Biochar-based composite probiotic agent and its role in promoting plant disease resistance

Abstract

The application provides a biochar-based composite probiotic agent and a role of the biochar-based composite probiotic agent in promoting plant disease resistance. Two probiotics, Bacillus subtilis NG330 and Xanthomonas campestris CG365, are used to manufacture a biochar-based composite probiotic agent with corn stalk biochar as a substrate. The Bacillus subtilis NG330 and the Xanthomonas campestris CG365 have been preserved in the Guangdong Microbial Digital Culture Collection Center, and the preservation numbers are GDMCC No. 63292 and GDMCC No. 63293, respectively. The biochar-based composite probiotic agent has the functions of strong adsorption capacity and improved activity of the probiotics, and the two probiotics can stably play a role and significantly enhance the growth and disease resistance of tomatoes. The composite agent helps to reduce the use amount and residual amount of chemical agents in agricultural products, is safer to use, and is easy to industrialize.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a biochar-based compound probiotic agent and its effect on plant disease resistance and growth promotion. Background Technology

[0002] As a major agricultural country, my country's agriculture relies heavily on chemical fertilizers and pesticides, leading to problems such as declining soil quality, environmental pollution, and pesticide residues in agricultural products. To effectively enhance the sustainable development of agriculture, my country has proposed the agricultural development slogan of "reducing fertilizer and pesticide use, and increasing efficiency." Obtaining beneficial functional microorganisms (probiotics) from the natural environment and leveraging their growth-promoting functions or disease-enhancing properties in crops can effectively reduce the use of chemical fertilizers and pesticides without causing significant damage to the original ecological environment, potentially contributing to improved agricultural sustainability.

[0003] Biochar is a carbonized biomass material produced by pyrolyzing agricultural waste (including crop straw, organic fertilizer, etc.) at high temperatures (300-800℃) in an oxygen-deficient environment. It is characterized by its porous, loose structure and large specific surface area. The indigenous peoples of the Amazon basin have long intentionally burned plants under oxygen-deficient conditions to produce biochar black, which helped them increase crop yields. Biochar's high adsorption capacity and stability make it suitable as a carrier for microorganisms, thus aiding in the production of microbial preparations. Biochar-based microbial preparations can leverage the different effects of biochar and probiotics, working synergistically to promote crop growth or enhance disease resistance.

[0004] Single probiotics or probiotic preparations are characterized by relatively limited functions, unstable efficacy, and weak resistance to environmental disturbances. This significantly restricts their effectiveness in practical applications. In recent years, the concept of complex microbial communities has gained increasing attention. Different types of probiotics may exert synergistic effects, enhancing their ability to withstand environmental disturbances and thus helping to ensure the efficacy of probiotic preparations. However, different types of microorganisms cannot be simply mixed and inoculated into a culture medium for large-scale cultivation, as this may lead to competition for nutrients, inhibiting the growth of certain types of probiotics. Summary of the Invention

[0005] To address the above problems, this invention provides a biochar-based compound probiotic agent, its preparation method, and its effects on plant disease resistance and growth promotion.

[0006] The first objective of this invention is to provide Bacillus subtilis NG330, with accession number GDMCC No: 63292.

[0007] Bacillus subtilis NG330 is a strain of Bacillus subtilis isolated and screened from tomato growing soil. It was deposited on March 27, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China. Its accession number is GDMCC No. 63292.

[0008] The second objective of this invention is to provide *Ensifer adhaerens* CG365, with accession number GDMCC No: 63293.

[0009] Ensifer adhaerens CG365 is an Ensifer adhaerens strain isolated and screened from tomato growing soil. It was deposited on March 27, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China. Its accession number is GDMCC No. 63293.

[0010] The two bacteria mentioned above can grow in tomatoes under drought stress, indicating that they have strong stress resistance and environmental adaptability. At the same time, these two bacteria may also help the host plant cope with drought stress and improve its resistance to adverse conditions.

[0011] The *Bacillus subtilis* NG330 is rod-shaped with rounded ends, mostly solitary, with a few growing in pairs or clusters, and is Gram-positive. On agar plates, it presents as rough, opaque plaques, dirty white or pale yellow, with slightly wrinkled edges. The *Strombus amygdalinus* CG365 is short rod-shaped or spindle-shaped, mostly growing oppositely or in chain-like clusters, with a few solitary, and is Gram-negative. On agar plates, it presents as a pale white, opaque strain, and when picked, it has a viscous consistency.

[0012] Preferably, the culture conditions for Bacillus subtilis NG330 and Adhesive Streptomyces CG365 are as follows: temperature 28-30℃, pH 6-8, and shaking speed 150-200 rpm.

[0013] A third objective of this invention is to provide a biochar-based compound probiotic agent comprising the aforementioned Bacillus subtilis NG330, the aforementioned Streptomyces CG365, and biochar.

[0014] Preferably, the biochar is biomass carbon black obtained by high-temperature pyrolysis and carbonization of crop straw under 500°C oxygen-deficient conditions.

[0015] Preferably, the crop is corn, rice, or wheat.

[0016] Preferably, in the biochar-based compound probiotic agent, the number of each type of beneficial probiotic is not less than 2 billion / g, and the total number of beneficial probiotics is not less than 10 billion / g.

[0017] The fourth objective of this invention is to provide a method for preparing a biochar-based compound probiotic agent, comprising the following steps: adding biochar to Bacillus subtilis NG330 bacterial solution and Streptomyces gracilis CG365 bacterial solution respectively, culturing and drying to obtain a biochar probiotic complex of Bacillus subtilis and a biochar probiotic complex of Streptomyces gracilis, and mixing the two biochar probiotic complexes to obtain a biochar-based compound probiotic agent.

[0018] Preferably, the biochar is porous biomass carbon black obtained by high-temperature pyrolysis and carbonization of corn stalks under 500°C oxygen-deficient conditions.

[0019] The mass-to-volume ratio of the biochar to the bacterial solution is 1 g: 10 mL;

[0020] The culture conditions are as follows: temperature 28-30℃, pH 6-8, shaking speed 150-200 rpm, 40 h-48 h;

[0021] The drying temperature is 37°C;

[0022] The mixing ratio of the biochar probiotic complex of Bacillus subtilis and the biochar probiotic complex of Streptomyces adhesus is 1:1 or 1:2 by mass.

[0023] Preferably, the method includes the following steps:

[0024] 1) Using crop straw such as corn, rice, and wheat as biomass raw materials, collect the dried and crushed raw materials, put them into a carbonization furnace, slowly increase the temperature to 500℃, and carry out high-temperature pyrolysis carbonization under oxygen-deficient conditions for 2-3 hours. After cooling to room temperature, the biochar is obtained by intermittent discharge through the discharge pipe.

[0025] 2) Two beneficial microorganisms, Bacillus subtilis NG330 and Brachymycium CG365, were initially cultured for 18-22 hours to obtain a preliminary fermentation broth with a viable cell count of approximately 5 × 10⁻⁶. 8 CFU / mL.

[0026] 3) Add biochar to the initial fermentation broth of the two probiotics at a dose of 0.1 g / mL and continue culturing for 40-48 h. After culturing, collect the biochar-probiotic complex by centrifugation, dry it at 37℃, and mix the biochar-probiotic complex of the two probiotics at a mass ratio of 1:1 or 1:2, so that the number of each beneficial probiotic is not less than 2 billion / g, and the total number of the compound beneficial bacteria agent is not less than 10 billion / g.

[0027] The fifth objective of this invention is to provide the application of the above-mentioned biochar-based compound probiotic agent in plant disease resistance and growth promotion.

[0028] Preferably, the plant is tomato, and the disease resistance is against scab fungus.

[0029] The sixth objective of this invention is to provide a method for promoting plant growth and enhancing its disease resistance, which involves planting plants in soil containing the aforementioned biochar-based compound probiotic agent.

[0030] The present invention has the following beneficial effects:

[0031] 1) The obtained biochar is loose and porous with a large specific surface area, which is conducive to the adsorption and preservation of probiotics.

[0032] 2) Experiments revealed that these two bacteria significantly inhibited the growth of *Scab mites*, a pathogenic bacterium affecting tomato leaves. A biochar-based compound probiotic agent, prepared by mixing these bacteria with biochar, significantly promoted tomato growth and enhanced tomato resistance to *Scab mites*. The biochar-based compound probiotic agent allowed the two probiotics to work synergistically, increasing the functional stability and environmental adaptability of the agent. This biochar-based compound probiotic agent significantly promoted tomato growth, with a biomass 2.63 times that of the control; simultaneously, it enhanced tomato resistance to pathogens such as *Scab mites*, reducing the disease index by 55.6%.

[0033] 3) The use of this biochar-based compound probiotic agent not only avoids environmental pollution caused by fertilization and pesticides, but is also simple and convenient to operate. At the same time, releasing Bacillus subtilis NG330, Brachymycium CG365, and an appropriate amount of biochar into the soil can enrich the beneficial microbial community of the soil, improve soil fertility, and enhance soil quality. Attached Figure Description

[0034] Figure 1 These are scanning electron microscope images of two bacteria: A, Bacillus subtilis B. substis NG330; B, E. adhaerens CG365, with a scale bar of 5 micrometers.

[0035] Figure 2 The fermentation broths of Bacillus subtilis NG330 (A) and E. adhaerens CG365 (B) showed the plate inhibition effect of the pathogenic bacterium Xanthomonas campestris 10048.

[0036] Figure 3This is a statistical chart showing the effects of biochar-based compound probiotics on disease resistance and growth promotion in tomatoes. C is the control group; B is the group with biochar; S is the group with probiotic liquid; and F is the group with biochar-based compound probiotics. Detailed Implementation

[0037] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0038] Example 1: Isolation and purification of rhizosphere bacteria from tomato

[0039] 1. Isolation culture medium

[0040] R2A agar medium (R2A solid medium): 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, and 15.0 g agar. Dissolve and bring the volume to 1 L with distilled water, adjusting the pH to 7.2. After preparation, sterilize by steaming at 121°C for 20 min.

[0041] 2. Experimental Procedure

[0042] (1) Select garden soil, which is red soil with a pH of 7.88 and organic matter of 10.23 g / kg. Construct a tomato planting system indoors and subject the tomatoes to a certain degree of drought stress (45% soil water holding capacity) by adjusting the soil moisture content. Pull out the drought-stricken tomato plants (6 weeks old), shake them off and collect the soil around the roots.

[0043] (2) Preparation of soil gradient dilution. Collect approximately 5 g of the above-mentioned rhizosphere soil and add it to a centrifuge tube containing 50 mL of sterile water. Incubate at 200 rpm for 2 h with shaking. After standing for 5 min, take 1 mL of the supernatant and add it to a test tube containing 9 mL of sterile water. Perform gradient dilutions to 10⁻⁶. -2 -10 -7 Prepare a soil suspension at a concentration of g / mL.

[0044] (3) Bacterial isolation: Take 150 μL of the diluted soil suspension (10 -2 -10 -7Add g / mL to R2A plates and spread evenly using a disposable plastic spreader. Set up three replicates for each concentration. After spreading, invert the plates and incubate at 30℃. Observe the colony growth on the plates the next day. Select single colonies with inconsistent morphology and streak them onto R2A plates for purification.

[0045] A total of 82 strains of bacteria were isolated.

[0046] Example 2: Plate antagonism experiment of the isolated strain against Tomato scab pathogen.

[0047] 1. Culture medium

[0048] NA solid medium: prepared by adding agar to nutrient broth (NB) medium (Guangdong Huankai Microbial Technology Co., Ltd., product number 022010), pH 7.2.

[0049] 2. Experimental Procedure

[0050] 2.1 Activation of the strain

[0051] X. campestris ACCC 10048, the scab causal agent, and 82 soil-isolated bacteria from Example 1 were transferred to NA solid medium and cultured at 30°C for 1-2 days for later use.

[0052] 2.2 Preparation of bacterial plates

[0053] A single colony of *X. campestris* ACCC 10048 was picked and inoculated into approximately 5 mL of NB medium (NA medium without agar, pH 7.2) and incubated at 30°C and 200 rpm for 24 h to prepare approximately 10... 10 Take 1 mL of the bacterial suspension (cfu / mL) and add it to 100 mL of NA medium cooled to about 45°C. Shake well and then pour the mixture onto a plate.

[0054] 2.3 Initial screening of antagonistic bacteria

[0055] The soil-isolated bacteria isolated and purified in Example 1 were streaked on NA plates for activation. The activated soil-isolated bacteria were picked up with a sterile pipette tip and spotted onto a bacterial plate containing bacteria. Eight test strains were spotted onto each plate. The plates were incubated at 30°C for 48 hours. The presence or absence of inhibition zones was observed, and their size was measured with a spiral micrometer. Antagonistic strains with inhibition zones were preserved for rescreening.

[0056] 2.4 Plate Antagonism Test - Secondary Screening

[0057] The antagonistic strains obtained from the initial screening were selected and activated as shown in 2.1. Single colonies were then inoculated into 100 mL of NB medium (placed in a 250 mL Erlenmeyer flask) and cultured at 28℃ and 200 rpm for 48 h to obtain the antagonistic bacterial fermentation broth. 150 μL of *X. campestris* ATCC 10048 bacterial suspension (prepared as shown in 2.2) was evenly spread onto NA solid medium. Wells were punched on the plates using a yellow pipette tip, and 100 μL of the antagonistic bacterial fermentation broth was added to each well. The plates were incubated at 28℃ for 1-3 days, and the presence of inhibition zones was observed. The results were photographed and recorded.

[0058] 3. Experimental Results

[0059] Five strains with obvious inhibition zones were obtained through screening, among which strains NG330 and CG365 were two strains with excellent antagonistic effects.

[0060] Figure 1 Images of strains NG330 (A) and CG365 (B) from a scanning electron microscope.

[0061] Figure 2 The diagram shows the antagonistic effect of strains NG330 (A) and CG365 (B). It can be seen that there is a clear inhibition zone, indicating that strains NG330 and CG365 have a significant antagonistic effect on scab pathogens.

[0062] Example 3: Identification of the isolated strain

[0063] The isolated strains were identified according to the *Handbook of Systematic Identification of Common Bacteria* (edited by Dong Xiuzhu, Science Press). The experimental methods are as follows: DNA was extracted from strains NG330 and CG365 by boiling in water. The 16S rDNA gene sequences of strains NG330 and CG365 were amplified using bacterial 16S rDNA-specific primers 27F and 1492R and Taq polymerase. Electrophoresis analysis of the amplification products produced bands of approximately 1500 bp. These bands were recovered from the gel and sent to Suzhou Genewiz Biotechnology Co., Ltd. for Sanger sequencing. The sequences were assembled using seqman software to obtain the 16S rDNA sequences. The nucleotide sequence of strain NG330 is shown in SEQ ID NO.1, and the nucleotide sequence of strain CG365 is shown in SEQ ID NO.2. The 16S rDNA sequences of these two strains were submitted to the EzBioCloud website (https: / / www.ezbiocloud.net / ) for identification.

[0064] The identification results showed that the 16S rDNA sequence of strain NG330 had 99.84% homology with Bacillus subtilis subsp. stercoris, and its morphological characteristics were most similar to those of Bacillus subtilis. Gram staining was positive, and the strain grew well in LB, NA, and R2A media, with the cells being rod-shaped. Figure 1 A). Based on the 16S rDNA sequence and morphological characteristics, the strain was classified as Bacillus subtilis NG330. The 16S rDNA sequence of strain CG365 showed 100% homology with Ensifer adhaerens, and its morphological characteristics were most similar to those of Ensifer adhaerens. Gram staining was negative, and the strain grew well in LB, NA, and R2A media. The cells were short rod-shaped or spindle-shaped. Figure 1 B). Based on the 16S rDNA sequence and morphological characteristics, the strain was classified as *Ensifer adhaerens* CG365.

[0065] Based on the identification results, strain NG330 was classified as Bacillus subtilis and named Bacillus subtilis NG330. It was deposited on March 27, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCCNo: 63292.

[0066] Based on the identification results, strain CG365 was classified as Ensifer adhaerens and named Ensifer adhaerens CG365. It was deposited on March 27, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCCNo: 63293.

[0067] Both strains mentioned above can be purchased from the Guangdong Provincial Microbial Culture Collection Center.

[0068] Example 4: Preparation of Biochar-Based Compound Probiotic Agent

[0069] (1) Preparation of biochar: Take 10 kg of corn stalks, air dry them, crush them with a pulverizer, put them into a carbonization furnace, slowly increase the temperature to 500℃, carry out high-temperature pyrolysis carbonization under oxygen-deficient conditions, and after carbonization for 3 hours, cool them to room temperature and discharge them intermittently through the discharge pipe to obtain biochar.

[0070] (2) Preparation of preliminary fermentation broth for two probiotics: Bacillus subtilis NG330 and Streptomyces adhesus CG365 were activated (as shown in step 2.1 of Example 2), and single colonies were inoculated into 400 mL of NB culture medium (in a 1 L Erlenmeyer flask) and cultured at 30℃ and 200 rpm for 18 h and 22 h respectively to obtain preliminary fermentation broth for the two probiotics. The number of probiotics was approximately 5 × 10⁻⁶. 8 CFU / mL.

[0071] (3) Mixed culture of biochar and probiotic preliminary fermentation broth: In a clean bench, approximately 40 g of sterilized biochar was added to the above-mentioned preliminary fermentation broths of Bacillus subtilis NG330 and Brachymyces CG365, respectively, and cultured at 30℃ and 200 rpm for 40 h and 48 h, respectively. After the culture was completed, the supernatant was discarded by centrifugation (8000×g, 5 min) to obtain the biochar-probiotic complex.

[0072] (4) Preparation of biochar-based compound probiotic agent: The biochar probiotic complex of Bacillus subtilis NG330 and Streptomyces adhesiveus CG365 was dried at a constant temperature of 37 degrees Celsius. Then, the two dried materials were mixed in a mass ratio of 1:1 and 1:2 to prepare the biochar-based compound probiotic agent. The number of each effective probiotic was not less than 2 billion / g, and the total number of effective probiotics in the compound probiotic agent was not less than 10 billion / g.

[0073] Example 5: Experiment on the growth-promoting and disease-resistant effects of biochar-based compound probiotics on tomatoes.

[0074] 1. Tomato growth substrate and treatment setup

[0075] Four treatments were set up, including control (C), biochar addition (B), probiotic solution addition (S), and biochar-based compound probiotic agent addition (F). Treatment C used sterile soil (garden soil sterilized at 121℃ for 20 min); treatment B used sterile soil containing 4% (w / w) sterilized corn straw biochar; treatment S used sterile soil and each tomato plant was watered with 4 mL of NB culture medium containing two types of probiotics with an OD600 of 0.5 (diluted to OD600 of 0.5 after shaking culture in NB medium for 24 hours), once a week; treatment F used sterile soil containing the biochar-based compound probiotic agent described in Example 4 mixed in a 1:1 mass ratio, with a biochar-based compound probiotic agent mass fraction of 4%.

[0076] 2. Seedling raising and transplanting of the tomato variety "Red Oriole"

[0077] Tomato seeds were placed in a 37°C water bath for 2 hours, soaked in 75% alcohol for 30 seconds, rinsed with deionized water, and then disinfected by soaking in a 5% NaClO solution for 10 minutes, followed by thorough rinsing with deionized water. The seeds were then evenly placed in a petri dish lined with two layers of gauze, and an appropriate amount of distilled water was added to keep the seeds and gauze moist. The dish was then placed in a 28°C constant temperature incubator for 2 days to germinate. Once the seeds had germinated and developed radicles of approximately 1-2 mm, they were sown into seedling trays containing perlite, one seed per cell. These trays were then placed in a 28°C constant temperature and light incubator with 80% humidity and a light / dark cycle of 16 hours / 8 hours. After 10 days of cultivation, uniformly growing tomato seedlings were carefully removed from the seedling trays and transplanted into soil or substrate containing different treatments. Ten parallel plants were prepared for each treatment, and each plant was grown in a large seedling tray containing 100 g of soil or substrate under the following conditions: 80% humidity and light / dark duration of 16 h / 8 h.

[0078] 3. Determination of the growth-promoting effect on tomatoes

[0079] After four weeks of cultivation under the same environmental conditions, five plants were randomly selected from each treatment for destructive sampling. Plant height was measured with a ruler, and plant biomass was measured with an electronic scale. Specific biomass was calculated as: Specific biomass = Biomass / Plant height. The results showed that the mean plant height of tomatoes under different treatments ranged from 7.56 cm to 8.44 cm, with no significant difference among the treatments. Figure 3 A). The mean tomato biomass ranged from 2.05 g to 5.39 g, with treatment F showing significantly higher values ​​than treatments C and S (pairwise t-test, P < 0.05), representing increases of 263% and 186%, respectively. Treatment B showed no significant difference from other treatments, and treatments S and C showed no significant difference. Figure 3 B). The mean specific biomass of tomatoes ranged from 0.266 g / cm³ to 0.691 g / cm³. Treatment F was significantly higher than treatments C and S (pairwise t-test, P < 0.05). Treatment B was significantly higher than treatment C, while there was no significant difference between treatments S and C. Figure 3 C). This part of the results shows that biochar treatment itself has a certain promoting effect on plant growth, but the effect is not strong. After adding biochar containing compound probiotics, the growth-promoting effect on plants is greatly enhanced. However, simply watering with probiotic liquid has a small promoting effect on plant growth, suggesting that watering with probiotic liquid is not conducive to its colonization in the rhizosphere, while the fixation of biochar helps the colonization and function of probiotics.

[0080] 4. Determination of resistance to tomato scab disease

[0081] 4.1 Activation of Tomato Scab Pathogen and Preparation of Fermentation Broth

[0082] Single colonies of X. campestris ATCC 10048 strain were picked and inoculated into 500 mL of NB medium (NA medium without agar) and cultured at 30℃ and 200 rpm for 24 h. The culture was then diluted with sterile physiological saline to obtain a diluted fermentation broth with an OD600 value of approximately 0.1 for use.

[0083] 4.2 Pathogen spraying experiment and disease index

[0084] Diluted fermented bacterial solution of scab pathogens was poured into a clean spray bottle, shaken well, and sprayed evenly onto the leaves of each plant transplanted into large seedling trays after the same treatment in step 2. Each plant was sprayed a total of 5 times. A second spray was applied on the 3rd day after the first spray. On the 14th day after the first spray, the disease incidence of each plant was observed, and the disease index was calculated. Disease incidence was evaluated on a leaf-by-leaf basis according to a grade of 0–4. Grade 0: No lesions; Grade 1: Lesion area less than 1 / 4 of the pinnate leaf area; Grade 2: Lesion area 1 / 4 to 1 / 2 of the pinnate leaf area; Grade 3: Lesion area 1 / 2 to 3 / 4 of the pinnate leaf area; Grade 4: Lesion area more than 3 / 4 of the pinnate leaf area or leaf death. Disease incidence = (Number of infected plants / Total number of plants) × 100%; Disease index = [(∑(Disease grade × Number of leaves at each disease grade)) / (4 × Total number of leaves)] × 100%. The disease index of each plant was calculated, and the results showed that the mean disease index of different treatments ranged from 0.8 to 1.8. The disease index of treatment F was significantly lower than that of the control, while there were no significant differences among the other treatments (pairwise t-test, P < 0.05). Figure 3 D). This result suggests that biochar-based compound probiotic agents have a better promoting effect on the resistance of tomatoes to scab disease than single biochar or probiotic fermentation broth.

Claims

1. Adhesive sword fungus ( Ensifer adhaerens CG365, with accession number GDMCC No: 63293.

2. A biochar-based compound probiotic agent, characterized in that, It includes Bacillus subtilis NG330, the Adhesive Streptomyces CG365 as described in claim 1, and biochar, wherein the preservation number of Bacillus subtilis NG330 is GDMCC No: 63292.

3. The biochar-based composite probiotic agent according to claim 2, characterized in that, The biochar is biomass carbon black obtained by high-temperature pyrolysis and carbonization of crop straw under 500℃ oxygen-deficient conditions, and the crop is corn, rice or wheat.

4. The biochar-based composite probiotic agent according to claim 2, characterized in that, The number of each type of beneficial probiotic is not less than 2 billion / g, and the total number of beneficial probiotics is not less than 10 billion / g.

5. A method for preparing the biochar-based composite probiotic agent according to claim 2, characterized in that, The process includes the following steps: adding biochar to Bacillus subtilis NG330 bacterial solution and Brachymycium CG365 bacterial solution respectively, culturing and drying to obtain Bacillus subtilis biochar probiotic complex and Brachymycium CG365 biochar probiotic complex, and mixing the two biochar probiotic complexes to obtain a biochar-based compound probiotic agent.

6. The method according to claim 5, characterized in that, The biochar is a porous biomass carbon black obtained by high-temperature pyrolysis and carbonization of corn stalks under anaerobic conditions at 500℃; the mass-to-volume ratio of the biochar to the bacterial solution is 1 g:10 mL; the culture conditions are as follows: temperature 28-30℃, shaking speed 150-200 rpm, 40 h-48 h; the drying temperature is 37℃; the mixing ratio of the biochar-probiotic complex of Bacillus subtilis and the biochar-probiotic complex of Streptomyces adoxoides is 1:1 or 1:2 by mass.

7. The application of the biochar-based compound probiotic agent according to claim 2 in plant disease resistance and growth promotion, wherein the plant is tomato, and the disease resistance is against scab pathogen.

8. A method for promoting plant growth and enhancing its disease resistance, characterized in that, Plants are grown in soil containing the biochar-based compound probiotic agent of claim 2, wherein the plant is a tomato and the disease resistance is against scab pathogens.

Images