A composition containing microbial inoculant and active peptides

Abstract

This invention belongs to the field of crop production technology, specifically relating to a composition containing microbial agents and active peptides. The composition is a compound of β-lupin globulin polypeptide and *Pythium oligandrum* or *Phellinus lacerum*. Specifically, the β-lupin globulin polypeptide is a 20% soluble concentrate; the *Pythium oligandrum* is a wettable powder with 1 million spores / g; and the *Phellinus lacerum* is a 15 million CFU / g technical material (GXMS1). This invention combines the microbial agent *Pythium oligandrum* or *Phellinus lacerum* with the active peptide β-lupin globulin polypeptide for the control of fungal diseases in crops. Indoor pot experiments have shown that this composition has a significant synergistic effect against wheat powdery mildew, improving its control efficacy, reducing pesticide dosage, lowering pesticide residues and environmental pollution, and providing support for the development and scientific use of green and efficient pesticides.

Description

Technical Field

[0001] This invention belongs to the field of agricultural production technology, and specifically relates to a composition containing microbial agents and active peptides. Background Technology

[0002] wheat transformed by the obligate parasitic fungus *Erysiphe graminearum* Blumeria grassinis f. sp. wheat Powdery mildew caused by Bgt (Brugia rotundifolia) is one of the most prevalent and serious diseases affecting wheat-producing regions worldwide. It is contagious and can occur at any stage of wheat development. In wheat production, powdery mildew leads to increased respiration and transpiration, reduced photosynthetic efficiency, and hinders normal growth and development, resulting in leaf wilting, decreased ear formation, reduced thousand-grain weight, and ultimately, lower yield.

[0003] Chemical control remains a crucial method for controlling wheat powdery mildew due to its effectiveness in controlling diseases that occur rapidly and with stable efficacy. Currently, fungicides used to control powdery mildew mainly include triazoles and methoxyacrylates, such as cyproconazole, tebuconazole, triadimefon, tebuconazole, azoxystrobin, and pyraclostrobin. However, with the long-term and extensive use of common fungicides, pathogen resistance has accumulated year by year, leading to a gradual decrease in the efficacy of these agents. While increasing the dosage per unit area can improve efficacy, this exacerbates the development of pathogen resistance and also results in excessive pesticide residues, worsening environmental pollution.

[0004] β-Lupinin polypeptide is a polypeptide extracted from the germination process of edible white lupin seeds through the hydrolysis of naturally stored proteins. It belongs to the broad-spectrum protective biofungicides. β-Lupinin polypeptide has no direct toxicity to its target organisms. Its well-established mechanism of action is that it has a very high affinity for glycoproteins on fungal cell membranes, and can non-specifically bind to the glycosylation products of these glycoproteins. Through this interaction, fungal cells die within hours due to the blockage or closure of many stomata on their cell membranes. In addition, other mechanisms of action have been identified or are being confirmed. For example, when β-Lupinin polypeptide acts on powdery mildew, it may open channels in the cell wall, leading to cell disintegration and death. It also exhibits chelating activity towards some metal ions, disrupting homeostasis within microbial cells.

[0005] *Pythium oligandrum* is a highly aggressive, hyperparasitic microorganism belonging to the genus *Pythium* in the family Pythaceae. Widely distributed in the rhizosphere and soil, it resists various pathogens and can antagonize or parasitize more than 20 fungi from 16 genera and other oomycetes, making it a potentially valuable antagonistic microorganism. The main biological control mechanisms of *Pythium oligandrum* are hyperparasitism and induction of systemic resistance in plants. For example, *Pythium oligandrum* can secrete cellulase and glucanase, thereby disrupting the hyphae of host fungi. It can also induce increased activity of a series of resistance-related enzymes, such as superoxide dismutase, peroxidase, polyphenol oxidase, catalase, and phenylalanine ammonia-lyase, enhancing plant resistance to pathogens and indirectly controlling pathogen infection. Furthermore, *Pythium oligandrum* is also a plant growth promoter, increasing rhizosphere development and crop yield. It enhances nitrogen, phosphorus, and potassium absorption, induces root development, increases the number of lateral roots, and promotes higher nutrient content in plants, among other growth-promoting effects.

[0006] *Inonotus laceratus* is a polypore fungus that produces functional substances containing chitinase, cellulase, and protease. These substances inhibit the germination of pathogenic spores and the growth of mycelia, destroy cell walls to allow mycelia to invade pathogens, causing mycelial distortion, deformity, and vacuolation, ultimately completely invading and covering the pathogen colony. Simultaneously, it degrades the internal structure of pathogens, providing nutrients for further infection expansion, thereby inhibiting pathogen growth or killing the pathogen. *Inonotus laceratus* can be used to control various fungal diseases such as Phytophthora blight of pepper, downy mildew of eggplant, vine blight of cucumber, and black shank of tobacco. Currently registered formulations include *Inonotus laceratus* GXMS1 technical material (15 million CFU / g) and *Inonotus laceratus* GXMS1 suspension concentrate (5 million CFU / g).

[0007] Combining different active ingredients is one of the effective and convenient ways to develop new pesticides. Rational combination of fungicides can delay the occurrence and development of drug resistance in pathogenic microorganisms, reduce pesticide residues, and broaden the control spectrum of pesticides. Through indoor screening experiments, the inventors discovered that the combination of β-lupin globulin peptide with *Pythium oligandrum* or *Porphyromonas laceratus* has a significant synergistic effect against wheat powdery mildew, thus improving the control efficacy against wheat powdery mildew. Summary of the Invention

[0008] The purpose of this invention is to provide a composition containing microbial agents and active peptides to address the problem that with the long-term and large-scale use of common pesticides, pathogen resistance accumulates year by year, leading to a gradual decrease in the efficacy of pesticides. Although increasing the dosage of pesticides per unit area can improve efficacy, this will exacerbate the development of pathogen resistance and also lead to excessive pesticide residues, worsening environmental pollution.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A composition containing microbial inoculants and active peptides, wherein the composition is composed of β-lupin globulin polypeptide and *Pythium oligandrum* or *Porphyromonas laceratus*; wherein the β-lupin globulin polypeptide is a 20% β-lupin globulin polypeptide soluble concentrate.

[0011] The *Pythium oligandrum* is a wettable powder containing 1 million spores per gram.

[0012] The *Phenothizoctonia solani* strain is a 15 million CFU / g *Phenothizoctonia solani* GXMS1 mother drug.

[0013] Preferably, the mass ratio of the β-lupin globulin polypeptide to *Pythium oligandrum* is 1-6:18-1.

[0014] Preferably, the mass ratio of the β-lupin globulin polypeptide to *Pheromus laceratus* is 1-9:45-1.

[0015] Preferably, the mass ratio of the β-lupin globulin polypeptide to *Pheromus laceratus* is 1:9.

[0016] This invention also provides the application of the composition containing microbial agents and active peptides in the prevention and control of crop diseases or the improvement of crop stress resistance. It is known in the prior art that *Pythium oligandrum* can induce plants to produce their own defense responses, enhancing plant resistance to diseases and improving plant stress resistance; in addition, *Phellinus thuringiensis* has the ability to solubilize phosphorus and potassium, which can help plants better absorb nutrients and enhance plant stress resistance. Therefore, it is expected that the composition containing microbial agents and active peptides of this invention will have the effect of improving crop stress resistance.

[0017] Preferably, the crop disease includes wheat powdery mildew.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention combines the microbial inoculant *Pythium oligandrum* or *Porphyromonas laceratus* with the active peptide β-lupin globulin polypeptide for the control of fungal diseases in crops. Indoor pot experiments revealed that this composition has a significant synergistic effect against wheat powdery mildew, improving its control efficacy, reducing pesticide dosage, and decreasing pesticide residues and environmental pollution. Furthermore, this composition is expected to contribute to improving crop resistance. Based on these findings, it can support the development and scientific application of green and efficient pesticides. Detailed Implementation

[0020] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0021] Example 1: Compatibility Test

[0022] 1. Test strains

[0023] 1 million spores / gram of Pythium oligandrum wettable powder (Czech Biological Products Co., Ltd.)

[0024] 15 million CFU / g of *Porphyromonas thuringiensis* GXMS1 technical material (Sichuan Jinzhu Ecological Agriculture Technology Co., Ltd.)

[0025] Single colonies were obtained by purifying and isolating Pythium oligospermum wettable powder (1 million spores / g) and Phytotrichum laceratum GXMS1 technical material (15 million CFU / g) on ​​PDA medium. The single colonies were then inoculated onto PDA medium and cultured at 25°C for further propagation.

[0026] 2. Test reagents

[0027] 20% β-Lupinin Polypeptide Soluble (Seves de Portugal)

[0028] 3. Test Methods

[0029] The compatibility between the test agent and the test strain was determined using the mycelial growth rate method. The test agent was diluted with 0.1% Tween-80 aqueous solution to prepare a series of concentrations of 0.625 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, and 20 mg / L. 1 mL of the drug solution was mixed thoroughly with 9 mL of pre-melted and cooled PDA medium to 45°C, and then poured into a 9 cm diameter petri dish. After cooling, the mixture formed a drug-containing plate. A treatment with an equal volume of sterile water was used as a blank control, and each treatment was replicated in five places.

[0030] Using a punch, take a 5mm diameter mycelial cake from the edge of the colony after propagation and inoculate it into the center of the plate. Cover the plate and continue to incubate at 25℃. Observe the growth of the colony after 48 hours.

[0031] Results: The tested strains grew normally on blank plates. On drug-containing plates, growth of the tested strains gradually increased with increasing drug concentration. At 10 mg / L, the tested strains could still grow on drug-containing plates, but the colony diameter was reduced compared to lower concentrations. At 20 mg / L, growth of the tested strains on drug-containing plates was significantly inhibited, but still possible. This indicates that β-lupin globulin peptides and *Pythium oligandrum* or *Phellinus laceratum* have good compatibility at low concentrations. Therefore, β-lupin globulin peptides can be combined with *Pythium oligandrum* or *Phellinus laceratum*.

[0032] Example 2: Combined Effect Test

[0033] 1. Test crop

[0034] Five seedlings of "Jingshuang 16" wheat were potted and cultivated to the 2-4 true leaf stage. Each pot contained five seedlings, which were then numbered for future use.

[0035] 2. Test pathogens

[0036] Leaves bearing fresh powdery mildew spores from wheat fields in the suburbs of Zhengzhou, Henan Province, were collected. In the laboratory, the fresh powdery mildew spores were brushed into a 0.1% Tween-80 aqueous solution using a brush to prepare a spore suspension. This spore suspension was then sprayed onto the test crops. Once fresh powdery mildew spores appeared on the leaves of the test crops, the fresh spores were washed off the leaves with a 0.1% Tween-80 aqueous solution, filtered through double-layered gauze, and prepared into 5×10⁻⁶ spores. 5 A spore suspension of 1 spore / mL is prepared for later use.

[0037] 3. Test reagents

[0038] 20% β-Lupinin Polypeptide Soluble (Seves de Portugal)

[0039] 1 million spores / gram of Pythium oligandrum wettable powder (Czech Biological Products Co., Ltd.)

[0040] 15 million CFU / g of *Porphyromonas thuringiensis* GXMS1 technical material (Sichuan Jinzhu Ecological Agriculture Technology Co., Ltd.)

[0041] The test reagent was prepared into a single-agent stock solution using a 0.1% Tween-80 aqueous solution. Then, multiple formulations were set up as needed. Each single agent and each formulation mixture was diluted with a 0.1% Tween-80 aqueous solution to create five mass concentration gradients. All reagents were prepared and used immediately.

[0042] 4. Test methods (Refer to NY / T 1156.4-2006 "Guidelines for Indoor Bioassay Tests of Pesticides - Fungicides Part 4: Pot Test for Control of Wheat Powdery Mildew")

[0043] The prepared spore suspension was sprayed onto the test crops. After air-drying for 24 hours, the solution was sprayed evenly onto the leaves of the test crops until they were completely wet. Five plants were used for each treatment, and a control group was set up by spraying with a 0.1% Tween-80 aqueous solution. After air-drying, the treated test crops were transferred to an incubator for further cultivation. Cultivation conditions: temperature 22℃, alternating light and dark cycles of 16 hours and 18 hours.

[0044] After 8 days of cultivation, the disease incidence was investigated at different levels for each treatment. The disease index and control effect of each treatment were calculated. The grading criteria are shown in Table 1.

[0045]

[0046]

[0047]

[0048] 5. Data Analysis

[0049] Data statistical analysis was performed using DPS software. Linear regression was conducted on the logarithmic value of fungicide concentration and the efficacy probability value to derive the virulence regression equation and the EC50 of the agent against the target pathogen. 50 The co-toxicity coefficient (CTC) of the mixture was calculated using the Sun Yunpei method, and the combined action type of the agents was evaluated based on the calculated CTC values: less than 80 indicates antagonistic effect; greater than 120 indicates synergistic effect; and between 80 and 120 indicates additive effect. The results are shown in Table 2-3.

[0050]

[0051] Table 2 shows that within a mass ratio range of 1-6:18-1, the combination of β-lupin globulin peptide and *Pythium oligandrum* exhibits a synergistic effect against wheat powdery mildew. This indicates that the combination of β-lupin globulin peptide and *Pythium oligandrum* in this invention can improve the control effect against wheat powdery mildew.

[0052]

[0053] Table 3 shows that within a mass ratio range of 1-9:45-1, the combination of β-lupin globulin peptide and *Phellinus lacerum* GXMS1 exhibits a synergistic effect against wheat powdery mildew. This indicates that the combination of β-lupin globulin peptide and *Phellinus lacerum* GXMS1 in this invention can improve the control effect against wheat powdery mildew.

[0054] In summary, this invention combines the microbial inoculant *Pythium oligandrum* or *Porphyromonas laceratus* with the active peptide β-lupin globulin polypeptide for the control of fungal diseases in crops. Indoor pot experiments revealed that this composition has a significant synergistic effect against wheat powdery mildew, improving its control efficacy, reducing the amount of pesticide used, and decreasing pesticide residues and environmental pollution.

[0055] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A composition containing microbial inoculant and active peptides, characterized in that, The composition containing microbial agents and active peptides is a compound of β-lupin globulin polypeptide and Pythium oligandrum; wherein, the β-lupin globulin polypeptide is a 20% β-lupin globulin polypeptide soluble concentrate, and the Pythium oligandrum is a Pythium oligandrum wettable powder with 1 million spores / gram. The mass ratio of the β-lupin globulin polypeptide to *Pythium oligandrum* is 1-6:18-1.

2. The application of the composition containing microbial agents and active peptides as described in claim 1 in the prevention and control of crop diseases or the improvement of crop stress resistance.

3. The application according to claim 2, characterized in that, The crop diseases mentioned include wheat powdery mildew.

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