A bio-organic fertilizer for reducing the incidence of tobacco root-knot nematodes and its preparation method
By preparing a bio-organic fertilizer with a specific ratio, and utilizing the synergistic effect of Bacillus subtilis, Trichoderma harzianum, and Bacillus mucilaginosus, the problem of high incidence of tobacco root-knot nematode disease was solved, resulting in improved root vitality and tobacco planting effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HAINAN TOBACCO CO DANZHOU CO
- Filing Date
- 2023-11-30
- Publication Date
- 2026-05-26
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of fertilizers, and in particular to a method for preparing a bio-organic fertilizer that reduces the incidence of tobacco root-knot nematodes. Background Technology
[0002] Tobacco root-knot nematode disease is a disease caused by root-knot nematodes. These nematodes belong to the phylum Nematoda, class Nematoda, order Nematoda, superfamily Nematodae, family Heterodermaceae, and genus *Strombyx*. Infected tobacco plants develop numerous root galls (insect galls) on their roots, with few fibrous roots. The tips and edges of the tobacco leaves wilt, and the leaves turn yellow, dry, and curl inwards. Initially, the root galls are white, gradually enlarging and becoming irregularly round or spindle-shaped. Sometimes the entire root system swells into a claw-like shape. Later, the roots rot and become hollow, negatively impacting the quality of tobacco cultivation. Summary of the Invention
[0003] Therefore, this invention proposes a method for preparing bio-organic fertilizer that reduces the incidence of tobacco root-knot nematodes, thus solving the above-mentioned problems.
[0004] The technical solution of this invention is implemented as follows:
[0005] A bio-organic fertilizer for reducing the incidence of tobacco root-knot nematodes comprises fermentation substrate, microbial agents, and *Smilax china* in a mass ratio of 10:0.2-0.4:2-3, wherein the microbial agents comprise *Bacillus subtilis*, *Trichoderma harzianum*, and *Bacillus mucilaginosus* in a mass ratio of 15-25:8-13:30-40.
[0006] Furthermore, by weight, the fermentation substrate comprises 20-30 parts cow dung, 10-15 parts wheat bran, 18-22 parts straw, and 3-5 parts earthworm castings.
[0007] Furthermore, the straw is rice straw.
[0008] Furthermore, the method for preparing the Bacillus subtilis bacterial solution involves inoculating Bacillus subtilis into liquid culture medium 1 and culturing it at 36-38℃ and 150-170 rpm for 18-22 hours to obtain the Bacillus subtilis bacterial solution.
[0009] Furthermore, the method for preparing the Trichoderma harzianum culture solution involves inoculating Trichoderma harzianum into PDA medium and culturing it at 25-27℃ and 110-130 rpm for 50-55 hours to obtain the Trichoderma harzianum culture solution.
[0010] Furthermore, the method for preparing the gelatinous Bacillus bacterial suspension involves inoculating gelatinous Bacillus into liquid culture medium 2 and culturing it at 36-38℃ and 150-170rpm for 18-22 hours to obtain the gelatinous Bacillus bacterial suspension.
[0011] Furthermore, the liquid culture medium 1 comprises, by weight percentage, 0.02-0.04% sorbic acid, 0.15-0.25% glucose, 0.4-0.6% dipotassium hydrogen phosphate, 0.003-0.005% tyrosine, 0.07-0.09% manganese sulfate, 0.2-0.4% peptone, 0.4-0.6% calcium carbonate, 0.2-0.3% agar, 2% soybean meal, and the balance being water.
[0012] Furthermore, the liquid culture medium 2 comprises, by weight percentage, 0.07-0.09% sodium gluconate, 0.15-0.25% glucose, 0.2-0.3% peptone, 0.003-0.005% tyrosine, 0.02-0.04% yeast extract, 0.4-0.6% potassium sulfate, 0.2-0.4% manganese sulfate, 0.4-0.6% potassium dihydrogen phosphate, 1-1.5% sweet potato flour, 0.8-1.2% hawthorn powder, and the balance being water.
[0013] Furthermore, the fermentation substrate and microbial inoculant are mixed and piled into a triangular pile 0.9-1.1m high, and fermented for 12-15 days to obtain fermented microbial fertilizer.
[0014] Furthermore, the fermented microbial fertilizer is mixed with snake stalk powder to produce bio-organic fertilizer.
[0015] The beneficial effects of this invention are:
[0016] This invention uses a microbial inoculum prepared from Bacillus subtilis, Trichoderma harzianum, and Bacillus mucilaginosus in a mass ratio of 20:11:35. The fermentation substrate and microbial inoculum are mixed and fermented. The fermentation substrate includes cow dung, straw, snake manure, and earthworm castings. Specifically, Bacillus subtilis is cultured using a medium prepared with sorbic acid, glucose, dipotassium hydrogen phosphate, tyrosine, manganese sulfate, peptone, calcium carbonate, agar, soybean meal, and water. Bacillus mucilaginosus is cultured using a medium prepared with sodium gluconate, glucose, peptone, tyrosine, yeast powder, potassium sulfate, manganese sulfate, potassium dihydrogen phosphate, sweet potato flour, hawthorn powder, and water. A suitable culture medium promotes microbial growth and cultivation, enhances the metabolic capacity and growth rate of the microbial preparation in organic fertilizer, accelerates organic fertilizer fermentation, and results in organic fertilizer containing more effective components that inhibit root-knot insect reproduction, thus improving the overall effectiveness of the organic fertilizer. This invention uses Bacillus subtilis, Trichoderma harzianum, and Bacillus mucilaginosus inoculum to prepare a microbial agent. The rational combination of microbial strains maximizes the utilization of nutrients in organic fertilizer, exerting a synergistic effect to accelerate fermentation. The resulting metabolites do not interfere with the microbial agent, allowing it to further grow and reproduce using these metabolites, thus improving the fermentation efficiency of the organic fertilizer. The fermentation substrate of this invention contains *Symplocos edulis* (a type of herb). After being crushed, dried, and fermented with the microbial inoculum, this invention selects appropriate microbial strains to extract the effective components from the *Symplocos edulis*, further enhancing the effect of the bio-organic fertilizer in inhibiting tobacco root-knot nematodes.
[0017] The bio-organic fertilizer prepared by this invention resulted in a 15% incidence rate of root-knot nematodes in tobacco, with a root dry weight of 39.2 g / plant. Experiments show that this bio-organic fertilizer effectively reduces the incidence of root-knot nematodes in tobacco, improves root vitality, promotes root growth, and further enhances the planting effect of tobacco leaves. Detailed Implementation
[0018] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.
[0019] The Bacillus subtilis, Trichoderma harzianum, Bacillus polymyxa, Paecilomyces lilacinus, and Bacillus mucilaginosus used in this invention were purchased from the Institute of Microbiology, Chinese Academy of Sciences.
[0020] Example 1
[0021] Liquid culture medium 1, by weight percentage, consists of 0.02% sorbic acid, 0.15% glucose, 0.4% dipotassium hydrogen phosphate, 0.003% tyrosine, 0.07% manganese sulfate, 0.2% peptone, 0.4% calcium carbonate, 0.2% agar, 2% soybean meal, and the balance water.
[0022] Liquid culture medium 2, by weight percentage, consists of 0.07% sodium gluconate, 0.15% glucose, 0.2% peptone, 0.003% tyrosine, 0.02% yeast extract, 0.4% potassium sulfate, 0.2% manganese sulfate, 0.4% potassium dihydrogen phosphate, 1% sweet potato powder, 0.8% hawthorn powder, and the balance being water.
[0023] Bacillus subtilis was inoculated into liquid culture medium 1 at an inoculation amount of 1% and cultured at 36℃ and 150rpm for 18h to obtain Bacillus subtilis bacterial suspension.
[0024] Trichoderma harzianum was inoculated into PDA medium at an inoculation rate of 1% and cultured at 25°C and 110 rpm for 50 h to obtain Trichoderma harzianum culture solution.
[0025] Bacillus jelly-like bacteria were inoculated into liquid culture medium 2 at an inoculation amount of 1% and cultured at 36℃ and 150rpm for 18h to obtain Bacillus jelly-like bacterial suspension.
[0026] Weigh out the fermentation substrate, microbial inoculant, and snake stalks in a mass ratio of 10:0.2:2.
[0027] Microbial inoculants were prepared by mixing Bacillus subtilis culture, Trichoderma harzianum culture, and Bacillus mucilaginosus culture in a mass ratio of 15:8:30.
[0028] The snake seeds were dried at 60℃ for 4 hours and then pulverized to obtain snake seed powder.
[0029] By weight, 20 parts cow dung, 10 parts wheat bran, 18 parts straw and 3 parts earthworm castings are mixed to prepare the fermentation substrate.
[0030] Microbial inoculants and fermentation substrates were mixed at a mass ratio of 10:0.2, piled into a triangular heap 0.9m high, and fermented for 12 days to obtain fermented microbial fertilizer.
[0031] Fermented microbial fertilizer and snake nut powder are mixed to produce bio-organic fertilizer.
[0032] Example 2
[0033] Liquid culture medium 1, by weight percentage, consists of 0.04% sorbic acid, 0.25% glucose, 0.6% dipotassium hydrogen phosphate, 0.005% tyrosine, 0.09% manganese sulfate, 0.4% peptone, 0.6% calcium carbonate, 0.3% agar, 2% soybean meal, and the balance water.
[0034] Liquid culture medium 2, by weight percentage, consists of 0.09% sodium gluconate, 0.25% glucose, 0.3% peptone, 0.005% tyrosine, 0.04% yeast extract, 0.6% potassium sulfate, 0.4% manganese sulfate, 0.6% potassium dihydrogen phosphate, 1.5% sweet potato powder, 1.2% hawthorn powder, and the balance being water.
[0035] Bacillus subtilis was inoculated into liquid culture medium 1 at an inoculation amount of 1% and cultured at 38°C and 170 rpm for 22 h to obtain Bacillus subtilis bacterial suspension.
[0036] Trichoderma harzianum was inoculated into PDA medium at an inoculation rate of 1% and cultured at 27°C and 130 rpm for 55 h to obtain Trichoderma harzianum culture solution.
[0037] Bacillus jelly-like bacteria were inoculated into liquid culture medium 2 at an inoculation amount of 1% and cultured at 38°C and 170 rpm for 22 h to obtain Bacillus jelly-like bacterial suspension.
[0038] Weigh out the fermentation substrate, microbial inoculant, and snake roe in a mass ratio of 10:0.4:3.
[0039] Microbial inoculants were prepared by mixing Bacillus subtilis culture, Trichoderma harzianum culture, and Bacillus mucilaginosus culture in a mass ratio of 25:13:40.
[0040] The snake seeds were dried at 60℃ for 4 hours and then pulverized to obtain snake seed powder.
[0041] By weight, 30 parts cow dung, 15 parts wheat bran, 22 parts straw, 13 parts snake manure, and 5 parts earthworm castings are mixed to prepare the fermentation substrate.
[0042] Microbial inoculants and fermentation substrates were mixed at a mass ratio of 10:0.4, piled into a triangular heap 1.1m high, and fermented for 15 days to obtain fermented microbial fertilizer.
[0043] Fermented microbial fertilizer and snake nut powder are mixed to produce bio-organic fertilizer.
[0044] Example 3
[0045] Liquid culture medium 1, by weight percentage, consists of 0.03% sorbic acid, 0.2% glucose, 0.5% dipotassium hydrogen phosphate, 0.004% tyrosine, 0.08% manganese sulfate, 0.3% peptone, 0.5% calcium carbonate, 0.25% agar, 2% soybean meal, and the balance water.
[0046] Liquid culture medium 2, by weight percentage, consists of 0.08% sodium gluconate, 0.2% glucose, 0.25% peptone, 0.004% tyrosine, 0.03% yeast extract, 0.5% potassium sulfate, 0.3% manganese sulfate, 0.5% potassium dihydrogen phosphate, 1.25% sweet potato powder, 1% hawthorn powder, and the balance being water.
[0047] Bacillus subtilis was inoculated into liquid culture medium 1 at an inoculation amount of 1% and cultured at 37°C and 160 rpm for 20 h to obtain Bacillus subtilis bacterial suspension.
[0048] Trichoderma harzianum was inoculated into PDA medium at an inoculation rate of 1% and cultured at 26°C and 120 rpm for 53 h to obtain Trichoderma harzianum culture solution.
[0049] Bacillus jelly-like bacteria were inoculated into liquid culture medium 2 at an inoculation amount of 1% and cultured at 37°C and 160 rpm for 20 h to obtain Bacillus jelly-like bacterial suspension.
[0050] Weigh out the fermentation substrate, microbial inoculum, and snake roe in a mass ratio of 10:0.3:2.5.
[0051] Microbial inoculants were prepared by mixing Bacillus subtilis culture, Trichoderma harzianum culture, and Bacillus mucilaginosus culture in a mass ratio of 20:11:35.
[0052] The snake seeds were dried at 60℃ for 4 hours and then pulverized to obtain snake seed powder.
[0053] By weight, 25 parts cow dung, 13 parts wheat bran, 20 parts straw, 11.5 parts snake manure, and 4 parts earthworm castings are mixed to prepare the fermentation substrate.
[0054] Microbial inoculants and fermentation substrates were mixed at a mass ratio of 10:0.3, piled into a triangular heap 1m high, and fermented for 15 days to obtain fermented microbial fertilizer.
[0055] Fermented microbial fertilizer and snake nut powder are mixed to produce bio-organic fertilizer.
[0056] Example 4
[0057] Liquid culture medium 1, by weight percentage, consists of 0.02% sorbic acid, 0.25% glucose, 0.5% dipotassium hydrogen phosphate, 0.004% tyrosine, 0.08% manganese sulfate, 0.4% peptone, 0.4% calcium carbonate, 0.2% agar, 2% soybean meal, and the balance water.
[0058] Liquid culture medium 2, by weight percentage, consists of 0.09% sodium gluconate, 0.15-0.25% glucose, 0.2% peptone, 0.005% tyrosine, 0.03% yeast extract, 0.5% potassium sulfate, 0.4% manganese sulfate, 0.6% potassium dihydrogen phosphate, 1% sweet potato powder, 1.2% hawthorn powder, and the balance being water.
[0059] Bacillus subtilis was inoculated into liquid culture medium 1 at an inoculation amount of 1% and cultured at 36-38℃ and 150-170rpm for 18-22h to obtain Bacillus subtilis bacterial suspension.
[0060] Bacillus subtilis was inoculated into liquid culture medium 1 at an inoculation amount of 1% and cultured at 37°C and 160 rpm for 20 h to obtain Bacillus subtilis bacterial suspension.
[0061] Trichoderma harzianum was inoculated into PDA medium at an inoculation rate of 1% and cultured at 26°C and 120 rpm for 53 h to obtain Trichoderma harzianum culture solution.
[0062] Bacillus jelly-like bacteria were inoculated into liquid culture medium 2 at an inoculation amount of 1% and cultured at 37°C and 160 rpm for 20 h to obtain Bacillus jelly-like bacterial suspension.
[0063] Weigh out the fermentation substrate, microbial inoculum, and snake roe in a mass ratio of 10:0.3:2.5.
[0064] Microbial inoculants were prepared by mixing Bacillus subtilis culture, Trichoderma harzianum culture, and Bacillus mucilaginosus culture in a mass ratio of 20:11:35.
[0065] The snake seeds were dried at 60℃ for 4 hours and then pulverized to obtain snake seed powder.
[0066] By weight, 20 parts cow dung, 15 parts wheat bran, 18 parts straw, 12 parts snake manure, and 5 parts earthworm castings are mixed to prepare the fermentation substrate.
[0067] Microbial inoculants and fermentation substrates were mixed at a mass ratio of 10:0.3, piled into a triangular heap 1m high, and fermented for 15 days to obtain fermented microbial fertilizer.
[0068] Fermented microbial fertilizer and snake nut powder are mixed to produce bio-organic fertilizer.
[0069] Comparative Example 1
[0070] The difference from Example 3 is that Bacillus subtilis, Trichoderma harzianum, and Bacillus colloidis were inoculated into PDA medium at an inoculation rate of 1% to prepare Bacillus subtilis culture, Trichoderma harzianum culture, and Bacillus colloidis culture, respectively.
[0071] Weigh out the fermentation substrate, microbial inoculum, and snake roe in a mass ratio of 10:0.3:2.5.
[0072] Microbial inoculants were prepared by mixing Bacillus subtilis culture, Trichoderma harzianum culture, and Bacillus mucilaginosus culture in a mass ratio of 20:11:35.
[0073] The snake seeds were dried at 60℃ for 4 hours and then pulverized to obtain snake seed powder.
[0074] By weight, 25 parts cow dung, 13 parts wheat bran, 20 parts straw, 11.5 parts snake manure, and 4 parts earthworm castings are mixed to prepare the fermentation substrate.
[0075] Microbial inoculants and fermentation substrates were mixed at a mass ratio of 10:0.3, piled into a triangular heap 1m high, and fermented for 15 days to obtain fermented microbial fertilizer.
[0076] Fermented microbial fertilizer and snake nut powder are mixed to produce bio-organic fertilizer.
[0077] Comparative Example 2
[0078] The difference in Example 3 is that the microbial agent consists of Paecilomyces lilacinus, Bacillus polymyxa, and Trichoderma harzianum.
[0079] Paecilomyces lilacinus was inoculated into liquid culture medium 1 at an inoculation amount of 1% and cultured at 37℃ and 160rpm for 20h to obtain Bacillus subtilis bacterial suspension.
[0080] Trichoderma harzianum was inoculated into PDA medium at an inoculation rate of 1% and cultured at 26°C and 120 rpm for 53 h to obtain Trichoderma harzianum culture solution.
[0081] Polymyxin Bacillus spores were inoculated into liquid culture medium 2 at an inoculation amount of 1% and cultured at 37℃ and 160rpm for 20h to obtain gelatinous Bacillus bacterial suspension.
[0082] Weigh out the fermentation substrate, microbial inoculum, and snake roe in a mass ratio of 10:0.3:2.5.
[0083] Microbial inoculants were prepared by mixing Paecilomyces lilacinus, Trichoderma harzianum, and Bacillus polymyxa in a mass ratio of 20:11:35.
[0084] The snake seeds were dried at 60℃ for 4 hours and then pulverized to obtain snake seed powder.
[0085] By weight, 25 parts cow dung, 13 parts wheat bran, 20 parts straw, 11.5 parts snake manure, and 4 parts earthworm castings are mixed to prepare the fermentation substrate.
[0086] Microbial inoculants and fermentation substrates were mixed at a mass ratio of 10:0.3, piled into a triangular heap 1m high, and fermented for 15 days to obtain fermented microbial fertilizer.
[0087] Fermented microbial fertilizer and snake nut powder are mixed to produce bio-organic fertilizer.
[0088] Experimental Example 1
[0089] The experiment was conducted in Danzhou City, Hainan Province. The bio-organic fertilizer prepared in Example 3 and Comparative Examples 1-2 was designated as treatment groups 1-3, with an application rate of 25 kg / mu. This was used to prepare the planting substrate. The control group used Wofuwo tobacco-specific fertilizer. When the tobacco seedlings reached 5-6 leaves, the roots were inoculated with 5 mL / plant of the tobacco root-knot worm pathogen. The treatment was repeated three times, with 100 plants each time. The root-knot worm incidence rate was recorded after 60 days.
[0090] Incidence rate = (Number of infected plants / 300) × 100%
[0091]
[0092] Experimental results show that the bio-organic fertilizer of this invention can effectively prevent the growth of tobacco root-knot worm pathogens. In Comparative Example 1, the culture medium for *Bacillus subtilis* and *Bacillus mucilaginosus* was modified. This invention, by culturing *Bacillus subtilis* and *Bacillus mucilaginosus* in a specific culture medium, can improve the metabolic capacity and growth rate of the microbial preparation in the organic fertilizer, promote organic fertilizer fermentation, and increase the content of more effective components that inhibit root-knot worm reproduction in the organic fertilizer, thus improving the effectiveness of the organic fertilizer. In Comparative Example 2, the combination was changed, using *Paecilomyces lilacinus*, *Bacillus polymyxa*, and *Trichoderma harzianum* to replace *Bacillus subtilis*, *Trichoderma harzianum*, and *Bacillus mucilaginosus* in the microbial preparation, resulting in a decrease in the inhibitory effect on root-knot worm disease. Experiments showed that the strains of this invention can exert a synergistic effect to accelerate organic fertilizer fermentation, and the resulting metabolites do not interfere with the microbial preparation. The microbial preparation can utilize the metabolites for further growth and reproduction, improving the fermentation efficiency of the organic fertilizer.
[0093] Experimental Example 2
[0094] Tobacco roots were collected from Experiment 1, dried, weighed, and the average weight was calculated.
[0095]
[0096] Experimental results show that the bio-organic fertilizer of this invention can improve root vitality, promote root growth, and further enhance the planting effect of tobacco leaves. The bio-organic fertilizer of this invention has a better planting effect than commercially available tobacco organic fertilizers, and can improve the economic benefits of tobacco planting.
[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A bio-organic fertilizer for reducing the incidence of tobacco root-knot nematode, characterized in that, The bio-organic fertilizer raw materials include fermentation substrate, microbial inoculants and snake stalks in a mass ratio of 10:0.3:2.
5. The microbial inoculants include Bacillus subtilis inoculum, Trichoderma harzianum inoculum and Bacillus thuringiensis inoculum in a mass ratio of 20:11:
35. The method for preparing Bacillus subtilis bacterial suspension is to inoculate Bacillus subtilis into liquid culture medium 1 and culture it at 37°C and 160 rpm for 20 h to obtain Bacillus subtilis bacterial suspension. The liquid culture medium 1, by weight percentage, comprises 0.03% sorbic acid, 0.2% glucose, 0.5% dipotassium hydrogen phosphate, 0.004% tyrosine, 0.08% manganese sulfate, 0.3% peptone, 0.5% calcium carbonate, 0.25% agar, 2% soybean meal, and the balance being water; The method for preparing Trichoderma harzianum culture solution is to inoculate Trichoderma harzianum into PDA medium and culture it at 26°C and 120 rpm for 53 h to obtain Trichoderma harzianum culture solution. The method for preparing the gelatinous Bacillus bacterial suspension is to inoculate gelatinous Bacillus into liquid culture medium 2 and culture it at 37°C and 160 rpm for 20 h to obtain gelatinous Bacillus bacterial suspension. The liquid culture medium 2 comprises, by weight percentage, 0.08% sodium gluconate, 0.2% glucose, 0.25% peptone, 0.004% tyrosine, 0.03% yeast extract, 0.5% potassium sulfate, 0.3% manganese sulfate, 0.5% potassium dihydrogen phosphate, 1.25% sweet potato powder, 1% hawthorn powder, and the balance being water.
2. The bio-organic fertilizer for reducing the incidence of tobacco root-knot nematodes as described in claim 1, characterized in that, By weight, the fermentation substrate comprises 25 parts cow dung, 13 parts wheat bran, 20 parts straw, 11.5 parts snake antlers, and 4 parts earthworm castings.
3. The method for preparing bio-organic fertilizer for reducing the incidence of tobacco root-knot nematodes as described in any one of claims 1-2, characterized in that, Microbial inoculants and fermentation substrates are mixed at a mass ratio of 10:0.3, piled into a triangular pile 1m high, and fermented for 15 days to obtain fermented microbial fertilizer. The fermented microbial fertilizer is then mixed with snake stalk powder to obtain bio-organic fertilizer.