtain carboxymethylated Ulva prolifera polysaccharide, which can also be called modified Ulva prolifera polysaccharide. A schematic diagram of the carboxymethylation of Ulva prolifera polysaccharide is shown below. Figure 4 As shown. The 1H NMR spectrum of *Ulva prolifera* polysaccharide is shown below. Figure 5 As shown, the 1H NMR spectrum of the modified *Ulva prolifera* polysaccharide is as follows: Figure 6 As shown.

[0039] Depend on Figures 4-6 It can be seen that, compared with the modified *Ulva prolifera* polysaccharide, the molecular structure of the modified *Ulva prolifera* polysaccharide has changed significantly, with a significant increase in carboxyl groups.

[0040] Example 3 Seed dressing experiment with *Ulva prolifera* polysaccharide and modified *Ulva prolifera* polysaccharide 1g of *Ulva prolifera* polysaccharide and 1g of modified *Ulva prolifera* polysaccharide were dissolved in 50g of deionized water to obtain aqueous solutions of *Ulva prolifera* polysaccharide and modified *Ulva prolifera* polysaccharide, respectively. These solutions were then diluted to a concentration of 50mg / L for seed dressing. The prepared solutions were poured into 1kg of corn seeds, stirred thoroughly, and then air-dried for seedling sowing. For the control, 50g of deionized water was poured into 1kg of corn seeds, stirred thoroughly, and then air-dried for seedling sowing. A 40cm diameter plastic pot was filled with soil, maintaining a soil moisture content of 0.18 cm³. 3 / cm 3 This was used for seed germination experiments. Twenty seeds were planted in each plastic pot at a depth of 5 cm. Biomass was measured on day 10 to analyze the growth status of maize under drought stress. The effects of each treatment group on maize seedling height and total root length under drought conditions are shown below. Figure 7 As shown in the figure, different letters indicate significant differences. Figure 7 Figure A shows the effect of each treatment group on the seedling height of maize under drought conditions; Figure B shows the effect of each treatment group on the total root length of maize seedlings under drought conditions.

[0041] Depend on Figure 7 It can be seen that, after comparing the seed dressing test, the modified seaweed polysaccharide treatment significantly promoted the growth of maize seeds. Compared with the seed dressing without modified seaweed oligosaccharide, the total root length increased by 15.7% and the plant height increased significantly by 8.3% under drought conditions, thus enhancing the drought resistance of maize.

[0042] Example 4 Screening of test strains The bacterial strain used in the experiment was Bacillus subtilis ( Bacillus subtilis ACCC 19742, Bacillus megaterium ( Bacillus magaterium ACCC 04296, Bacillus mucilaginosa ( Paenibacillus mucilaginosus ACCC 10013 and fast-growing rhizobium ( Rhizobium ACCC 15082 was obtained by the Agricultural Culture Collection of China.

[0043] The following are the culture media: The composition of each liter of nitrogen-fixing medium is as follows: mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.5 g, NaCl 0.2 g, CaSO4·2H2O 0.1 g, CaCO3 5 g, agar 15 g, pH 7.0~7.2, sterilized at 121℃ for 15 min.

[0044] The composition of each liter of rhizobium nitrogen-fixing medium is as follows: 1.0 g yeast extract, 10 g mannitol, 0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 3 g CaCO3, 15 g agar, pH 6.8~7.2, sterilized at 121℃ for 15 min.

[0045] The composition of each liter of phosphorus-solubilizing medium is as follows: 10 g glucose, 5 g Ca3(PO4)2, 5 g MgCl2, 0.5 g MgSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, pH 6.8~7.2, sterilized at 121℃ for 15 min.

[0046] The composition of potassium-solubilizing medium per liter is as follows: 5 g glucose, 0.5 g (NH4)2SO4, 0.5 g yeast extract, 0.3 g MgSO4, 2 g NaH2PO4, 0.03 g FeSO4, 0.03 g MnSO4, 2 g potassium feldspar, 15 g agar, pH 7.2, sterilized at 121℃ for 15 min.

[0047] The composition of each liter of iron-carrier culture medium is as follows: 0.0605 g of Chromium Azurite S, 0.0729 g of Hexadecyl Trimethylammonium Bromide (HDTMA), 0.002645 g of FeCl3·6H2O, 0.29525 g of NaH2PO4·2H2O, 1.2135 g of Na2HPO4·12H2O, 0.125 g of NH4Cl, 0.0375 g of KH2PO4, 0.0625 g of NaCl, 9 g of agar, pH 6.7~6.9, sterilized at 121℃ for 15 min.

[0048] The composition of each liter of LB liquid medium is: 10 g tryptone, 5 g yeast extract, 10 g NaCl, pH 7.0~7.2, sterilized at 121℃ for 15 min.

[0049] The composition of each liter of nutrient meat broth liquid culture medium is: 3 g beef extract, 10 g peptone, 5 g NaCl, pH 7.4~7.6, sterilized at 121℃ for 15 min.

[0050] The composition of each liter of TSA medium is: 15 g of tryptone, 5 g of soybean peptone, 5 g of NaCl, pH 7.3~7.5, sterilized at 121℃ for 15 min.

[0051] The composition of each liter of YMA liquid culture medium is: 10 g mannitol, 1 g yeast extract, 0.5 g KH2PO4, 0.1 g NaCl, 0.2 g MgSO4·7H2O, pH 7.0, sterilized at 121℃ for 15 min.

[0052] Salkowski colorimetric solution: 50 mL 35% HClO4 + 1 mL 0.5 mol / L FeCl3.

[0053] Test methods The following functional tests were performed on the test strains of Bacillus subtilis, Bacillus megaterium, Bacillus mucilaginosus, and Bacillus rhizobium, with three replicates for each functional test: 1. Nitrogen fixation function test Using an inoculation loop, Bacillus subtilis, Bacillus megaterium, and Bacillus mucilaginosus were inoculated onto nitrogen-fixing medium plates. Rapid-growing rhizobia were inoculated onto rhizobia nitrogen-fixing medium plates. After incubation at 28°C for 4-7 days, the large, raised, mucilaginous colonies were observed. The large colonies were those with nitrogen-fixing ability.

[0054] 2. Potassium solubilization function test Inoculate the test strain onto a potassium-solubilizing medium plate using an inoculation loop, and incubate at 28°C for 3 days. Observe whether transparent oil droplet-like colonies appear in the medium plate. If they do, it proves that the strain has potassium-solubilizing ability.

[0055] 3. Phosphorus solubilization function test Inoculate the test strain onto a phosphate-solubilizing medium plate using an inoculation loop, and incubate at 30°C for 3-7 days. Observe whether a transparent halo is formed around the colony; if so, it proves that the strain has phosphate-solubilizing ability.

[0056] 4. Ferrocarrier Functional Testing Inoculate the test strain onto a siderophore medium plate using an inoculation loop, and incubate at 33°C for 2 days. Observe whether there is an orange iron precipitate band around the target colony on the medium. If there is, it proves that the strain has the ability to generate siderophores.

[0057] 5. IAA Functional Testing The bacterial strain was inoculated into LB liquid medium containing 0.1 g / L L-tryptophan, with three replicates for each strain. After incubation on a shaker (30℃, 180 r / min) for 1 day, the bacterial suspension was added to test tubes along with an equal volume of Salkowski colorimetric solution. A mixture of uninoculated LB liquid medium and an equal volume of colorimetric solution served as a control. The test tubes were incubated at room temperature in the dark for 30 minutes and then observed. A pink color indicated a positive result, meaning the bacteria could secrete IAA; the deeper the color, the greater the secretion. No color change indicated a negative result, meaning the bacteria could not secrete IAA.

[0058] 6. Drought resistance test All bacterial strains except for *Rhizobium brevicornum* were inoculated into LB liquid medium containing 0%, 30%, and 50% PEG6000. *Rhizobium brevicornum* was inoculated alone into YMA liquid medium containing 0%, 30%, and 50% PEG6000. The cultures were incubated at 37°C and 170 r / min on a shaker for 1–3 days. The OD values ​​of the bacterial solutions were read using a UV-Vis spectrophotometer. 600 The values ​​were analyzed to assess the growth of the experimental strains under different concentrations of PEG6000, and to conduct a comprehensive evaluation of their drought resistance.

[0059] The functional test results are shown in Table 1.

[0060] Table 1 Functional table of different strains

[0061] Note: An asterisk represents the ability to secrete IAA and the ability to resist drought and grow. This means p < 0.001. This means p < 0.01. A √ indicates p < 0.05; - indicates no corresponding ability or inability to produce the corresponding substance; √ indicates having the corresponding ability or being able to produce the corresponding substance.

[0062] Table 1 shows that Bacillus subtilis possesses potassium-solubilizing, phosphorus-solubilizing, siderophore-generating, and IAA-secreting capabilities, as well as the ability to synthesize OD values ​​at different PEG concentrations. 600 The drought resistance of *Bacillus subtilis* was significantly higher than that of other strains. *Bacillus megaterium* possessed potassium-solubilizing, phosphorus-solubilizing, siderophore-generating, and IAA-secreting abilities. While *Bacillus megaterium* had the second-highest drought resistance after *Bacillus subtilis*, its IAA-secreting ability was the strongest. Considering both drought resistance and IAA-secreting ability, both strains could promote seed germination under drought conditions. *Bacillus subtilis* and *Bacillus megaterium* were selected as the next experimental strains, and no antagonistic effect was observed between them.

[0063] Example 5 The strains used in the experiment were from the same source as in Example 4.

[0064] Single-cell expansion culture: Bacillus subtilis was cultured on TSA medium at 30℃ and 180 r / min for 24–36 h until the viable count reached 1 × 10⁻⁶. 9 CFU / mL (OD) 600 =1.0), to obtain a Bacillus subtilis suspension, which is the Bacillus subtilis seed coating agent.

[0065] Bacillus megaterium: Cultured in nutrient broth liquid medium at 30℃ and 150 r / min for 36-48 h until the viable count reaches 5×10⁻⁶. 8 CFU / mL (OD) 600 =0.8), to obtain a suspension of Bacillus megaterium, which is the Bacillus megaterium seed coating agent.

[0066] Bacterial suspension mixing: Mix Bacillus subtilis and Bacillus megaterium bacterial suspensions in a volume ratio of 1:1, stir for 10-15 minutes at a speed of 200 r / min, add 0.5% glycerol as a protectant to improve bacterial stability, and obtain a compound bacterial suspension, which is the compound bacterial coating agent.

[0067] The air-dried soil was prepared according to a soil volumetric moisture content of 0.18 cm. 3 / cm 3 Add water, and after the water is evenly absorbed, add water according to the soil bulk density of 1.3 g / cm³. 3 Each layer is 10 cm thick, and the seeds are layered and filled into a pot with an inner diameter of 19 cm and a height of 20 cm. Select seeds of uniform size and plumpness for coating. Then select 30 seeds per pot, sow 1 seed per hole at a depth of 2 cm, and then cover with transparent mulch to prevent water evaporation. Set up 4 corn seed coating treatments: (1) No coating treatment (control); (2) Bacillus subtilis coating: The above Bacillus subtilis seed coating agent is mixed with corn seeds at a mass ratio of 1:20 and the seeds are soaked for 10 min at a temperature of 30℃; (3) Bacillus megaterium coating: The above Bacillus megaterium seed coating agent is mixed with corn seeds at a mass ratio of 1:20 and the seeds are soaked for 10 min at a temperature of 30℃; (4) Compound microbial coating: The above compound microbial seed coating agent is mixed with corn seeds at a mass ratio of 1:20 and the seeds are soaked for 10 min at a temperature of 30℃.

[0068] Each treatment was performed in four replicates (four flowerpots), with the flowerpots placed at 25°C and a radiation intensity of 240 µmol / m². 2 In an artificial climate chamber with a capacity of / s, light is provided for 12 hours daily from 6:00 to 18:00.

[0069] Germination was defined as corn seedlings emerging 0.5 cm from the ground. Starting from the third day, the germination status of seeds in each treatment was observed at the same time every day. The germination rate was the percentage of germinated seeds to the total number of seeds tested.

[0070] Seed germination rate of maize on days 3 and 7 after sowing under four drought stress treatments is as follows: Figure 8 As shown. Figure 8The group consisting of Bacillus subtilis seed coating agent, Bacillus megaterium seed coating agent, and compound Bacillus megaterium seed coating agent is a compound bacterial seed coating agent. Figure 8 The germination rate is the seedling emergence rate. Figure 8 The left graph in the figure shows the germination rate of each treatment group on day 3; the right graph shows the germination rate of each treatment group on day 7. Different letters in the graph indicate significant differences.

[0071] Figure 8 The results showed that on the 3rd day after sowing, the germination rate of the compound microbial seed coating treatment reached the highest at 56.0%, which was significantly higher than that of the control and other treatments. The next highest germination rates were achieved by the Bacillus megaterium seed coating treatment, followed by the Bacillus subtilis seed coating treatment and the control. On the 7th day, the germination rates of all treatments increased significantly, with the compound microbial seed coating treatment reaching the highest at 98.0%, followed by the Bacillus megaterium seed coating treatment at 90.0%. There was no significant difference between the Bacillus subtilis seed coating treatment and the control.

[0072] Example 6 2000 corn seeds were ultrasonically treated with mixed frequency ultrasound for 40 seconds, the same method as in Example 1, referred to as ultrasonic treatment.

[0073] Take 1000 ultrasonically treated seeds and coat them with the compound microbial coating agent of Example 5. The method is as follows: mix the compound microbial coating agent with corn seeds at a mass ratio of 1:30 and soak the seeds for 10 minutes at a temperature of 30°C. This is recorded as ultrasonic + compound microbial treatment.

[0074] Take 500 seeds treated with ultrasound and compound bacteria and treat them with modified seaweed polysaccharide using the method described in Example 3. Specifically, 1g of modified seaweed polysaccharide was dissolved in 50g of deionized water and then diluted to 50mg / L for seed dressing. The prepared solution was poured into 1kg of corn seeds, stirred evenly, and then air-dried naturally. This was recorded as: ultrasound + compound bacteria + modified seaweed polysaccharide treatment.

[0075] Untreated seeds were used as a control.

[0076] Soil volumetric water content was set at 0.18 cm³. 3 / cm 3 Seed germination experiments were conducted in the soil; one seed was planted in each small pot, with 5 pots forming a group, and 3 groups were set up for each treatment, for a total of 4 treatments.

[0077] The results of maize growth monitoring on days 7, 10, and 20 after sowing under four drought stress treatments are as follows: Figure 9 As shown in Table 2. Figure 9(1) shows the growth status of each treatment group on day 7, (2) shows the growth status of each treatment group on day 10, and (3) shows the growth status of each treatment group on day 20.

[0078] Table 2. Maize growth under different treatments

[0079] As shown in Table 2 and Figure 9 As shown, the treatment with ultrasound + compound bacteria and ultrasound + compound bacteria + modified Ulva polysaccharide showed better results, especially the treatment with ultrasound + compound bacteria + modified Ulva polysaccharide. Due to poor soil structure, large spatial variability in soil moisture and nutrients, and numerous stress conditions faced by seeds, drought stress experiments indicated that the treatment with ultrasound + compound bacteria + modified Ulva polysaccharide significantly promoted maize seedling growth, achieving uniform and robust seedling emergence.

[0080] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of modified *Ulva prolifera* polysaccharides in improving seed vigor and / or seed drought resistance, wherein the modified *Ulva prolifera* polysaccharides comprise: Modified *Ulva prolifera* polysaccharide was prepared by carboxymethylation.

2. The application according to claim 1, characterized in that, The preparation method of the modified Ulva prolifera polysaccharide includes: The polysaccharide solution of Ulva prolifera was mixed with the NaOH aqueous solution to activate the hydroxyl groups of the polysaccharide, thus obtaining activated Ulva prolifera polysaccharide. The activated Ulva polysaccharide was mixed with a chloroacetic acid solution and subjected to a carboxymethylation reaction to obtain carboxymethylated Ulva polysaccharide, which is a reaction system containing modified Ulva polysaccharide.

3. The application according to claim 2, characterized in that, The mass percentage of *Ulva prolifera* polysaccharide in the aqueous solution is 10-20 wt%; the mass percentage of NaOH in the aqueous solution is 10-30 wt%; the mass ratio of the aqueous solution of *Ulva prolifera* polysaccharide to the aqueous solution of NaOH is 1:(1-2); the activation temperature of the polysaccharide hydroxyl groups is 20-40℃; and the activation time of the polysaccharide hydroxyl groups is 2-3 h.

4. The application according to claim 2, characterized in that, The chloroacetic acid solution contains 20-40% chloroacetic acid by mass; the mass ratio of the *Ulva prolifera* polysaccharide aqueous solution to the chloroacetic acid solution is 10:(1-2); the temperature of the carboxymethylation reaction is 50-80℃; and the time of the carboxymethylation reaction is 1-2 hours.

5. The application according to claim 2, characterized in that, The preparation method further includes the separation and purification of modified Ulva polysaccharide: the separation and purification method of modified Ulva polysaccharide includes: mixing the reaction system containing modified Ulva polysaccharide with ethanol to precipitate the modified Ulva polysaccharide.

6. A method for improving seed vigor, characterized in that, include: The modified *Ulva prolifera* polysaccharide used in any one of claims 1 to 5 is mixed with the seeds for seed dressing.

7. The method according to claim 6, characterized in that, The seed dressing method includes: mixing modified seaweed polysaccharide aqueous solution with seeds and then drying; the mass concentration of modified seaweed polysaccharide in the modified seaweed polysaccharide aqueous solution is 20~50mg / L; the mass ratio of modified seaweed polysaccharide aqueous solution to seeds is (50~100)g:1kg.

8. The method according to claim 6, characterized in that, Before seed dressing, the process also includes: Seeds are subjected to ultrasonic treatment to obtain ultrasonically treated seeds; The seeds treated with ultrasound were coated with a compound microbial coating agent.

9. The method according to claim 8, characterized in that, The ultrasonic treatment includes a mixed ultrasonic treatment; the frequency of the first ultrasonic wave in the mixed ultrasonic treatment is 20~40kHz, and the frequency of the second ultrasonic wave is 60~90kHz; the duration of the mixed ultrasonic treatment is 30~40s. The compound bacterial coating agent includes Bacillus subtilis and Bacillus megaterium.

10. The application of the method according to any one of claims 6 to 9 in improving the drought resistance of plants.

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