Application of a bacterial sugar composition in stress resistance regulation in saline-alkali soil

Abstract

The application discloses application of a bacteria-sugar composition in stress resistance regulation of saline-alkali soil and belongs to the technical field of plant growth regulation. The microorganism is Bacillus, and the plant source extract is a salvia miltiorrhiza-safflower composite polysaccharide. The biological preservation number of the Bacillus in the composition is CGMCC No. 25566, the bacterial concentration is 8.5*10 5 CFU / mL, and the salvia miltiorrhiza-safflower composite polysaccharide is prepared by mixing salvia miltiorrhiza and safflower as raw materials according to a mass ratio of (10~1):(1~10) and adopting polysaccharide extraction, and the concentration is 1 ug / mL. The Bacillus with the biological preservation number of CGMCC No. 25566, which is screened from medicinal plant salvia miltiorrhiza symbiotic microorganism, is combined with the salvia miltiorrhiza-safflower composite polysaccharide to obtain a bacteria-sugar composition for regulating crop tolerance to moderate to severe saline-alkali soil, and the application of salvia miltiorrhiza endophyte and plant source polysaccharide is further developed.

Description

Technical Field

[0001] This invention relates to the field of plant growth regulation technology, specifically to the application of a fungal sugar composition in the regulation of stress resistance in saline-alkali land. Background Technology

[0002] Coastal saline-alkali land is widely distributed in my country, accounting for nearly 40% of the total saline soil area. It is mainly distributed in the eastern coastal areas of the North China Plain, such as Liaodong Bay, Bohai Bay, Laizhou Bay, and the coastal areas of Jiangsu and Zhejiang provinces. These coastal areas generally receive over 500 mm of annual precipitation and have abundant groundwater resources with great potential for development and utilization. Due to constant seawater intrusion, excessive soluble salts accumulate in the surface layer or soil of these coastal areas, with chlorides being the dominant salt. Current treatment methods for coastal saline-alkali soil include planting salt-tolerant plants, freshwater leaching, chemical amendments, and biological amendments. Chemical and biological amendments have received more research. Chemical amendments use acidic salts to improve the properties of saline-alkali land, reducing soil pH, increasing cation exchange capacity, and lowering salinity. Biological amendments introduce biological or microbial agents to improve soil structure, increase organic matter, and promote salt degradation. However, reports on the direct regulation of crop salt tolerance using these amendments are limited.

[0003] According to the classification and grading standards for soil salinity from the Third National Soil Survey, the salinity classification and grading of saline-alkali cultivated land in coastal areas is based on the salt content (g / kg) of the topsoil layer (0-20 cm): slightly saline 1-2, moderate saline 2-4, severely saline 4-6, and saline / alkali soil ratio > 6. Calculated in per‰, soil with a salt content of 3-6‰ is classified as moderately to severely saline-alkali soil. Currently, most saline-alkali land improvement efforts focus on slightly and moderately saline-alkali land. Patent application number CN202310461661.3 discloses a multifunctional endophytic bacterium of *Salvia miltiorrhiza* and its application. This bacterium, with good antifungal activity against plant pathogenic fungi, was screened from epiphytic microorganisms of the medicinal plant *Salvia miltiorrhiza*. It can also be used to increase maize yield in slightly to moderately saline-alkali land, even under conditions of lower salt tolerance. This strain was used on saline-alkali land with a salinity of 3‰ to increase corn yield. However, for moderately to severely saline-alkali land, where the salt content is generally greater than 3‰, it is difficult to achieve salt tolerance in such land using only the strain. Therefore, it is necessary to develop a microbial agent composition containing endophytic bacteria from *Salvia miltiorrhiza* to regulate crop tolerance to moderately to severely saline-alkali conditions, and to further explore the application of *Salvia miltiorrhiza* endophytic bacteria. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of a bacterial saccharide composition in the regulation of stress resistance in saline-alkali land. This invention uses Bacillus subtilis (CGMCC No. 25566), screened from epiphytic microorganisms of the medicinal plant *Salvia miltiorrhiza*, in combination with *Salvia miltiorrhiza* and safflower polysaccharides to obtain a bacterial saccharide composition that regulates crop tolerance to moderate to severe salinity and alkali conditions, further expanding the application of *Salvia miltiorrhiza* endophytic bacteria and plant-derived polysaccharides.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides an application of a microbial saccharide composition in the regulation of stress resistance in saline-alkali land, wherein the microbial saccharide composition comprises microbial bacteria and plant-derived extracts;

[0007] The microorganism is Bacillus; the plant-derived extract is a complex polysaccharide of Salvia miltiorrhiza and Carthamus tinctorius.

[0008] Preferably, the biological preservation number of the Bacillus is CGMCC No. 25566.

[0009] Preferably, the tanshinone and safflower complex polysaccharide is prepared by mixing tanshinone and safflower in a mass ratio of (10~1):(1~10) and extracting the polysaccharide.

[0010] Preferably, the preparation method of the Danshen-safflower complex polysaccharide is as follows:

[0011] The danshen and safflower were pulverized and sieved, and then extracted with deionized water using ultrasonic-assisted extraction. After centrifugation and filtration, an aqueous extract was obtained. Sevage reagent was added to the aqueous extract to remove proteins. The upper aqueous phase was taken and ethanol was added for alcohol precipitation. After refrigeration and standing, the mixture was filtered and freeze-dried to obtain the danshen and safflower complex polysaccharide.

[0012] Preferably, the mass ratio of Salvia miltiorrhiza to Carthamus tinctorius is 3:1.

[0013] Preferably, the concentration of the Bacillus is 8.5 × 10⁻⁶. 5 CFU / mL.

[0014] Preferably, the concentration of the Danshen-safflower complex polysaccharide is 1 μg / mL.

[0015] Preferably, the saline-alkali land has a salt content of 3-6‰.

[0016] Preferably, the application includes increasing the net photosynthetic rate, transpiration rate, stomatal conductance, single-plant dry weight and yield of crops in saline-alkali land, and reducing the intercellular CO2 concentration of crops in saline-alkali land.

[0017] Preferably, the bacterial sugar composition is sprayed onto the leaves of crops grown in saline-alkali soil at a rate of 225-375 L / hm. -2 .

[0018] The beneficial effects of this invention are:

[0019] (1) The present invention uses Bacillus spp. with biological preservation number CGMCCNo.25566, which was screened from the epiphytic microorganisms of the medicinal plant Salvia miltiorrhiza, in combination with Salvia miltiorrhiza and Carthamus tinctorius polysaccharide to obtain a bacterial sugar composition that regulates the crop's tolerance to moderate to severe salinity and alkali, further expanding the application of Salvia miltiorrhiza endophytic bacteria and plant-derived polysaccharides.

[0020] (2) Compared with chemically synthesized agents, the tanshinone and safflower polysaccharide components in the bacterial sugar composition of the present invention are safer and have fewer side effects. The bacterial sugar composition of the present invention acts on the leaves of crops and can effectively activate the plant's defense mechanism and alleviate crop damage caused by moderate to severe salt stress. Salt tolerance tests on maize show that foliar spraying once each at the tasseling and tasseling stages can significantly improve crop salt tolerance: increase net photosynthetic rate, transpiration rate, stomatal conductance, single-plant dry weight and yield, and reduce intercellular CO2 concentration in crops grown in saline-alkali soil. Attached Figure Description

[0021] Figure 1 Dry weight of individual maize plants in different treatment groups. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] As introduced in the background section, current research on crop salt tolerance mostly involves adding various soil conditioners to saline-alkali soils, while reports on applying agents directly to crops to regulate their salt tolerance are relatively few. Furthermore, most reports on salt tolerance focus on mild to moderate salt tolerance; achieving tolerance to moderate to severe salt tolerance remains a significant challenge.

[0024] Therefore, the purpose of this invention is to provide an application of a bacterial polysaccharide composition in the regulation of stress resistance in saline-alkali land. This invention combines Bacillus subtilis (CGMCC No. 25566) with a complex polysaccharide from Salvia miltiorrhiza and Carthamus tinctorius, with the Bacillus subtilis concentration being 8.5 × 10⁻⁶. 5 The concentration of CFU / mL and the concentration of Danshen-safflower polysaccharide compound polysaccharide was 1 μg / mL; it was sprayed on the crop leaves. Taking corn as an example, foliar spraying once each at the large trumpet stage and the tasseling stage can significantly improve the crop's salt tolerance, and its effect is significantly higher than using Bacillus or Danshen-safflower polysaccharide compound polysaccharide alone.

[0025] When crops are subjected to abiotic stresses such as high temperature, salinity, and drought, they are highly susceptible to imbalances in reactive oxygen species metabolism and cell membrane damage, leading to damage to the photosynthetic system and causing wilting. This invention combines microbial agents with functional plant polysaccharides. The microorganisms enhance crop stress resistance through growth hormone secretion and improved nutrient absorption; the polysaccharides effectively scavenge the accumulation of reactive oxygen free radicals caused by salinity stress, enhancing the crop's resistance to saline-alkali soils. This combination of microbial agents and polysaccharides significantly improves the crop's salt and alkali resistance, promoting increased yield.

[0026] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0027] Note: The Bacillus strain used in this invention has the biological accession number CGMCC No. 25566, and the depository institution is the China General Microbiological Culture Collection Center. This strain is described in the patent application CN202310461661.3, entitled "A Multifunctional Endophytic Bacterium of Salvia miltiorrhiza and Its Application".

[0028] According to the "Classification and Grading Standards for Soil Salinity in the Third National Soil Census" in the "Implementation Plan for the Special Survey of Saline-Alkali Land Soil," the salinity classification and grading of saline-alkali cultivated land in coastal areas is based on the salt content (g / kg) of the topsoil layer (0-20 cm): slightly saline 1-2, moderate saline 2-4, severely saline 4-6, and saline / alkali soil ratio > 6. Therefore, calculated in per‰, soil with a salt content of 3-6‰ is classified as moderately to severely saline-alkali soil.

[0029] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0030] Example 1: Preparation of Danshen and Safflower Complex Polysaccharide

[0031] Take 3 g of Salvia miltiorrhiza and 1 g of Carthamus tinctorius, pulverize them and pass them through a 40-mesh sieve; add 400 g of deionized water; perform ultrasonic-assisted extraction of polysaccharides at a frequency of 30 kHz, a power of 150 W, and a time of 30 min. After extraction, centrifuge and filter to obtain an aqueous extract. Add Sevage reagent (prepared by mixing n-butanol and chloroform at a volume ratio of 1:4) to the aqueous extract to remove proteins. The mass ratio of the aqueous extract to Sevage reagent is 4:1. Take the upper aqueous phase and add ethanol to make the final ethanol concentration about 70 wt%. Refrigerate at 4℃ and let stand for 24 h. After filtration, freeze-dry at -30℃ for 48 h to obtain Salvia miltiorrhiza and Carthamus tinctorius complex polysaccharides.

[0032] Example 2: Culture of Bacillus

[0033] Bacillus (CGMCC No. 25566) was inoculated into liquid PDA medium and cultured at 200 rpm and 30°C for 3 days to obtain a bacterial concentration of 8.5 × 10⁻⁶. 5 CFU / mL bacterial suspension.

[0034] Example 3

[0035] Take the bacterial culture prepared in Example 2, add the tanshinone and safflower complex polysaccharide prepared in Example 1 to a concentration of 1 μg / mL. Mix well to obtain the saline-alkali land stress-regulating bacterial polysaccharide composition.

[0036] Comparative Example

[0037] The tanshinone and safflower complex polysaccharide prepared in Example 1 was added to water to a concentration of 1 μg / mL to obtain a saline-alkali land stress-regulating polysaccharide.

[0038] Test case

[0039] 1. Experimental setup

[0040] A field experiment was conducted at the Yibang Agricultural Technology Experimental Base in Kenli District, Dongying City, Shandong Province (37°5'N, 118°36'E), which has a warm temperate monsoon climate with four distinct seasons and abundant sunshine. Soil samples were collected on June 15, 2022, and the soil pH was measured to be 7.63, with an average salinity of 4‰, classifying it as moderately to severely saline-alkali coastal land.

[0041] The experimental material used was the maize variety Denghai 605, sown on June 15th each year, with a planting density of 67,500 plants per hectare. -2 The row spacing was 60 cm, and the plant spacing was 24.5 cm. The fertilizer used in the experiment was active humic acid compound fertilizer (N:P2O5:K2O=26:11:14), and the application rate was 750 kg / hm². -2 Apply the fertilizer once before sowing. Before sowing, prepare the land thoroughly and ensure adequate soil moisture. Thin the seedlings to the designed density at the 3-leaf stage. Irrigate according to weather conditions and soil moisture, and promptly control pests, diseases, and weeds. Other cultivation and management practices follow local farmers' habits. When the corn reaches the designated growth stage, apply exogenous substances via foliar spraying.

[0042] The experiment was divided into four groups: the water control group (denoted as CK), the Example 3 group (denoted as T), the Example 2 group (denoted as SNMB1), and the Comparative Example 1 group (denoted as 3S1C).

[0043] The experiment involved a single foliar spraying at the tasseling and monsoon stages (V9) and tasseling stages of maize, using an electric sprayer at a rate of 300 L / hm². -2No other chemical control agents are used during the growing season. Each group is planted with dimensions of 5 m long and 6 m wide, covering an area of ​​30 m². 2 .

[0044] 2. Measurement Items and Methods

[0045] 2.1 Leaf photosynthetic performance

[0046] 2.1.1 Leaf area index and leaf photosynthetic potential

[0047] Five plants with uniform growth were selected in the field at the flowering (R1), milk stage (R3), and maturity stage (R6) of each treatment group. The leaf length and width of all green leaves were measured, the leaf area index (LAI) was calculated, and the leaf photosynthetic potential (LAD) was calculated.

[0048] Fully unfolded leaf area (cm²) 2 = Leaf length (cm) × Leaf width (cm) × 0.75;

[0049] Leaf area of ​​a single leaf that is not fully expanded (cm²) 2 = Leaf length (cm) × Leaf width (cm) × 0.5;

[0050] Leaf Area Index (LAI) = (Leaf area per plant × Number of plants per unit land area) / Unit land area.

[0051] LAD=(LA2+LA1) / 2×(t2-t1);

[0052] In the formula, LA1 and LA2 are the leaf area indices per unit land area during time periods t1 and t2, respectively.

[0053] 2.1.2 Gas exchange parameters

[0054] Each treatment group underwent R1 and R3 using a CIRAS-II photosynthesis system (PP Systems, USA) on a sunny day from 10:00 to 12:00, at a concentration of 1600 µmol / m³. -2 s -1 Under light intensity conditions, 10 representative plants from each plot were selected to measure the photosynthetic performance of the ear-position leaves: net photosynthetic rate of ear-position leaves (Pn, μmol m). -2 s -1 ), transpiration rate (Tr, mmol m -2 s -1 Stomatal conductance (Gs, mmol m) -2 s -1 ) and intercellular CO2 concentration (Ci, μmol mol) -1 ).

[0055] 2.2 Dry matter accumulation

[0056] Five plants of uniform growth were selected from each treatment at the flowering (R1), milk stage (R3), and maturity stage (R6). The above-ground parts were separated into stems (including leaf sheaths), leaves, female ears (column + grains), male ears, and husks and placed in kraft paper bags. The bags were placed in an oven at 105℃ for 30 min to kill the green, and then dried at 80℃ to constant weight. After cooling, the dry weight was measured.

[0057] 2.3 Output and its components

[0058] During the corn harvest season, select plots with uniform growth of 5 m in length and 3 rows in width for harvesting and yield measurement. Count the number of plants per unit area, the number of ears, and the double ear rate. After natural air drying, conduct indoor seed testing to count the number of ear rows, the number of ears per hectare, and the thousand-grain weight (moisture content 14%), and calculate the yield per hectare.

[0059] Yield (kg / hm) 2 = Number of ears per hectare × Grain weight per ear × Thousand grains weight

[0060] 3. Test Results

[0061] 3.1 Leaf photosynthetic performance

[0062] The photosynthetic performance of the leaves is shown in Tables 1 and 2.

[0063] Table 1 Leaf area index and photosynthetic potential

[0064]

[0065] As shown in Table 1, after spraying the T, SNMB1, and 3S1C treatments, the leaf area index (LAI) at stage R1 increased by 22.8%, 9.3%, and 11.4% compared to the control (CK); at stage R3, the LAI increased by 22.8%, 10.4%, and 12.0% respectively; and at stage R6, the LAI increased by 27.2%, 12.9%, and 14.2% respectively. Notably, the LAI of the 3S1C group was slightly higher than that of the SNMB1 group, indicating that the effect of using polysaccharides alone was slightly better than that of using inoculants alone. It can be seen that foliar spraying of the saline-alkali land stress-regulating inoculant-sugar composition prepared in Example 3 significantly improved the LAI of summer maize under salt stress in saline-alkali land compared to using inoculants or polysaccharides alone, thus maintaining a higher green leaf area throughout the growth period.

[0066] Compared with the control (CK), the total photosynthetic potential during the R1, R3, and R6 growth stages increased by 23.88%, 10.23%, and 13.03%, respectively, after foliar spraying of the T, SNMB1, and 3S1C treatments. The increase in leaf photosynthetic potential improved plant photosynthetic performance, which is beneficial for plant biomass accumulation. This indicates that foliar spraying of the saline-alkali soil stress-regulating bacterial-sugar composition prepared in Example 3 significantly improved plant biomass accumulation compared to using bacterial agents or polysaccharides alone.

[0067] Table 2 Blade Gas Exchange Parameters

[0068]

[0069] As shown in Table 2, compared with the control (CK), the net photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) were all increased after spraying the T, SNMB1, and 3S1C treatments. Notably, the transpiration rate of the 3S1C group was higher than that of the T group in the R1 stage, but decreased significantly in the R3 stage. The intercellular CO2 concentration (Ci) in leaves decreased after spraying the T, SNMB1, and 3S1C treatments, with the most significant decrease in the T group. The overall gas exchange parameters showed a pattern of T > 3S1C > SNMB1 > CK, with the 3S1C group slightly higher than the SNMB1 group. This indicates that foliar spraying of the saline-alkali land stress-regulating bacterial sugar composition prepared in Example 3 can significantly increase Pn, Tr, and Gs in summer maize leaves in saline-alkali land, and reduce Ci, thus providing a guarantee for biomass accumulation and yield improvement.

[0070] 3.2 Dry matter accumulation

[0071] The amount of dry matter accumulation is shown in Table 3 and Figure 1 .

[0072] according to Figure 1 It can be seen that, compared with the control (CK), the dry weight of individual plants in the R6 stage increased by 31.18%, 14.24%, and 15.16% respectively after spraying the T, SNMB1, and 3S1C treatments.

[0073] Table 3. Dry weight of maize per plant under different treatments

[0074]

[0075] As shown in Table 3, compared with the control group, the dry matter content of maize in stages R1 to R6 was improved after spraying with the T, SNMB1, and 3S1C treatments. The increase in the T group was significantly higher than that in the SNMB1 and 3S1C groups, indicating that the addition of the bacterial saccharide composition significantly improves the dry matter content of maize compared to using SNMB1 bacteria or the tanshinone and safflower polysaccharide alone. The bacterial saccharide composition of this invention is beneficial for the accumulation of dry matter.

[0076] 3.3 Output and its components

[0077] Table 4 shows the maize yield and yield components of different treatment groups.

[0078] Table 4. Maize yield and its components in different treatment groups

[0079]

[0080] As shown in Table 4, the yield of treatment T was 11651 kg / hm² compared to treatment CK. 2 The yield of the SNMB1 treatment was 10603 kg / hm², an increase of 18.34% compared to the control (CK). 2 The yield of the 3S1C treatment was 10710 kg / hm², an increase of 7.69% compared to the control (CK). 2 The yield increased by 8.78% compared to the control (CK). Example 3 shows that the yield of maize tolerant to moderate to severe salinity and alkalinity can be significantly increased by using a combination of microbial agents and polysaccharides, indicating that the microbial-polysaccharide composition of the present invention can significantly improve the maize's tolerance to moderate to severe salinity and alkalinity.

[0081] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of a bacterial saccharide composition in the regulation of stress resistance in saline-alkali land, characterized in that, The saline-alkali land has a salt content of 4‰; the bacterial sugar composition is a microbial and plant-derived extract; the application is to improve the net photosynthetic rate, transpiration rate, stomatal conductance, single-plant dry weight and yield of crops in saline-alkali land, and reduce the intercellular CO2 concentration of crops in saline-alkali land. The microorganism is Bacillus; the plant-derived extract is a complex polysaccharide of Salvia miltiorrhiza and Carthamus tinctorius. The biosecurity number of the Bacillus is CGMCC No. 25566; the concentration of the Bacillus is 8.5 × 10⁻⁶. 5 CFU / mL; The Danshen-Carthamus tinctorius complex polysaccharide is prepared by mixing Danshen and Carthamus tinctorius in a mass ratio of 3:1 and then extracting the polysaccharide. The preparation method of the Danshen and Safflower complex polysaccharide is as follows: The danshen and safflower were pulverized and sieved, and then subjected to ultrasonic-assisted extraction with deionized water. After centrifugation and filtration, an aqueous extract was obtained. Sevage reagent was added to the aqueous extract to remove proteins. The upper aqueous phase was precipitated with ethanol, refrigerated and allowed to stand, filtered, and then freeze-dried to obtain the danshen and safflower complex polysaccharide. The concentration of the danshen and safflower complex polysaccharide was 1 μg / mL.

2. The application according to claim 1, characterized in that, The bacterial sugar composition was sprayed onto the leaves of crops grown in saline-alkali soil at a rate of 225-375 L / hm. -2 .

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