Bio-organic fertilizer for preventing and treating wasabi root and stem diseases and its application

Abstract

The present invention relates to the technical field of microorganisms and bio-organic fertilizers, and specifically discloses a bio-organic fertilizer for preventing and treating wasabi root diseases and its application. The bio-organic fertilizer comprises the following components: Component A: Bacillus velezensis agent with a deposit number of CCTCC NO: M 2022923 and Trichoderma yunnanensis agent with a deposit number of CCTCC NO: M 2025645; Component B: soybean meal, vinegar dregs, biochar, wollastonite powder, and allicin. The bio-organic fertilizer of the present invention can increase the content of growth hormones such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), and the phytoalexin content in the wasabi root system, as well as the activity of chitinase and β-1,3-glucanase, and has an effect on the growth of stem mold of wasabi ( Phoma wasabiae Yokog) infection and carrot soft rot caused by Pectobacterium carrot subspecies ( Pectobacterium carotovorum subsp .carotovorum, It has a good control effect on soft rot caused by Pcc) infection.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and organic fertilizers, and in particular relates to a biological organic fertilizer for preventing and treating wasabi root and stem diseases and application thereof. Background Art

[0002] Wasabi ( Wasabia japonica ) As a global high-end spice crop, it is the core raw material for Japanese cuisine, seasoning foods and functional foods. Its unique spicy flavor comes from the natural active ingredient isothiocyanate, which has antibacterial, anti-inflammatory, antioxidant and other physiological effects. Its economic value far exceeds that of ordinary vegetable crops.

[0003] During the wasabi planting cycle, rhizome diseases have become the main bottleneck restricting the development of the industry. Black heart disease ( Phoma wasabiae ) and soft rot ( Pectobacterium carotovorum ) and other rhizome diseases are becoming increasingly severe year by year, with incidence rates reaching 40%-60% in continuously cropped plots. In severe cases, this can lead to total crop failure, severely impacting wasabi yield and quality, and causing enormous economic losses to wasabi growers. Currently, the common method for preventing and controlling wasabi rhizome diseases is the use of chemical agents. While these agents can have a certain inhibitory effect on the disease in the short term, long-term use can bring numerous disadvantages. On the one hand, chemical residues can affect the quality and safety of wasabi products, potentially posing a potential threat to consumer health. On the other hand, the extensive use of chemical agents can also pollute the soil and water environments, disrupting the ecological balance and posing a serious threat to environmental safety. These shortcomings make it difficult for chemical control technology to meet the needs of sustainable development in the wasabi cultivation industry.

[0004] Given the limitations of chemical control technology, the research and development of microbial agents based on the functional application of microorganisms and their application technologies has become particularly urgent. As a green, environmentally friendly and sustainable biological control method, microbial agents have the advantages of being environmentally friendly, not prone to resistance, and promoting crop growth. By screening microbial strains with functions such as antagonizing pathogens and improving the soil microecological environment, composite microbial agents have been developed. They can not only effectively prevent and control rhizomes of wasabi, reduce the use of chemical agents, and ensure product quality and environmental safety, but also provide a sustainable disease control solution for the wasabi planting industry and promote the healthy development of the wasabi industry. Therefore, conducting research on microbial agents based on the functional application of microorganisms and their application technologies is of great practical significance for solving the problem of rhizomes in the wasabi planting process and promoting the sustainable development of the wasabi planting industry. Summary of the Invention

[0005] The present invention aims to address the problems of chemical pesticides used in the prior art for preventing and controlling wasabi root diseases (blackheart and wasabi soft rot), which are poorly effective during crop cultivation, pose high environmental risks, and fail to meet consumer demand for food safety. The present invention provides a bio-organic fertilizer for preventing and controlling wasabi root diseases and its use. The bio-organic fertilizer provided by the present invention can effectively enhance wasabi disease resistance and exhibits excellent disease control efficacy.

[0006] To achieve the above object, the present invention provides a first aspect of a bio-organic fertilizer for preventing and treating wasabi root diseases (blackheart disease and wasabi soft rot), wherein the microbial agent comprises the following components:

[0007] Component A: Bacillus velez with a deposit number of CCTCC NO: M 2022923 and Trichoderma yunnanensis with a deposit number of CCTCC NO: M 2025645;

[0008] Component B: soybean meal, biochar, wollastonite powder, vinegar grains and allicin.

[0009] Furthermore, in component A, the effective viable bacteria count in the Bacillus Velez bacteria agent is not less than 5×10 10 CFU / g, the effective viable bacteria count in the Trichoderma yunnanensis agent is not less than 5×10 9 CFU / g.

[0010] Furthermore, the dosage of the Bacillus Velez subtilis agent and the Trichoderma yunnanensis agent is 1-2% by weight of component B.

[0011] Furthermore, in component B, the weight ratio of soybean meal, biochar, wollastonite powder, vinegar grains and allicin is 20-30:20-30:20-30:0.4-1:109-139.6.

[0012] Furthermore, the soybean meal, based on dry matter, has a protein content of 40-50% by weight, a carbohydrate content of 30-40% by weight, and a fat content of 2-4% by weight. Furthermore, / or the wollastonite powder, based on dry matter, has a CaO content of 35-45% by weight and a SiO2 content of 40-50% by weight. Furthermore, / or the vinegar lees, based on dry matter, has a cellulose content of 30-40% by weight, a protein content of 10-15% by weight, a fat content of 10-15% by weight, and an ash content of 10-15% by weight.

[0013] The second aspect of the present invention provides the use of the above-mentioned bio-organic fertilizer in increasing the content of root growth hormone and phytoalexin and the activity of defense enzymes in crops, and / or preventing and controlling root diseases of wasabi, wherein the crop is wasabi; the growth hormone is at least one of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET); the phytoalexin is Camalexin, and the defense enzyme is at least one of chitinase and β-1,3-glucanase; the root disease is at least one of wasabi black heart disease and wasabi soft rot, wherein the pathogen causing the wasabi black heart disease is stem spot mold ( Phoma wasabiae Yokog and wasabi soft rot is caused by Pectobacterium carrot subspecies ( Pectobacterium carotovorum subsp .carotovorum, Pcc).

[0014] A third aspect of the present invention provides a method for increasing the content of root growth hormones and phytoalexins and the activity of defense enzymes and / or preventing and controlling root and tuber diseases in crops, the method comprising applying the above-mentioned bio-organic fertilizer to the soil, the amount of the bio-organic fertilizer applied to the soil being 100-200 kg / mu / time, and the application frequency being once during the entire growth period.

[0015] Beneficial effects of the present invention:

[0016] (1) Bacillus velezensis inhibits pathogens by producing antimicrobial substances, promotes plant growth (such as nitrogen fixation and phosphorus solubilization), induces plant systemic resistance, improves soil microbial communities, enhances crop stress resistance, and reduces the occurrence of diseases; Trichoderma yunnanensis inhibits plant pathogens by secreting antibiotics, enzymes and competitive effects, promotes plant root development, induces systemic resistance, improves soil microbial structure, and assists in degrading organic matter, thereby enhancing crop stress resistance. Bacillus velezensis relies on the secretion of antimicrobial proteins (such as subtilisin), peptides and volatile substances to fight bacterial diseases (soft rot ( Pectobacterium carotovorum ) has a significant control effect. Yunnan Trichoderma destroys the cell wall of pathogens by secreting cellulase and chitinase, and directly infects pathogens by using antibiotics (such as trichoderma) and heavy parasitism, which has a significant effect on fungal diseases (black heart disease ( Phoma wasabiae) has a significant control effect. The combined biocontrol agents Bacillus Velez and Trichoderma yunnanensis can exert a synergistic effect of "1+1>2", which is manifested in a broader spectrum of disease control, more significant growth promotion and stress resistance, and a more stable microbial community. At the same time, it reduces the dosage of a single bacterial agent and improves the reliability and sustainability of biological control. Specifically, Bacillus Velez inhibits bacteria and some fungal diseases by secreting antimicrobial proteins (such as subtilisin) and volatile substances, while Trichoderma yunnanensis effectively controls fungal diseases through the production of chitinase and heavy parasitic mechanisms. The combination of the two forms a double line of chemical and physical defense, covering multiple types of pathogens such as bacteria and fungi, and significantly reducing the risk of mixed infection. In terms of growth promotion, the nitrogen fixation, phosphate solubilization and ACC deaminase activity of Bacillus Velez can alleviate plant stress, while the hyphal network of Trichoderma yunnanensis improves soil structure and activates the jasmonic acid signaling pathway, synergistically improving plant biomass and stress resistance. Furthermore, the spore structure of Bacillus velezensis complements the hyphal expansion ability of Trichoderma yunnanensis, enhancing its adaptability in arid or humid environments, reducing the dosage of a single agent while extending its effectiveness. The combined agent also optimizes the rhizosphere microbial community, competitively excluding pathogens and promoting the expansion of beneficial bacteria (such as actinomycetes), forming a stable ecological barrier. This synergistic effect not only improves disease control efficiency and reduces reliance on chemical pesticides, but also promotes the sustainable development of agricultural ecosystems by improving soil health and microbial diversity, providing a highly effective and environmentally friendly solution for green plant protection.

[0017] (2) The soybean meal is rich in protein and can be decomposed to provide plants with nutrients such as nitrogen, phosphorus, potassium, iron, and zinc. The high porosity of biochar can improve soil aeration and water retention, regulate acidity and alkalinity, reduce nutrient loss, and improve fertilizer efficiency. Wollastonite powder contains mineral nutrients such as silicon and calcium. Silicon can strengthen plant cell walls and enhance crop disease resistance. Vinegar dregs are rich in organic matter, a small amount of nitrogen, phosphorus, potassium, and amino acids. After composting, they decompose to provide plants with slow-release nutrients. Allicin, as the sulfur-containing active ingredient in garlic, can directly inhibit the metabolism and reproduction of pathogenic bacteria cells, induce plants to produce systemic resistance and antioxidant effects, and effectively prevent and control a variety of fungal and bacterial diseases. It is an important functional component of natural plant-derived fungicides. The above-mentioned composition achieves the synergy of structural improvement, nutrient regulation, and pollution remediation at the soil level through multi-component functional coupling, and achieves the unity of pathogen inhibition, resistance induction, and healthy growth at the crop level, constructing a benign ecosystem of "soil-plant-microorganism" interaction, and providing soil health protection for the prevention and control of root and stem diseases.

[0018] (3) Biocontrol Bacillus Velez and Trichoderma yunnanensis colonize the rhizosphere, seize the living space and nutrition of pathogens, and combine with the direct antibacterial effect of allicin to form a "space-nutrition-chemical" triple pathogen barrier; microorganisms secrete lipopeptides, hydrolases, etc. to cooperate with allicin thiosulfinate to destroy the cell structure of pathogens at multiple targets, expand the antibacterial spectrum and enhance the bactericidal ability; bacterial components and metabolites activate the plant defense enzyme system, and combined with the physical barrier of silicified cell walls of wollastonite powder, build a "biochemical defense + mechanical resistance" dual protection; biochar and organic matter (soybean meal, vinegar residue) provide carriers and carbon sources for beneficial bacteria, promote their large-scale reproduction, inhibit pathogens, and optimize the structure of soil microbial communities; microbial agents synergistically decompose nutrients (such as mineral elements in wollastonite powder), promote root development and balanced nutrient absorption, enhance the plant's disease resistance, and reduce the risk of disease caused by nutrient deficiency and weak seedlings; the overall "pathogen inhibition-induction resistance-optimization microecology-strong plants" The three-dimensional network significantly improves the effect of disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony morphology diagram of the YNK-FG0001 strain cultured on a plate in Example 1;

[0020] Figure 2 This is the phylogenetic tree constructed based on the ITS gene sequence of the YNK-FG0001 strain in Example 1.

[0021] Biological Deposits

[0022] The Bacillus Velezii used in the present invention is classified and named Bacillus velezensis SH-1471 was deposited with the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, on June 28, 2022, with the deposit number CCTCC NO: M 2022923. It has been disclosed in the invention patent application number CN202211479280.X.

[0023] The Yunnan Trichoderma used in the present invention is classified and named Trichoderma yunnanense YNK-FG0001 was deposited in the China Center for Type Culture Collection on March 31, 2025, at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with the deposit number CCTCC NO: M 2025645. DETAILED DESCRIPTION

[0024] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0025] In the present invention, Bacillus velez SH-1471 and Bacillus velez CCTCC NO: M 2022923 are the same strain, have the same meaning, and their names (numbers) can be used interchangeably. Trichoderma yunnanensis YNK-FG0001 and Trichoderma yunnanensis CCTCC NO: M 2025645 are the same strain, have the same meaning, and their names (numbers) can be used interchangeably.

[0026] The inventors of the present invention isolated and obtained a strain of Bacillus velezensis ( Bacillus velezensis ), named SH-1471, deposited in China Center for Type Culture Collection on June 28, 2022, with the deposit number CCTCC NO: M 2022923, and a strain of Trichoderma yunnanensis ( Trichoderma yunnanense The strain, designated YNK-FG0001, was deposited with the China Center for Type Culture Collection on March 31, 2025, with the accession number CCTCC NO: M2025645. Research has shown that the strains Bacillus velezensis SH-1471 and Trichoderma yunnanensis YNK-FG0001 exhibit strong biological activity, demonstrating potent antagonism against fungal and bacterial pathogens that cause crop diseases.

[0027] The inventors of the present invention found in their research that when a biological organic fertilizer made by mixing soybean meal, vinegar grains, biochar, wollastonite powder and allicin in a certain proportion is applied to the soil, the content of growth hormone and phytoalexin in the root system of wasabi, the activity of defense enzymes and the effect of preventing and controlling root and rhizome diseases are further improved.

[0028] Based on the above findings, the present invention provides a bio-organic fertilizer on one hand, the bio-organic fertilizer comprising:

[0029] Component A: Bacillus velez bacteria agent with a deposit number of CCTCC NO: M 2022923 and Trichoderma yunnanensis bacteria agent with a deposit number of CCTCC NO: M 2025645;

[0030] Component B: soybean meal, biochar, wollastonite powder, vinegar grains, and allicin.

[0031] That is, the bio-organic fertilizer formula provided by the present invention is any one of the following:

[0032] (1) Bacillus velezensis SH-1471 and Trichoderma yunnanensis YNK-FG0001, soybean meal, biochar, wollastonite powder, vinegar grains, and allicin;

[0033] (2) Soybean meal, biochar, wollastonite powder, vinegar grains, and allicin. In component B, the weight ratio of soybean meal, biochar, wollastonite powder, vinegar grains, and allicin is 20-30:20-30:20-30:0.4-1:109-139.6.

[0034] In the present invention, there is no particular limitation on the raw materials used for the above-mentioned bio-organic fertilizer (such as soybean meal, biochar, wollastonite powder, vinegar grains, allicin, etc.). They can be any related products in the art that can be used to prepare bio-organic fertilizers, and can be commercially available products or related products prepared by themselves according to existing technologies.

[0035] The inventors discovered that using specific raw materials resulted in a bio-organic fertilizer that increased the content of growth hormones and phytoalexins in wasabi roots, increased the activity of defense enzymes, and achieved better disease control. Therefore, according to a preferred embodiment of the present invention, the soybean meal, calculated on a dry matter basis, comprises 40-50% protein, 30-40% carbohydrates, and 2-4% fat.

[0036] Preferably, the soybean meal has a protein content of 43-50% by weight, a carbohydrate content of 33-40% by weight, and a fat content of 2.5-4% by weight, calculated on a dry matter basis.

[0037] More preferably, the soybean meal has a protein content of 45-50% by weight, a carbohydrate content of 35-40% by weight, and a fat content of 3-4% by weight, calculated on a dry matter basis.

[0038] According to a preferred embodiment of the present invention, the wollastonite powder has a CaO content of 35-45% by weight and a SiO2 content of 40-50% by weight, calculated on a dry matter basis.

[0039] Preferably, the wollastonite powder has a CaO content of 37-45% by weight and a SiO2 content of 43-50% by weight, calculated on a dry matter basis.

[0040] More preferably, the wollastonite powder has a CaO content of 40-45% by weight and a SiO2 content of 45-50% by weight, calculated on a dry matter basis.

[0041] According to a preferred embodiment of the present invention, the vinegar dregs, calculated on a dry matter basis, have a cellulose content of 30-40% by weight, a protein content of 10-15% by weight, a fat content of 10-15% by weight, and an ash content of 10-15% by weight.

[0042] Preferably, the vinegar dregs have, based on dry matter, a cellulose content of 33-40% by weight, a protein content of 11.5-15% by weight, a fat content of 11.5-15% by weight, and an ash content of 11.5-15% by weight.

[0043] More preferably, the vinegar lees have, based on dry matter, a cellulose content of 35-40% by weight, a protein content of 13-15% by weight, a fat content of 13-15% by weight, and an ash content of 13-15% by weight.

[0044] According to a preferred embodiment of the present invention, the dosage of the Bacillus Velez SH-1471 and Trichoderma yunnanensis YNK-FG0001 agents are both 1-2 weight % of component B, based on the total weight of the bio-organic fertilizer.

[0045] The present invention further provides a method for preparing the above-mentioned bio-organic fertilizer. According to a preferred embodiment of the present invention, the method comprises mixing the soybean meal, biochar, wollastonite powder, vinegar grains and allicin of the above-mentioned component B and composting them to obtain a basic bio-organic fertilizer, and then adding the optional raw materials such as Bacillus velezensis SH-1471 and Trichoderma yunnanensis YNK-FG0001 (or its bacterial agent) to the basic fertilizer according to the above-mentioned dosage ratio according to the above-mentioned dosage ratio, and mixing them evenly.

[0046] Among the present invention, there is no particular restriction for the concrete storage and use form of described bio-organic fertilizer.Because the raw material adopted in this bio-organic fertilizer is solid substantially, preparation method is only each component is mixed in proportion.Therefore, this bio-organic fertilizer can directly store or use with the form of the solid preparation obtained by preparation.

[0047] The second aspect of the present invention provides the use of the above-mentioned bio-organic fertilizer in increasing the content of growth hormones and phytoalexins and the activity of defense enzymes in crop roots and / or preventing and controlling root and tuber diseases.

[0048] In the present invention, crop root growth hormones refer to salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), phytoalexin refers to Camalexin, and defense enzymes refer to chitinase and β-1,3-glucanase; and controlling crop diseases refers to preventing or reducing the occurrence of crop diseases, or reducing the losses caused by diseases after the diseases occur.

[0049] According to a preferred embodiment of the present invention, the crop is wasabi, and the root disease is selected from at least one of wasabi blackheart disease and wasabi soft rot; wherein the pathogen causing the wasabi blackheart disease is stem mold of wasabi ( Phoma wasabiae Yokog and wasabi soft rot is caused by Pectobacterium carrot subspecies ( Pectobacterium carotovorum subsp .carotovorum, Pcc).

[0050] The third aspect of the present invention provides a method for increasing the content of root growth hormones and phytoalexins and the activity of defense enzymes and / or preventing and controlling root and tuber diseases in crops, the method comprising applying the bio-organic fertilizer described in the second aspect to the soil.

[0051] In the present invention, the crop is wasabi.

[0052] Preferably, the pathogen causing the wasabi rhizogenes is Stemomyces wasabi ( Phoma wasabiae Yokog and wasabi soft rot is caused by Pectobacterium carrot subspecies ( Pectobacterium carotovorum subsp .carotovorum, Pcc).

[0053] The specific characteristics of the Bacillus Velezii and Trichoderma yunnanensis used in the method provided by the present invention and the bio-organic fertilizer are as described above and will not be repeated here.

[0054] In the present invention, there is no particular limitation on the specific usage of Bacillus velezensis, Trichoderma yunnanensis and bio-organic fertilizer, as long as they can enhance the activity of defense enzymes and prevent and control crop diseases.

[0055] The bio-organic fertilizer is applied to crop planting soil in an amount of 100-200 kg / mu / time. For example, it can be 100 kg / mu / time, 120 kg / mu / time, 140 kg / mu / time, 160 kg / mu / time, 180 kg / mu / time, 200 kg / mu / time, or any intermediate value between any two of the above values.

[0056] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0057] Example 1

[0058] This example is used to illustrate the acquisition, identification and preservation of Trichoderma yunnanensis CCTCC NO: M 2025645.

[0059] (1) Strain isolation and purification

[0060] In August 2024, a fungus was isolated from the soil of a flue-cured tobacco farmland in Yiliang County, Zhaotong City, Yunnan Province, China (104°21'E, 27°37'N) and deposited in the General Microbiology Center of the China Culture Collection Administration with the deposit number CCTCC NO: M 2025645.

[0061] PDA medium was used in the isolation and purification of the strain. The preparation method was as follows: 200 g of potato, 20 g of glucose, 15-20 g of agar, 1000 mL of deionized water, adjusted to pH 7.0 ± 0.1, and sterilized by high pressure at 121°C for 25 min.

[0062] A fungus was isolated and purified from soil samples collected from flue-cured tobacco fields in Yiliang County, Zhaotong City, Yunnan Province by using the dilution spread plate method and tip hyphae picking method. It was named Trichoderma yunnanensis.

[0063] (2) Strain identification

[0064] 1. Identification of fungal morphological characteristics and molecular genetic classification

[0065] Colony: Yunnan Trichoderma strain YNK-FG0001 was inoculated on PDA medium and cultured at 28℃. The colony on the PDA medium showed the following changes: at the beginning, the strain was white, dense, and round, and expanded to the surrounding areas; after growing for a period of time, light green spores were produced from the center of the colony, and the center turned green; finally, the entire colony turned green. Figure 1 As shown. Trichoderma yunnanense) strain was inoculated into PDA solid medium and cultured at 30°C for 48 hours. The strain was then sent to the Chongqing branch of Beijing Qingke Biotechnology Co., Ltd. for ITS sequencing. The sequence result was: (SEQ ID NO.1). The sequencing results were imported into NCBI for comparison and compared with the sequence of a standard strain with a homology of more than 80%. It was also compared with the sequence of Trichoderma yunnanensis ( Trichodermayunnanense , NR134419) with a homology of up to 98.98% (see Figure 2 ), so it was identified as a microorganism of the subphylum Deuteromycotina, class Hypomycetes, order Hypomycetales, family Hypomycetaceae, and genus Trichoderma. A phylogenetic tree of the strain was constructed using MEGA7. Figure 2 The drawn phylogenetic tree of the strains is shown.

[0066] 3. Identification results

[0067] Combined with the molecular detection results of strain YNK-FG0001 and the fungal morphological characteristics detection results, the strain was identified as Trichoderma yunnanensis ( Trichoderma yunnanense )

[0068] (3) Strain preservation

[0069] Name the above obtained classification as: Trichoderma yunnanense The Yunnan Trichoderma fungus YNK-FG0001 was deposited in the China Center for Type Culture Collection on March 31, 2025, at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with the deposit number CCTCCNO: M 2025645.

[0070] Example 2

[0071] The soybean meal used in this example was purchased from Kunming Tengze Industry and Trade Co., Ltd., the vinegar residue was purchased from Yunnan Lufeng Dingxin Vinegar Co., Ltd., the wollastonite powder was purchased from Yunnan Shijing Silicon Industry Co., Ltd., the biochar was purchased from Yunnan Yongde Kangwei Biocarbon Industry Co., Ltd., and the allicin was purchased from Yunnan Dadong Biopharmaceutical Co., Ltd.

[0072] Table 1 shows the test results of the raw material components used for the preparation of basic organic fertilizer. Among them, the protein content in soybean meal was determined by Kjeldahl method, the carbohydrate content was determined by enzymatic hydrolysis method, and the fat content was determined by Soxhlet extraction method; the cellulose content in vinegar lees was determined by enzymatic hydrolysis method, the crude protein content was determined by Kjeldahl method, the crude fat content was determined by Soxhlet extraction method, and the crude ash content was determined by acid washing method; the CaO content in wollastonite powder was determined by acid-base titration method, and the SiO2 content was determined by phosphomolybdic acid spectrophotometry.

[0073] Table 1 Basic organic fertilizer material composition

[0074]

[0075] Preparation of the Bacillus Velez and Trichoderma yunnanensis agents of the present invention:

[0076] The inoculum was prepared by expanding the liquid culture of Bacillus velezensis 1471 (see 202211479280.X for the method of obtaining it) to a concentration of 5×10 10 cfu / g.

[0077] The liquid culture solution obtained by expanding the Yunnan Trichoderma YNK-FG0001 was 5×10 9 cfu / g. The method comprises the following steps: (1) solid culturing the above-mentioned Trichoderma yunnanensis in a solid culture medium to obtain test tube seeds; (2) preparing a liquid seed culture medium, inoculating the test tube seeds therein for liquid culture to obtain liquid seeds; and (3) preparing a liquid fermentation medium, inoculating the liquid seeds therein for fermentation.

[0078] In step (1), the Trichoderma yunnanensis is inoculated by slant inoculation, and the conditions for culturing the slant solid culture medium are: culturing at a temperature of 25-30°C for 120-144 hours; the solid culture medium comprises: 200 g / L potato, 20 g / L glucose, 15-20 g / L agar, and a pH of 6.2-6.6.

[0079] In step (2), the test tube seeds are inoculated into a liquid seed culture medium, and the liquid culture is treated for 96-120 hours at a temperature of 25-30°C and a rotation speed of 150-200 r / min; the liquid seed culture medium comprises: 5 g / L peptone, 2 g / L yeast extract powder, 20 g / L glucose, 1 g / L potassium hydrogen phosphate, 0.5 g / L magnesium sulfate, and a pH of 6.2-6.6.

[0080] In step (3), the liquid seeds are inoculated in a liquid fermentation medium at a volume ratio of 0.05-0.1:1 and cultured at a temperature of 25-30°C and a rotation speed of 150-200 r / min for 72-96 hours; the liquid fermentation medium comprises: 20 g / L malt extract, 20 g / L glucose, and a pH of 6.2-6.6. In this step, the liquid fermentation medium is preferably sterilized at 120-125°C for 20-30 minutes, cooled, inoculated with the liquid seeds, and cultured preferably at 28-30°C on a shaker at a rotation speed of 150-200 r / min for 72-96 hours to obtain a fermentation liquid.

[0081] The bio-organic fertilizer was prepared according to the formula in Table 2. The specific preparation method included: weighing the raw materials according to the proportions in Table 2, and mixing them evenly to obtain the bio-organic fertilizer (solid preparation).

[0082] Table 2 Microbial agent formula

[0083]

[0084] According to a preferred embodiment of the present invention, the application frequency of the Bacillus Velez subtilis and Trichoderma yunnanensis bio-organic fertilizer is once per crop.

[0085] According to the formula of bio-organic fertilizer in Table 2, fertilizer was applied at a rate of 100-200 kg / mu / time (100 plants were planted in each experimental group); the activity of plant defense enzymes was measured 30 days after transplanting; and the disease control effect was investigated at the harvest period.

[0086] Determination of growth hormone and phytoalexin content in wasabi roots: Salicylic acid (SA), jasmonic acid (JA), ethylene (ET), fusaric acid and Camalexin contents were determined by high performance liquid chromatography (HPLC).

[0087] Method for determining the activity of defense enzymes in wasabi roots: Chitinase and β-1,3-glucanase were determined using kits purchased from Suzhou Gres Biotechnology Co., Ltd. The method of use was referred to the instructions.

[0088] Wasabi Blackheart Disease Survey and Grading Standards:

[0089] Level 0: The whole plant is disease-free; Level 1: The lesions on the stem do not exceed 1 / 3 of the stem circumference, and some leaves wilt; Level 3: The lesions on the stem do not exceed 1 / 2 of the stem circumference, or less than half of the leaves are slightly withered, or lesions appear on a few leaves at the bottom; Level 5: The lesions on the stem exceed 1 / 2 of the stem circumference, but do not completely surround the stem circumference, or more than half of the leaves are slightly withered; Level 7: The lesions on the stem surround the stem circumference, or more than 2 / 3 of the leaves are withered; Level 9: All leaves of the diseased plant wilt or die.

[0090] Wasabi soft rot investigation and grading standards:

[0091] Level 0: Disease-free, the plant is completely healthy and does not have any symptoms of soft rot.

[0092] Level 1: Single or a few small water-soaked lesions appear on the outer leaves or top leaves of the plant, and the area of ​​lesions accounts for less than 10% of the total leaf area; Level 3: There are more lesions on the outer leaves or top leaves, and some leaves begin to wilt slightly, and the area of ​​lesions accounts for 20%-30% of the total leaf area; Level 5: Most of the outer leaves or top leaves wilt, and the area of ​​lesions accounts for 40%-50% of the total leaf area. The lesions at the base of the stem expand and begin to show signs of rot, but have not spread to the whole plant; Level 7: More than 1 / 2 of the leaves on the whole plant are soft rotted, the base of the stem is severely rotted, most of the plant collapses, but some leaves still remain green; Level 9: The whole plant rots, leaving only some residual roots and some incompletely rotten tissues, and the field appears soft and rotten.

[0093] The control effect is calculated using the following two formulas:

[0094] Disease index = [(Σ(number of diseased plants × representative level)) / (total number of plants × highest representative level)] × 100

[0095] Preventive and therapeutic effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%

[0096] It can be seen from the data in Tables 3 and 4 that when the biological organic fertilizer provided by the present invention is applied, the content of growth hormone and phytoalexin and the activity of defense enzymes in the wasabi root system are significantly higher than those in the control, and the prevention and control effect on wasabi rhizome diseases is further improved and significantly higher than that in the control.

[0097] Table 3 Test conditions and results of the test on the control effect of biological organic fertilizer on wasabi rhizome diseases

[0098]

[0099] *Blank control refers to the blank control group without application of any bio-organic fertilizer.

[0100] Table 4 Test conditions and results of the test on growth hormone and phytoalexin content and defense enzyme activity of the tested bio-organic fertilizer

[0101]

[0102] *Blank control refers to the blank control group without application of any bio-organic fertilizer.

[0103] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A bio-organic fertilizer for preventing and treating wasabi root diseases, characterized in that: The bio-organic fertilizer comprises the following components: Component A: Bacillus velezensis with a deposit number of CCTCC NO: M 2022923 ( Bacillus velezensis) Bacterial agent and Trichoderma yunnanensis with a preservation number of CCTCC NO: M 2025645 ( Trichoderma yunnanense ) microbial agents; Component B: soybean meal, biochar, wollastonite powder, vinegar grains and allicin.

2. A biological organic fertilizer for preventing and treating wasabi root diseases according to claim 1, characterized in that: In component A, the effective viable bacteria count in the Bacillus Velez bacteria agent is not less than 5×10 10 CFU / g, the effective viable bacteria count in the Trichoderma yunnanensis agent is not less than 5×10 9 CFU / g.

3. A biological organic fertilizer for preventing and treating wasabi root diseases according to claim 1, characterized in that: The dosage of the Bacillus Velez subtilis agent and the Trichoderma yunnanensis agent is 1-2% by weight of component B.

4. A biological organic fertilizer for preventing and treating wasabi root diseases according to claim 1, characterized in that: In component B, the weight ratio of soybean meal, biochar, wollastonite powder, vinegar grains and allicin is 20-30:20-30:20-30:0.4-1:109-139.

6.

5. A biological organic fertilizer for preventing and treating wasabi rhizome diseases according to claim 4, characterized in that: The soybean meal, calculated on a dry matter basis, has a protein content of 40-50% by weight, a carbohydrate content of 30-40% by weight, and a fat content of 2-4% by weight; and / or the wollastonite powder, calculated on a dry matter basis, has a CaO content of 35-45% by weight, and a SiO2 content of 40-50% by weight; and / or the vinegar lees, calculated on a dry matter basis, has a cellulose content of 30-40% by weight, a protein content of 10-15% by weight, a fat content of 10-15% by weight, and an ash content of 10-15% by weight.

6. Use of the bio-organic fertilizer according to any one of claims 1 to 5 for increasing the content of root growth hormones, phytoalexins, and defense enzyme activities in crops, wherein the crop is wasabi; the growth hormone is at least one of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET); the phytoalexin is camalexin; and the defense enzyme is at least one of chitinase and β-1,3-glucanase.

7. Use of the bio-organic fertilizer according to any one of claims 1 to 5 in preventing and treating wasabi root diseases, wherein the root diseases are selected from at least one of wasabi blackheart disease and wasabi soft rot, wherein: The pathogen causing the wasabi blackheart disease is stem mold of wasabi ( Phoma wasabiae Yokog), wasabi soft rot is caused by Pectobacterium carrot subspecies ( Pectobacterium carotovorum subsp .carotovorum, Pcc).

8. A method for increasing the content of growth hormones, phytoalexins and defense enzyme activities in crop roots, characterized in that: The method comprises applying the bio-organic fertilizer according to any one of claims 1 to 4 to the soil, wherein the amount of the bio-organic fertilizer applied to the soil is 100-200 kg / mu / time, and the application frequency is once during the entire growth period.

9. A method for preventing and controlling rhizome diseases, characterized in that: The method comprises applying the bio-organic fertilizer according to any one of claims 1 to 4 to the soil, wherein the amount of the bio-organic fertilizer applied to the soil is 100-200 kg / mu / time, and the application frequency is once during the entire growth period.

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