Compound microbial inoculant for improving stress resistance of fruit trees and application thereof

Abstract

This invention provides a compound microbial agent, the compound microbial agent comprising methyltrophic Bacillus (Bacillus) Bacillus methylotrophicus ), with accession number CGMCC No. 20908 and Bacillus cereus ( Bacillus cereus The compound microbial agent, with accession number CGMCC No.24485 and a ratio of methyltrophic Bacillus to Bacillus cereus of 1~5:1~5, can be used to improve the cold and drought resistance of fruit trees.

Description

Technical Field

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

[0002] Methyltrophic Bacillus is widely distributed in various natural habitats, including soil, plant roots, and aquatic environments. In agriculture, methyltrophic Bacillus is often used for biological control or soil improvement. This bacterium can effectively inhibit the growth of soil-borne and foliar pathogens by competing for ecological niches and secreting various active substances such as antimicrobial peptides and lysins. It can also break up soil compaction and improve soil aggregate structure through its metabolic products.

[0003] Bacillus cereus is widely found in nature and shows broad application prospects in agricultural biological control, environmental remediation, and animal husbandry. In agriculture, it is commonly used for crop disease control and soil remediation. Bacillus cereus can inhibit various plant pathogens, such as cotton wilt, cucumber wilt, rice sheath blight, and gray mold. Its metabolites also help plants fix nitrogen and decompose harmful substances in the soil.

[0004] As perennial economic crops, fruit trees are significantly affected by environmental stresses in terms of growth, development, yield, and quality. Low temperatures and drought are two core abiotic stressors globally, restricting the expansion of fruit tree cultivation areas, yield stability, and fruit quality improvement. With the intensification of global climate change, the frequency and intensity of extreme low-temperature events and drought disasters are increasing, causing increasingly severe economic losses to the fruit tree industry.

[0005] Chinese patent CN108064271A discloses a method for applying an effective amount of methyltrophic Bacillus or a combination thereof to gramineous crops to improve their drought resistance; Chinese patent CN103667132B discloses a strain of Bacillus cereus for improving the cold resistance of walnuts. Although various Bacillus strains have been disclosed in the prior art for improving crop stress 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, antagonistic effects may exist between different strains, and when strains are compounded into inoculants, their efficacy may be affected. Therefore, screening compound inoculants that can improve the stress resistance of fruit trees 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 to improve the cold and drought resistance of fruit trees. The technical solution of this invention is as follows: A compound microbial agent, said compound microbial agent comprising methyltrophic Bacillus (Bacillus) Bacillus methylotrophicus ), with accession number CGMCC No. 20908 (Chinese Patent CN202011482541.4) and Bacillus cereus ( Bacillus cereus The accession number is CGMCC No.24485 (Chinese Patent CN202211033651.1), and the ratio of methyltrophic Bacillus to Bacillus cereus is 1~5:1~5.

[0007] Preferably, the ratio of methyltrophic Bacillus to Bacillus cereus is 3:2.

[0008] Preferably, the methyltrophic Bacillus and Bacillus cereus account for 0.1% to 20% of the weight of the compound bacterial agent.

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

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

[0011] Preferably, the fruit tree is an apple tree.

[0012] Preferably, the resilience includes cold resistance and drought resistance.

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

[0014] 2. The compound microbial agent provided by this invention can improve the drought resistance of fruit trees. 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 Methyltrophic Bacillus technical (100 billion CFU / g), 3%; Bacillus cereus technical grade (100 billion CFU / g), 3%; Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0019] Example 2 Methyltrophic Bacillus technical (100 billion CFU / g), 4%; Bacillus cereus technical grade (100 billion CFU / g), 2%; Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0020] Example 3 Methyltrophic Bacillus technical material (100 billion CFU / g), 4.5%, Bacillus cereus technical grade (100 billion CFU / g), 1.5%; Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0021] Example 4 Methyltrophic Bacillus technical (100 billion CFU / g), 5%, Bacillus cereus technical grade (100 billion CFU / g), 1%; Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0022] Example 5 Methyltrophic Bacillus technical (100 billion CFU / g), 3.6%, Bacillus cereus technical grade (100 billion CFU / g), 2.4%; Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0023] Comparative Example 1 Methyltrophic Bacillus technical (100 billion CFU / g), 6%, Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0024] Comparative Example 2 Bacillus cereus technical grade (100 billion CFU / g), 6%; Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0025] Comparative Example 3 Methyltrophic Bacillus technical grade (100 billion CFU / g), 0.75%, Bacillus cereus technical grade (100 billion CFU / g), 5.25%; Sodium dodecyl polyoxyethylene ether sulfate, 2%; Polycarboxylate dispersant T-36, 6%; Trehalose, 2%; Monosodium glutamate, 2%; Light calcium carbonate, 11%; Diatomaceous earth was replenished to 100%.

[0026] II. Fruit Tree Cold Resistance Test Test reagents: Example 1, Example 2, Example 3, Example 4, Example 5, Comparative Example 1, Comparative Example 2, Comparative Example 3.

[0027] Test crops: apple trees, Qin Guan.

[0028] Each test agent was prepared into a 2000-fold dilution for later use. Apple trees with uniform growth were selected and sprayed evenly. Water was used as control 1 (CK1), and branches without test agents and kept at 4℃ were used as control 2 (CK2). Seven days after application, one current-year branch with a length of more than 30cm was selected from the east, south, west, and north directions of the apple trees. The branches were uniform in thickness, vigorous, and free from mechanical damage and pests. After cutting each branch, the cut ends were immediately wrapped with plastic wrap to prevent water loss. The branches were then placed in self-sealing bags and stored in an insulated box for later use.

[0029] Low-temperature freezing treatment was carried out on the branches using a high-low temperature alternating test chamber. Two low-temperature treatment gradients were set: -10℃ and -20℃. The temperature was lowered at a rate of 5℃ / h until the preset treatment temperature was reached and held for 12 hours. Then, the temperature was raised at the same rate until the temperature reached 4℃ and held for 2 hours. After the branches were removed, they were placed at room temperature for 2 hours before testing.

[0030] Avoiding buds, cut each treated branch into 1-2 mm thick slices using a microtome and quickly freeze in liquid nitrogen for later analysis. Weigh 1 g of branch and place it in a pre-cooled mortar, add an appropriate amount of pre-cooled phosphate buffer (pH=7.8), and grind into a paste on an ice bath. Rinse the mortar 2-3 times with buffer to a final volume of 10 mL. Pour the homogenate into centrifuge tubes and centrifuge at 10,000 rpm for 10 min at 4°C. Collect the supernatant and store it at 4°C. Extract the residue again with 10 mL of pre-cooled phosphate buffer (pH=7.8), and combine the two supernatants for a total extract volume of 20 mL for analysis.

[0031] The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) in the tested branches were determined using a kit (Shanghai Jianglai Biotechnology Co., Ltd.) and in accordance with the methods described in the kit instructions.

[0032] 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 the branches, and can effectively reduce the content of MDA. Among them, Example 5 has the best effect, that is, the effect is best when the ratio of methyltrophic Bacillus to Bacillus cereus is 3:2.

[0033] Table 1. Data on cold resistance of fruit trees

[0034] When fruit trees are subjected to low-temperature stress, free radicals accumulate, leading to membrane lipid peroxidation and cell damage. The content of MDA, a product of membrane lipid peroxidation, can reflect the degree of cell damage. Furthermore, under low-temperature stress, MDA can further damage biological membranes and has a toxic effect on the plasma membrane.

[0035] 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 fruit tree's ability to scavenge superoxide anion free radicals and H2O2, thereby improving the fruit tree's cold resistance.

[0036] III. Experiment on the drought resistance of fruit trees Test reagents: Example 1, Example 2, Example 3, Example 4, Example 5, Comparative Example 1, Comparative Example 2, Comparative Example 3.

[0037] Test crops: apple trees, Fuji apples.

[0038] Different drought levels were treated using a potted plant water control method. The experiment was divided into two treatment levels: mild stress 50%~55% (percentage of maximum water holding capacity in the soil); severe stress 40%~45%.

[0039] Fruit trees were managed normally, with water control implemented before the experiment began. The experiment commenced once three water gradients were achieved. Each test agent was prepared at a 2000-fold dilution and uniformly sprayed onto the apple trees. The water treatment served as control 1 (CK1), and the leaves of fruit trees under normal management (without drought stress) and without test agent application served as control 2 (CK2). After the experiment began, daily water loss was calculated by weighing and replenished to maintain the set water content level for each treatment.

[0040] Sampling was conducted 30 days after the treatment. Leaves from the 3rd to 9th leaf positions of the first branch on potted seedlings were randomly selected for the experiment. Each treatment was repeated 4 times. The sampled leaves were ground in liquid nitrogen and then stored in liquid nitrogen for later use.

[0041] Superoxide dismutase (SOD) activity was determined according to Gao Junfeng's NBT photoreduction method (Gao Junfeng, ed., *Experimental Guide to Plant Physiology*, 2006). 0.1 mL of supernatant (0.1 mL of distilled water was added to both the control and zeroing tubes), and 1.5 mL of 50 mmol / L phosphate buffer, 0.3 mL of 130 mmol / L methionine (Met) solution, 0.3 mL of 750 μmol / L NBT solution, 0.3 mL of 100 μmol / L EDTA-Na2 solution, 0.3 mL of 20 μmol / L riboflavin solution, and 0.5 mL of distilled water were added to each tube. The zeroing tube was placed in the dark, while the others were reacted under 4000 lx fluorescent light for 20 min, and the results were measured at 560 nm. Results were expressed as A per gram of fresh tissue per minute. 240 A decrease of 0.1 represents one enzyme activity unit (U).

[0042] SOD activity (U / g FW) = (A0 - A S )×Vt×60 / A0×0.5×FW×Vs×t In the formula: A0 is the absorbance of the control tube under light; A S Vt is the absorbance of the sample test tube; Vs is the total volume of the sample extract (mL); t is the amount of crude enzyme solution taken during the test (mL); FW is the light exposure time for the colorimetric reaction (min); and FW is the fresh weight of the sample (g).

[0043] Peroxidase (POD) activity was determined according to Sun Qun's guaiacol method (Sun Qun, ed., *Technology of Plant Physiological Research*, 2005). 3 mL of the reaction mixture was taken, and 0.3 mL of enzyme extraction buffer (phosphate buffer was used as the control) was added. During the measurement, a stopwatch was immediately started after each addition of enzyme solution, and the A value was read at 470 nm for 0–3 minutes. 470 Values ​​were read every minute. Results were expressed as A per gram of fresh tissue per minute. 470 A decrease of 0.1 represents one enzyme activity unit (U).

[0044] POD activity (U / g·min FW) = A 470 ×Vt / 0.1×Vs×t×FW In the formula: FW is the fresh weight of the sample (g); Vt is the total volume of the enzyme extract (mL); Vs is the volume of enzyme solution used in the assay (mL); t is the assay time (min). A 47 The difference is 0-3 minutes.

[0045] Catalase (CAT) activity was determined according to Chen Tianlong's method (Effects of inoculation with arbuscular mycorrhizal fungi on apple tree growth and fruit quality, 2024). 0.1 ml of enzyme solution was added to 2.5 ml of CAT reaction solution, and colorimetric readings were taken at 240 nm every 1 min using a spectrophotometer, for a total of 3 readings.

[0046] Malondialdehyde (MDA) content was determined according to Wang Yirou's method (The Influence of Low Temperature on Membrane Lipid Peroxidation in Photosynthetic Organs of Rice Seedlings under Light and Dark Conditions, 1986). 0.5 g of fresh leaf weight was weighed into a pre-cooled mortar, 2 mL of 10% trichloroacetic acid (TCA) and a small amount of quartz sand were added, and the mixture was ground into a homogenate. Another 2 mL of TCA was added, and the homogenate was centrifuged at 4000 rpm for 10 min. 2 mL of the supernatant was collected and 2 mL of 0.62% 2-thiobarbituric acid solution was added. After mixing, the mixture was placed in a boiling water bath for 20 min. After the time was up, the mixture was quickly placed in an ice-cold water bath to cool. After cooling, the mixture was centrifuged at 4000 rpm for 10 min, and the absorbance of the supernatant was measured at wavelengths of 532 nm, 600 nm, and 450 nm. The 0.62% 2-thiobarbituric acid solution was used as a blank.

[0047] MDA content (nmol / g FW) = [6.45 × (A 532 -A 600 -0.56×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 (mL); FW is the fresh weight of the sample (g).

[0048] The experimental results are shown in the table below. Compared with CK1, Examples 1 to 5 can all improve the activity of SOD, POD and CAT in the leaves, and can effectively reduce the content of MDA. Among them, Example 5 has the best effect, that is, the effect is best when the ratio of Bacillus methyltrophicus to Bacillus cereus is 3:2.

[0049] Table 2. Data on drought resistance of fruit trees

[0050] Under drought stress, SOD activity is positively correlated with the antioxidant capacity of fruit trees. POD and CAT play important roles in scavenging H2O2 in fruit trees, decomposing it into O2 and H2O to protect cells from damage. The three defensive enzymes work synergistically to reduce cell damage under drought stress. Membrane lipid peroxidation can damage membrane function and cause membrane phase separation. MDA, a product of membrane lipid peroxidation, can cause enzyme molecules to polymerize and cross-link, leading to enzyme inactivation and direct toxicity to plant cells. Higher MDA content indicates more severe biomembrane damage and weaker plant resistance.

[0051] 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 fruit tree's ability to scavenge superoxide anion free radicals and H2O2, thereby improving the drought resistance of the fruit tree.

[0052] The compound microbial agent provided by this invention can increase the activity of defense enzymes in fruit tree branches and leaves under adverse conditions and reduce the content of MDA, thereby improving the stress resistance of fruit trees.

[0053] 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 methyltrophic Bacillus (Bacillus) Bacillus methylotrophicus ), with accession number CGMCC No. 20908 and Bacillus cereus ( Bacillus cereus The accession number is CGMCC No. 24485, and the ratio of methyltrophic Bacillus to Bacillus cereus is 1~5:1~5.

2. The compound microbial agent as described in claim 1, characterized in that, The ratio of methyltrophic Bacillus to Bacillus cereus is 3:

2.

3. The compound microbial agent as described in claim 1, characterized in that, The methyltrophic Bacillus and Bacillus cereus account for 0.1% to 20% of the weight of the compound bacterial agent.

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 the stress resistance of fruit trees.

6. The use as described in claim 5, characterized in that, The fruit tree in question is an apple tree.

7. The use as described in claim 5, characterized in that, The resilience mentioned includes cold resistance and drought resistance.

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