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CN-121974744-A - Preparation method of co-culture metabolic solution carbon-based biostimulant based on different combinations of trichoderma, bacillus and metarhizium anisopliae

CN121974744ACN 121974744 ACN121974744 ACN 121974744ACN-121974744-A

Abstract

The invention discloses a preparation method of co-culture metabolic solution carbon-based biostimulant based on different combinations of trichoderma, bacillus and metarhizium anisopliae. The synergistic effect of the carbon-based preparation of the co-cultured metabolic liquid of different combinations is obvious compared with that of the carbon-based preparation of the co-cultured metabolic liquid of a single microorganism on the aspects of promoting the growth of wheat and improving the stress resistance, and the synergistic effect of the rice straw biochar on adsorbing the optimal proportion and time of the co-cultured metabolic liquid of different combinations of trichoderma, bacillus belicus and metarhizium anisopliae is obvious in promoting the germination of wheat seeds and the growth of seedlings and improving the stress resistance of the wheat. Different combinations of co-culture metabolic solution and biochar compound preparations can be developed into novel carbon-based bio-stimulation hormone, and have important significance in promoting the growth of wheat and corn, increasing stress resistance and disease resistance and the like.

Inventors

  • CHEN JIE
  • Yang Congzhou
  • BAI ZHENXU
  • ZHANG CHENG
  • ZHANG XIFEN
  • WU HAOYANG
  • WANG YONGKUN
  • WANG XINHUA

Assignees

  • 上海大井生物工程有限公司
  • 上海交通大学

Dates

Publication Date
20260505
Application Date
20260119

Claims (10)

  1. 1. The carbon-based biological hormone based on the biological carbon adsorption microorganism co-culture metabolic solution is characterized in that the carbon-based biological hormone is prepared from the biological carbon adsorption microorganism co-culture metabolic solution, and the microorganism co-culture metabolic solution is a microorganism co-culture metabolic solution of the same genus or different genera selected from trichoderma, bacillus and metarhizium anisopliae.
  2. 2. The charcoal-based bio-stimulus according to claim 1, wherein the bio-charcoal is rice straw bio-charcoal, the specific surface area (BET) of the rice straw bio-charcoal is 100-350 m2 g - < 1 >, the Cation Exchange Capacity (CEC) is 150-250 cmol kg - < 1 >, the micropores are 1-2 nm, and the mesopores are 3-5 nm.
  3. 3. The charcoal-based bio-stimulus according to claim 1, wherein the same genus microorganism is co-cultured with Trichoderma asperellum and Trichoderma harzianum, denoted Trichoderma asperellum-Trichoderma harzianum co-culture, and the different genus microorganism is co-cultured with Trichoderma asperellum and Bacillus bailii, co-cultured with Trichoderma harzianum and Bacillus bailii, or co-cultured with Trichoderma asperellum and Metarhizium anisopliae, denoted Trichoderma asperellum-Bacillus bailii co-culture, trichoderma harzianum-Bacillus bailii co-culture, and Trichoderma asperellum-Metarhizium anisopliae co-culture, respectively.
  4. 4. The char-based microbial stimulus according to claim 3, wherein the trichoderma asperellum is trichoderma asperellum (Trichoderma asperelum) GDSF CGMCC No.9512; The trichoderma harzianum is trichoderma harzianum (Trichoderma harzianum) 10569 CGMCC No.40246; The bacillus belgium is bacillus belgium (Bacillus velezensis) 15006 CGMCC No.22176; the Metarhizium anisopliae is Metarhizium anisopliae (Metarhizium anisopliae) M CGMCC No.3.11962.
  5. 5. The char-based bio-stimulus according to claim 3 or 4, characterized in that the preparation of the microbial co-culture metabolic solution comprises the steps of: s1, preparation of microbial co-culture bacterial liquid S1-1, mixing trichoderma asperellum spore suspension and trichoderma harzianum spore suspension according to the ratio of 1:1 (v/v), inoculating the mixed solution into a trichoderma asperellum-trichoderma harzianum co-fermentation medium according to the inoculation amount of 3-5% (v/v), and fermenting to obtain trichoderma asperellum-trichoderma harzianum co-culture bacterial liquid, co-culturing for 6-7 days, wherein the comprehensive spore concentration in the co-culture bacterial liquid is 2-3X 10 8 cfu/mL; Or S1-2, adopting a sequential inoculation method, inoculating a trichoderma spore suspension into a trichoderma-bacillus belicus co-fermentation medium according to the inoculum size of 2-3% (v/v), culturing for 36-48 hours, then inoculating a bacillus belicus spore suspension, and co-culturing for 66-76 hours to obtain trichoderma-bacillus belicus co-culture bacterial liquid, wherein trichoderma is trichoderma asperellum or trichoderma harzianum, the trichoderma spore content in the co-culture bacterial liquid is 2-3 multiplied by10 8 cfu/mL, and the bacillus belicus spore content is 3-5 multiplied by10 8 cfu/mL; Or S1-3, mixing trichoderma asperellum spore suspension and metarhizium anisopliae spore suspension according to a ratio of 1:1 (v/v), inoculating the mixed solution into trichoderma asperellum-metarhizium anisopliae co-fermentation culture medium according to an inoculum size of 2-3% (v/v), co-culturing for 5-6 days, and fermenting to obtain trichoderma viride-metarhizium anisopliae co-culture bacterial solution, wherein the trichoderma asperellum spore content is 2-3 multiplied by 10 8 cfu/mL, and the metarhizium anisopliae spore content is 2-3 multiplied by 10 8 cfu/mL; s2, centrifuging and membrane filtering the microbial co-culture bacterial liquid to obtain spore-free pure metabolic liquid, namely the microbial co-culture metabolic liquid.
  6. 6. The char-based biostimulation hormone according to claim 4, wherein trichoderma asperellum spore suspension Is prepared by inoculating Trichoderma asperellum GDSF to Potato Dextrose Agar (PDA), activating at 25-28deg.C for 3-5 days, suspending Trichoderma asperellum conidium with sterile water, and preparing into 10 5 -10 8 /mL Trichoderma asperellum spore suspension; trichoderma harzianum spore suspension is prepared by inoculating Trichoderma harzianum 10569 on Potato Dextrose Agar (PDA), activating at 25-28deg.C for 3-5 days, suspending Trichoderma conidium with sterile water to obtain 10 5 -10 8 Trichoderma harzianum spore suspension/mL; bacillus bailii spore suspension is prepared by streaking Bacillus bailii 15006 in LBA medium, culturing at 30-37deg.C for 18-24 hr, preparing into spore suspension with spore concentration of 2×10 7 c-2×10 8 cfu/mL with sterile water; The Metarhizium anisopliae spore suspension is prepared by inoculating Metarhizium anisopliae M on Potato Dextrose Agar (PDA), activating at 25-28deg.C for 3-5 days, suspending Trichoderma conidium with sterile water, and preparing into 10 5 -10 7 /mL Metarhizium anisopliae spore suspension.
  7. 7. The carbon-based microbial hormone of claim 3 or 4, The trichoderma asperellum-trichoderma harzianum co-fermentation medium is 15-17 g/L of corn flour, 0.8-1.2 g/L of dipotassium hydrogen phosphate, 0.1-0.3 g/L of tryptophan, 28-31 g/L of seaweed residues, 0.8-1.2 g/L of ferrous sulfate heptahydrate and 1.3-1.6 g of ammonium sulfate; the trichoderma asperellum or trichoderma harzianum-bacillus bailii co-fermentation medium is 1.4-1.6 g/L of monopotassium phosphate, 9-11 g/L of molasses, 1.3-1.6 g/L of ammonium sulfate, 28-31 g/L of galactose, 3-5 g/L of yeast extract and 1.3-1.6 g/L of magnesium sulfate heptahydrate; The trichoderma asperellum-metarhizium anisopliae co-fermentation culture medium comprises 5.0-7.0 g/L of soybean peptone, 23-26 g/L of molasses, 0.5-0.8 g/L of ammonium sulfate and 0.8-1.0 g/L of magnesium sulfate.
  8. 8. The charcoal-based bio-stimulus according to claim 5, wherein, In the step S1-1, fermentation parameters are that the rotating speed is 185r/min-190r/min, the dissolved oxygen is 60 percent, the pH is 5-6, and the culture is carried out for 6-7 days; in the step S1-2, fermentation parameters are 200r/min-214r/min, the temperature is 28 ℃, the aeration rate is 0.5L-1.0L/(L.min), the pH is 6-7, and the co-culture is carried out for 5-6 days; In the step S1-3, fermentation parameters are 28 ℃ and 180 r/min, ventilation is 0.5L-1.0L/(L.min), pH is 6-7, and co-culture is carried out for 5-6 days; in the step S2, the co-culture bacterial liquid is centrifuged for 15-20 minutes at 5000 Xg, spores are removed, and then the co-culture bacterial liquid is filtered by an ultrafiltration membrane (10-50 kDa) to obtain 5-time concentrated liquid of the co-culture metabolic liquid without spores.
  9. 9. The char-based biostimulation hormone of claim 1, further comprising at least one of the following technical features: A1, mixing biochar and microorganism co-culture metabolic solution according to a mass-volume ratio of 5:5-10:5 g/mL; A2, adsorbing the spore-free co-culture metabolic solution by using the biochar for 15-30 minutes; and A3, standing for 15-30 minutes at 25-45 ℃ after the adsorption is finished, and drying for 7-10 hours at 40-60 ℃.
  10. 10. Use of a char-based microbial stimulation agent according to any of claims 1-9 for promoting wheat growth or improving stress resistance.

Description

Preparation method of co-culture metabolic solution carbon-based biostimulant based on different combinations of trichoderma, bacillus and metarhizium anisopliae Technical Field The invention belongs to the technical field of biology, relates to a preparation method of a carbon-based bio-hormone of a co-culture metabolic solution based on different combinations of trichoderma, bacillus and metarhizium anisopliae, and in particular relates to a preparation method of a bio-carbon preparation (namely bio-hormone) of a microbial co-culture metabolic solution taking bio-carbon as a carrier and application of the bio-carbon preparation in wheat growth and stress resistance induction, wherein the microbial co-culture metabolic solution comprises a bio-carbon adsorption trichoderma aspergilli-trichoderma harzianum co-culture metabolic solution, a bio-carbon adsorption trichoderma aspergilli or trichoderma harzianum-bacillus besii co-culture metabolic solution, a bio-carbon adsorption trichoderma aspergilli-metarhizium anisopliae co-culture metabolic solution and the like. Background Biochar (Biochar) is a porous carbonaceous material prepared from biomass (such as wood, straw, fruit shell, livestock manure, etc.) by Pyrolysis (Pyrolysis) under anoxic or oxygen-limited conditions. The modified carbon material has stable chemical structure, rich pores and surface functional groups, and is widely applied to the fields of environmental remediation, agricultural improvement, carbon sequestration and energy sources. In general, the adsorption function of biochar refers to its ability to capture and immobilize contaminants (e.g., heavy metals, organic contaminants, gases, etc.) by physical or chemical action. The method mainly comprises the steps of ⑴ physical adsorption, including pore structure adsorption (micropores, mesopores and macropores), van der Waals force or capillary action adsorption, specific Surface Area (SSA) adsorption (more than 1000 m < 2 >/g), chemical adsorption, surface functional group adsorption (the surface of biochar contains functional groups such as-OH, -COOH, -C=O and the like), and electrostatic attraction (the surface of charged biochar). Compared with the traditional fertilizer, the biochar-based slow release fertilizer taking the biochar as the carrier has the advantages of reducing the loss of nutrient substances and improving the utilization rate of crop nutrients by utilizing the complex reticular pore structure and fibrous surface characteristics of the biomass charcoal. Research shows that the biochar can quickly adsorb metabolites such as L-asparagine, L-glutamine, L-arginine and the like (Rebecca A. Hill et al Eect of Biochar on Microbial Growth: A Metabolomics andBacteriological Investigation in E. coli: Environ. Sci. Technol. 2019, 53, 26352646) Biochar has a high specific surface area and a porous structure (especially mesopores and micropores), can adsorb metabolites of microorganisms through physical actions such as Van der Waals force, pore interception and the like, for example, organic acids (such as oxalic acid and citric acid) generated by microorganisms, can be combined with microbial metabolites through hydrogen bonds and electrostatic actions through carboxyl (-COOH), hydroxyl (-OH) and the like on the surface of the biochar, can adsorb positively charged metabolites (such as certain amino acids) when the pH is higher, and can adsorb hydrophobic substances (such as lipids and hydrophobins) of microorganisms. Research shows that the biochar is combined by Van der Waals force, electrostatic acting force and hydrophobicity, Adsorption of bacillus behenryis under the combined action of ion exchange and the like, and adsorption of bacteria by biochar and humic acid Is a spontaneous exothermic process, but the adsorption mechanism of biochar on spore metabolite is not clear from studies (Shao Yaxu, 2024). The patent 'a carbon-based composite microorganism metabolic solution rice seed soaking agent and application thereof' previously reported by the subject group is the recombination of metabolic solutions of three microorganisms which are fermented by single bacteria respectively, and is only applicable to rice seed soaking, the co-culture of two different microorganisms in the same pot is not performed, and the co-culture of two microorganisms can mutually induce silent gene expression, so that new compounds are generated (references). The study shows that the combined treatment of trichoderma and biochar significantly improves the root length (9.23 cm), the plant height (26.03 cm) and the root quality (43.33 g) of the chickpea plants, can reduce the incidence of chickpea wilt by 27%, and can reduce the incidence of stem basal rot by 33% (Ranjna Kumari et al., frontier in Microbiology, 10.3389/fmib.2025.1583114). However, this study is a combination of live trichoderma cells and biochar, whose mechanism of action is quite different from that of biochar adsorption of a micro-