Compound microbial inoculant for improving drought resistance of crops and application thereof

Abstract

The application provides a compound microbial agent, which comprises bacillus velezensis (Bacillus velezensis) Bacillus vezelensis ) with a preservation number of CGMCC No.24516 and pseudomonas fluorescens (Pseudomonas fluorescens) Pseudomonas fluorescens ) with a preservation number of CGMCC No.14105, and the ratio of the bacillus velezensis and the pseudomonas fluorescens is 1-3:1-6, and the compound microbial agent can be used for improving drought resistance of crops.

Description

Technical Field

[0001] This invention belongs to the field of plant growth regulators, specifically relating to a compound microbial agent for improving crop drought resistance and its application. Background Technology

[0002] Bacillus belyssus, a newly discovered species of Bacillus that has attracted considerable attention in recent years, has shown great application potential in agricultural biological control, environmental remediation, aquaculture, and feed additives due to its broad-spectrum antibacterial activity, strong environmental adaptability, and metabolic diversity. In agriculture, Bacillus belyssus is commonly used for biological control and promoting plant growth. It can effectively control several important diseases such as anthracnose in peppers, powdery mildew in cucumbers, and black shank in tobacco. It can also secrete hormones such as indoleacetic acid, which directly stimulate crop roots and promote crop growth.

[0003] *Pseudomonas fluorescens*, with its highly efficient metabolic capacity, diverse secretion systems, and strong environmental adaptability, shows broad application prospects in agricultural biological control, environmental remediation, protein expression, and industrial biotechnology. In agriculture, it is commonly used for biological control and promoting plant growth. *Pseudomonas fluorescens* can inhibit the growth of pathogens through mechanisms such as secreting antibacterial substances and competing for nutrient space, thereby achieving disease control. It can also promote plant growth through various pathways, including secreting plant hormones and activating soil nutrients.

[0004] As two core global food crops, maize and wheat yield stability is directly related to food security and sustainable agricultural development. Drought is the primary abiotic stress factor restricting maize and wheat production, with global drought stress causing maize yield reductions of 20% to 50% and wheat yield reductions of 15% to 40%. With the intensification of global climate change, the frequency and duration of extreme drought events have increased significantly, posing a serious threat to the growth, development, photosynthetic efficiency, and yield formation of maize and wheat.

[0005] Chinese patent CN108004185B discloses the application of Bacillus belyceae E6 in improving plant drought resistance. Although various Bacillus strains have been disclosed in the prior art for improving crop drought resistance, differences still exist between different strains of the same type, and bacteria of the same type do not necessarily have the same capabilities. Furthermore, different strains may exhibit antagonistic effects, which can affect efficacy when combined into inoculants. Therefore, screening for compound inoculants that can improve crop stress resistance is of great significance. Summary of the Invention

[0006] Based on the above, the purpose of this invention is to provide a compound microbial agent for improving the drought resistance of wheat and corn. The technical solution of this invention is as follows: A compound microbial agent, said compound microbial agent comprising Bacillus belye ( Bacillus vezelensis ), with accession number CGMCC No. 24516 (Chinese Patent CN202210901172.0) and Pseudomonas fluorescens ( Pseudomonas fluorescens The accession number is CGMCC No.14105 (Chinese Patent CN201710480486.7), and the ratio of Bacillus belysae to Pseudomonas fluorescens is 1~3:1~6.

[0007] Preferably, the ratio of Bacillus belyi to Pseudomonas fluorescens is 1:3.

[0008] Preferably, the weight percentage of Bacillus belyi and Pseudomonas fluorescens in the compound bacterial agent is 0.1% to 20%.

[0009] Preferably, the compound microbial agent further includes one or more of wetting agents, dispersants, stabilizers, and fillers by weight percentage.

[0010] The compound microbial agent is used to improve the stress resistance of crops.

[0011] Preferably, the crops are wheat and corn.

[0012] Preferably, the resilience is drought resistance.

[0013] The beneficial effects of this invention are: 1. The compound microbial agent provided by this invention can improve the drought resistance of wheat.

[0014] 2. The compound microbial agent provided by this invention can improve the drought resistance of corn. Detailed Implementation

[0015] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0017] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available.

[0018] I. Formulation Examples Example 1 Bacillus vesiculosus (100 billion CFU / g), 3%; Fluorescent Pseudomonas (100 billion CFU / g), 3%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0019] Example 2 Bacillus vesiculosus (100 billion CFU / g), 2%; Fluorescent Pseudomonas (100 billion CFU / g), 4%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0020] Example 3 Bacillus vesiculosus (100 billion CFU / g), 1.5%; Fluorescent Pseudomonas (100 billion CFU / g), 4.5%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0021] Example 4 Bacillus vesiculosus (100 billion CFU / g), 1.2%; Fluorescent Pseudomonas (100 billion CFU / g), 4.8%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0022] Example 5 Bacillus belyssus (100 billion CFU / g), 2.4%; Fluorescent Pseudomonas (100 billion CFU / g), 3.6%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0023] Comparative Example 1 Bacillus belyssus (100 billion CFU / g), 6%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0024] Comparative Example 2 Fluorescent Pseudomonas (100 billion CFU / g), 6%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0025] Comparative Example 3 Bacillus belyssus (100 billion CFU / g), 0.75%; Fluorescent Pseudomonas (100 billion CFU / g), 5.25%; Alkyl naphthalene sulfonate, 3%; Sodium lignosulfonate, 6%; Trehalose, 3%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0026] II. Wheat drought resistance test Test reagents: Example 1, Example 2, Example 3, Example 4, Example 5, Comparative Example 1, Comparative Example 2, Comparative Example 3.

[0027] The tested crop was wheat, specifically Xinong 389.

[0028] Select uniformly sized, plump, and disease-free wheat seeds. Treat them with 70% ethanol for 1 minute, then rinse with sterile water. Next, treat them with 1% sodium hypochlorite for 10 minutes, rinse with sterile water, and then place them on a petri dish lined with sterile, damp gauze. Germinate them at 28°C for later use. Plant 6 seeds per plastic pot containing sterile soil and place them in a greenhouse for conventional cultivation for 20 days.

[0029] Drought stress was treated using a pot-based controlled water method. The soil moisture content in the drought stress treatment group was set at 50% of field capacity. Water was controlled before the experiment began, and the experiment commenced once the required moisture content was reached. The test agent was prepared at a 2000-fold dilution and sprayed evenly onto the plants. The water treatment served as control 1 (CK1), and the normal management (no drought stress, soil moisture content at 70% of field capacity) and no application of the test agent served as control 2 (CK2). Wheat leaf samples were collected 30 days after application for testing.

[0030] Superoxide dismutase (SOD) activity was determined according to the Sun Qun method (Sun Qun, ed., *Techniques for Plant Physiological Research*, 2005). 0.5 g of wheat material was ground in a mortar with 2 ml of SOD extraction medium, and the volume was adjusted to 10 ml in a stoppered test tube. 5 ml of the extract was centrifuged at 10,000 rpm for 15 min to obtain the crude SOD extract. Two control tubes (without the crude extract) and one dark control tube were prepared and placed together with the test tube containing the crude extract under four 40W fluorescent lamps for color development, reacting for 15–20 min. The dark control tube was used as a blank, and the absorbance of the reaction solution was measured at 560 nm.

[0031] SOD activity (U / g·h FW)=[(A0-As)×Vt×60] / (A0×0.5×FW×Vs×t) In the formula: A0 is the absorbance of the control tube under light; As is the absorbance of the sample test tube; Vt is the total volume of the sample extract (ml); Vs is the volume of crude enzyme solution taken during the test (ml); FW is the fresh weight of the sample (g); t is the light exposure time for the colorimetric reaction (min).

[0032] Peroxidase (POD) activity was determined according to the Sun Qun method (Sun Qun, ed., *Techniques for Plant Physiological Research*, 2005). 0.5 g of wheat material was ground in a mortar with 20 mmol of KH₂PO₄, and the mixture was brought to a 10 ml stoppered test tube. 5 ml of the extract was centrifuged at 5000 g for 15 min to obtain the enzyme extract. A test tube without enzyme extract was used as a blank for zeroing the instrument. A stopwatch was started immediately after each addition of enzyme extract, and absorbance values ​​were read at 470 nm for 0–3 min, with readings taken every 1 min.

[0033] POD activity (μg / g·min FW) = (X×Vt) / (FW×Vs×t) In the formula: FW is the fresh weight of the sample (g); Vs is the volume of crude enzyme solution taken during the determination (ml); Vt is the total volume of the enzyme solution (ml); t is the enzyme reaction time (min); X is A 240 The difference between 0 and 3 minutes.

[0034] Catalase (CAT) activity was determined according to the Sun Qun method (Sun Qun, ed., *Techniques for Plant Physiological Research*, 2005). 1.00 g of wheat material was ground in a mortar with an appropriate amount of phosphate buffer and the mixture was brought to a 10 ml stoppered test tube. 5 ml of the extract was centrifuged at 10000 rpm for 15 min to obtain the enzyme extract. A test tube without enzyme extract was used as a blank for zeroing the instrument. A stopwatch was started immediately after each addition of enzyme extract, and absorbance readings were taken at 240 nm every 30 seconds from 0 to 3 minutes.

[0035] CAT activity (μg / g·min FW) = (X×Vt) / (FW×Vs×t) In the formula: FW is the fresh weight of the sample (g); Vs is the volume of crude enzyme solution taken during the determination (ml); Vt is the total volume of the enzyme solution (ml); t is the enzyme reaction time (min); X is A 240 The difference between 0 and 3 minutes.

[0036] Malondialdehyde (MDA) content was determined according to the Sun Qun method (Sun Qun, ed., *Techniques for Plant Physiological Research*, 2005). 0.3 g of leaves from different leaf positions on wheat plants were weighed, and extracted in a mortar with a small amount of quartz sand and 5 ml of 5% TCA solution in an ice bath. The homogenate was centrifuged at 3000 g for 15 min, and 2 ml of the supernatant was discarded. 5 ml of 0.5% thiobarbituric acid solution was added and boiled in boiling water for 10 min. After cooling, the solution was centrifuged for 15 min, and the absorbance of the supernatant was measured at wavelengths of 532 nm, 600 nm, and 450 nm.

[0037] MDA content (mmol / g FW) = [6.452 × (A 532 -A 600 -0.559×A 450 ]×[Vt / (Vs×FW)] In the formula: Vt is the total volume of the extract (ml); Vs is the volume of the extract used for determination; FW is the fresh weight of the sample.

[0038] The experimental results are shown in the table below. Compared with CK1, Examples 1 to 5 can all increase the activity of SOD, POD and CAT in wheat leaves, and can effectively reduce the content of MDA. Among them, Example 3 has the best effect, that is, the effect is best when the ratio of Bacillus belye and Pseudomonas fluorescens is 1:3.

[0039] Table 1. Data on wheat drought resistance measurement

[0040] Wheat often experiences membrane lipid peroxidation under drought stress. This peroxidation damages membrane function and causes membrane phase separation. MDA, a product of membrane lipid peroxidation, has a direct toxic effect on plant cells. Higher MDA levels indicate more severe membrane damage and weaker plant resistance. Increased activity of SOD, POD, and CAT helps to remove membrane lipid peroxidation products from wheat plants, reducing their damaging effects on plant cells and thus improving the drought resistance of wheat.

[0041] The above experimental results show that the compound microbial agent provided by the present invention can effectively reduce the content of MDA and reduce its damage to cells. At the same time, the compound microbial agent provided by the present invention can also increase the activity of SOD, POD and CAT, and improve the wheat's ability to scavenge superoxide anion free radicals and H2O2, thereby improving the wheat's drought resistance.

[0042] III. Experiment on the drought resistance of maize Test reagents: Example 1, Example 2, Example 3, Example 4, Example 5, Comparative Example 1, Comparative Example 2, Comparative Example 3.

[0043] Test crop: Maize, Zhengdan 958.

[0044] Select a suitable amount of corn seeds of uniform size and plump kernels. First, disinfect them with 10% sodium hypochlorite for 10 minutes, then rinse them three times with sterile water for 5 minutes each time. Finally, place them in a dark room to germinate. When the sprouts grow to 2-3 cm, select seeds with uniform sprout length and transplant them into plastic pots containing sterilized soil. Cultivate them under normal management conditions.

[0045] When the maize reached the three-leaf stage, a pot-based water-controlled treatment was used to induce drought stress. The soil moisture content in the drought stress treatment group was 50% of the field capacity. Water was controlled before the experiment began, and the experiment commenced once the required moisture content was reached. The test agent was prepared at a 2000-fold dilution and sprayed evenly onto the plants. The water treatment served as control 1 (CK1), and the normal management (no drought stress, soil moisture content at 70% of field capacity) and no application of the test agent served as control 2 (CK2). Maize leaf samples were collected 7 days after application for testing.

[0046] Superoxide dismutase (SOD) activity was determined according to Li Gang's method (Regulatory effect of S-inducer on maize growth under drought stress, 2017). The assay solution was prepared by adding 2 mL each of 130 mmol / L methionine solution, 750 μmol / L nitroblue tetrazolium solution, 100 μmol / L sodium ethylenediaminetetraacetate solution, and 20 μmol / L riboflavin solution to 2.5 mL of deionized water and 15 mL of phosphate buffer (0.05 mol / L, pH=7.8).

[0047] Take 0.5g of corn leaves to be tested, place them in a pre-cooled mortar, add a small amount of liquid nitrogen, a little quartz sand, and 2mL of pre-cooled pH=7.8, 0.5mmol / L phosphate buffer, and grind into a homogenate. Then add 3mL of buffer solution and centrifuge at 5000r / min for 15min at 4℃. Take the supernatant as the crude enzyme solution. Add 20μL of enzyme solution and 3mL of reaction solution to a test tube, and irradiate with 4000Lux light for 30min. There are 3 test tubes for each treatment and 1 control tube (buffer solution is used instead of enzyme solution). The control tube is placed in the dark, and the treatment tubes are irradiated with 4000Lux light for 30min. Store in the dark, zero the instrument with a blank tube, and measure the absorbance at 560nm.

[0048] Peroxidase (POD) activity was determined according to Li Gang's method (Regulatory effect of S-inducer on maize growth under drought stress, 2017). 0.5 g of maize leaves were placed in a pre-cooled mortar, and a small amount of liquid nitrogen, a small amount of quartz sand, and 2 mL of pre-cooled 0.2 mol / L, pH 7.0 phosphate buffer (containing 0.1% polyvinylpyrrolidone) were added. The mixture was ground into a homogenate, and then 6 mL of buffer was added to rinse the mortar. The mixture was centrifuged at 5000 g for 10 min at 4 °C, and the supernatant was used as the crude enzyme solution. A 5 mL reaction system contained 2 mL of 0.2 mol / L, pH 5.0 acetate buffer, 1.0 mL of 0.1% guaiacol, 1.0 mL of 2.0% H2O2, and 1.0 mL of crude enzyme solution diluted 5 times. The enzyme solution boiled for 5 min was used as a control. After adding the enzyme solution to the reaction system, the system was immediately incubated in a 34 °C water bath for 3 min, and H2O2 was added to initiate the reaction. The activity of A was rapidly measured within 5 min. 470 The absorbance value was measured every 30 seconds to monitor changes in absorbance. The absorbance was recorded in units of absorbance per minute (A). 470 An increase of 0.01 represents one enzyme activity unit. The specific activity of enzymes in fresh material is expressed as U / g·min.

[0049] Catalase (CAT) activity was determined according to Li Gang's method (Regulatory effect of S-inducin on maize growth under drought stress, 2017). 0.5 g of maize leaves were placed in a pre-cooled mortar, and a small amount of liquid nitrogen, a small amount of quartz sand, and 2 mL of pre-cooled 0.2 mol / L, pH 7.0 phosphate buffer (containing 0.1% polyvinylpyrrolidone) were added. The mixture was ground into a homogenate, and then 6 mL of buffer was added to rinse the mortar. The mixture was centrifuged at 5000 g for 10 min at 4 °C, and the supernatant was used as the crude enzyme solution. 2.0 mL of 0.1 mol / L, pH 7.0 phosphate buffer, 1.0 mL of 0.1% H2O2, and 1.0 mL of the crude enzyme solution diluted 30 times were added to a test tube. After preheating to 25 °C, H2O2 was added to initiate the reaction, and the activity of catalase (CAT) within 4 min was measured. 240 Changes in absorbance values. One enzyme activity unit is defined as a change in absorbance of 0.01 per gram of sample per minute. The specific activity of enzymes in fresh material is expressed as U / g·min.

[0050] Malondialdehyde (MDA) content was determined according to Li Gang's method (Regulatory effect of S-inducer on maize growth under drought stress, 2017). Three to five maize leaves were taken, washed, dried, and cut into 0.5 cm segments. 0.2 g of the leaves were mixed, and 2 mL of 0.1% TCA and a small amount of quartz sand were added. The mixture was ground into a homogenate, and then further ground with an appropriate amount of TCA. The homogenate was transferred to a 5 mL centrifuge tube and centrifuged at 5000 rpm for 10 min. The supernatant was used as the MDA extract. 1 mL of the supernatant (with 1 mL of 0.1% TCA added as a blank) was taken, and 3 mL of 0.6% TBA solution was added. The mixture was incubated in a boiling water bath for 30 min, rapidly cooled, and then centrifuged at 5000 rpm for 10 min. For MDA content determination, 0.6% thiobarbituric acid was used as a blank. The absorbance of the supernatant was measured at 532 nm, 600 nm, and 450 nm.

[0051] MDA content (μmol / g FW) = [6.452 × (A 532 -A 600 -0.559×A 450 )×[Vt / (1000×FW)) In the formula: Vt is the total volume of the extract (mL); FW is the fresh weight of the sample (g).

[0052] The experimental results are shown in the table below. Compared with CK1, Examples 1 to 5 can all increase the activity of SOD, POD and CAT in maize leaves, and can effectively reduce the content of MDA. Among them, Example 3 has the best effect, that is, the effect is best when the ratio of Bacillus belye and Pseudomonas fluorescens is 1:3.

[0053] Table 2. Data on drought resistance of maize

[0054] When maize is subjected to drought stress, membrane lipid peroxidation often occurs within the plant. The products of membrane lipid peroxidation damage the membrane system, leading to cell damage in maize plants. MDA is a product of membrane lipid peroxidation; the higher its content, the more severe the damage to the biomembrane. Increased activity of SOD, POD, and CAT helps to clear membrane lipid peroxidation products from maize plants, reducing their damaging effects on plant cells and thus improving the drought resistance of maize.

[0055] The above experimental results show that the compound microbial agent provided by the present invention can effectively reduce the content of MDA and reduce its damage to cells. At the same time, the compound microbial agent provided by the present invention can also increase the activity of SOD, POD and CAT, and improve the corn's ability to scavenge superoxide anion free radicals and H2O2, thereby improving the corn's drought resistance.

[0056] The compound microbial agent provided by this invention can increase the activity of defense enzymes in crop leaves under drought stress and reduce the content of MDA, thereby improving the drought resistance of crops.

[0057] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent includes Bacillus vesiculosus (B. vesiculosus) Bacillus vezelensis ), with accession number CGMCC No. 24516 and Pseudomonas fluorescens ( Pseudomonas fluorescens The accession number is CGMCC No.14105, and the ratio of Bacillus belyssus and Pseudomonas fluorescens is 1~3:1~6.

2. The compound microbial agent as described in claim 1, characterized in that, The ratio of Bacillus belyi to Pseudomonas fluorescens was 1:

3.

3. The compound microbial agent as described in claim 1, characterized in that, The weight percentage of Bacillus belyi and Pseudomonas fluorescens in the compound bacterial agent is 0.1% to 20%.

4. The compound microbial agent as described in claim 1, characterized in that, The compound microbial agent, by weight percentage, also includes one or more of wetting agents, dispersants, stabilizers, and fillers.

5. The use of the compound microbial agent as described in any one of claims 1 to 4 for improving crop stress resistance.

6. The use as described in claim 5, characterized in that, The crops mentioned are wheat and corn.

7. The use as described in claim 5, characterized in that, The resilience mentioned refers to drought resistance.

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