CN101974462B - Bacillus subtilis for preventing and controlling Chinese herbal medicine soil-borne diseases and preparation method of bactericide thereof - Google Patents
Bacillus subtilis for preventing and controlling Chinese herbal medicine soil-borne diseases and preparation method of bactericide thereof Download PDFInfo
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Abstract
The invention relates to a bacillus subtilis for preventing and controlling Chinese herbal medicine soil-borne diseases and a preparation method of a bactericide thereof, in particular to a bacillus subtilis and a preparation method of the bactericide thereof. The bacillus subtilis X1 for controlling the Chinese herbal medicine soil-borne diseases is Bacillus with a preservation number of CGMCC No.4037 and a preservation date of 26th, July 2010. The bactericide is prepared by the following steps: inoculating an activated strain to a NYD fluid nutrient medium for enrichment culture to obtain asterilized solution with 2-4 billion/millimetre in bacterial count concentration. The bacillus subtilis X1 of the invention is a bio-control bacterial strain which is provided with high control effect on the soil-borne diseases and the bactericide can enjoy a prevention rate of 60-80% on the Chinese herbal medicine soil-borne diseases including ginseng root rot, sclerotiniose, North China fritillary sclerotiniose, Schisandra chinensis root rot, seedling blight and the like. The bactericide bio-control preparation is safe and environment-friendly and does not damage drug resistance of nontarget organisms and inductive pathogenic bacteria.
Description
Technical field
The present invention relates to a kind of subtilis and fungicide preparation thereof.
Background technology
The modernization of Chinese medicine to the Eight Terms Guidelines of traditional medicine development is: " safety, effective, stable, controlled "; The standard of weighing Chinese medicinal materials quality such as genseng, flat shellfish, Schisandra chinensis also comprises the pollution of external contaminants such as chemical pesticide and heavy metal except that the effective constituent of Chinese medicine self.Agricultural residual and heavy metal contamination in China's genseng is the important factor that causes the genseng downgrade, and becomes the major obstacle that restriction China genseng moves towards the world.In the introducing culture process of genseng, can suffer the harm of various disease and pests, the main at present chemical pesticide control that relies on.Chinese medicinal materialss such as genseng, flat shellfish, Schisandra chinensis are the specialty goods that people are used for diseases prevention, cure the disease, keep healthy, and excessive applying pesticides not only makes the permission standard that pesticide residue surpass in the world or China stipulates in the Chinese medicinal materials, directly damages HUMAN HEALTH; And research shows that some medicinal plant applying pesticides is improper, and its active constituent content is reduced.Therefore, carrying out Chinese medicinal materialss such as genseng, flat shellfish, Schisandra chinensis and produce pollution-free Research on New, produce nuisanceless, fine Green Ginseng, is the important step that improves Chinese medicinal materials output and quality.
Medicinal plant receives harmful microbe infringement through regular meeting in growth and development process, cause the output of plant medicinal part to reduce, quality degenerates so that lose pharmaceutical use.Genseng, flat shellfish, Schisandra chinensis etc. all are northern China authentic medicinal herbses.In recent years, because authentic medicinal herbs is being cultivated when ground warp for many years, pathogenic bacteria causes disease serious through accumulation for many years, especially shows on the soil-borne disease.Like genseng damping-off, blight, root rot, sclerotium disease, equal serious day by day trend of being of shellfish sclerotium disease and soil-borne diseases such as shizandra berry root-rot, base rot disease.Through chemical bactericide seed and soil are handled, played mitigate the disease and the effect that stops pathogen infection, but also bring problems such as the Chinese medicinal materials farming of being used as medicine with root, rhizome, bulb etc. residually exceeds standard, edatope deterioration simultaneously.Therefore, the biological and ecological methods to prevent plant disease, pests, and erosion of highly effective and safe technology is introduced in the control to above-mentioned several kinds of authentic medicinal herbs soil-borne diseases, treated both principal and secondary aspect of disease playing, the multiple action of disease-resistant yield-increasing upgrading, for the high-efficiency high-quality production of authentic medicinal herbs is given security.
In the medicinal plant cultivation process, often suffer the harm of various disease and pests, influence the quality and the output of medicinal material, cause great financial loss.Therefore the effectively harm of control disease and pest is the gordian technique measure that guarantees the Chinese medicinal materials high yield and high quality.At present, therefore when the generation disease and pest, can only transplant the method for preventing and treating on the farm crop because the producer lacks relevant technical know-how and Pesticidal products safely and effectively; Cause indiscriminate use of pesticide, misuse, not only disease and pest can not get effective control, and the resistance of pathogenic bacteria, insect constantly increases; And pesticide residue exceed standard; Seriously restrict the production of high yield, high-quality, low residue Chinese medicinal materials, influenced the reputation of China's Chinese medicine, reduced the competitive power on the world market.21 century, the healthful Chinese crud drug advocated was meant pollution-free, safety, fine Chinese medicinal materials.According to the characteristics of medicinal plant self, carry out pollution-free new technology, novel methods such as biological control and have a extensive future.
Summary of the invention
The invention provides a kind of subtilis and fungicide preparation thereof of preventing and treating the herbal medicine soil-borne disease; Purpose is in order to solve present employing chemical pesticide control Chinese medicinal materials disease, and bring out pathogenic bacteria or sick worm resistance, cause the problem of pesticide residual contamination and injury non-target organism.
The subtilis of control herbal medicine soil-borne disease; It is the subtilis X1 of control herbal medicine soil-borne disease; For subtilis (Bacillus subtilis), belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; Deposit number is CGMCC No.4037, and preservation date is on July 26th, 2010; It is gram-positive microorganism, and is shaft-like, and length is 1.2 μ m～1.4 μ m, and wide is 0.6 μ m～0.8 μ m, give birth in the gemma, and ellipse, atrichia does not produce pigment; On beef-protein medium, form the oyster white bacterium colony, gauffer, the edge is irregular, and the bacterium colony umbo is carinate.
The subtilis X1 starch hydrolysis of control herbal medicine soil-borne disease is positive among the present invention; Fat hydrolysis is negative; Gelatin hydrolysis is positive; The litmus milk test is positive; Hydrolysis of urea is negative; Hydrogen sulfide production test is negative; Indole test is positive; Methyl red test is negative; The Fu Pu test is positive; Citrate test is positive; Peroxidase test is positive; Oxidase test is negative; Can utilize dextrose plus saccharose to produce not aerogenesis of acid; The hippurate test is positive; Nitrate reduction test is positive; Good health is long; Growth pH value is 6.0～8.0; Growth temperature is 20～40 ℃.
The subtilis of control herbal medicine soil-borne disease among the present invention; Bacterial strain number is X1; For subtilis (Bacillus subtilis), belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; Deposit number is CGMCC No.4037, and preservation date is on July 26th, 2010.
The method of the bacillus subtilis microbial agent of preparation control herbal medicine soil-borne disease is carried out according to the following steps: the bacterium liquid of one, getting the subtilis X1 of the control herbal medicine soil-borne disease that-70 ℃ of glycerine preserve; Streak inoculation is on the NYDA solid medium; Cultivate 18～20h in 30 ℃ of thermostat containers; Picking list colony inoculation is in the NYD of 5ml liquid nutrient medium, and 30 ℃, frequency are that 18～20h is cultivated in concussion under the 150r/min condition, obtain activation bacterium liquid; Two, get 0.1ml activation bacterium liquid and coat on the eggplant bottle of dress NYDA substratum, in 30 ℃ of thermostat containers, cultivate 18～20h, get activated spawn; Three, activated spawn is inoculated in the fermentor tank that the NYD liquid nutrient medium is housed; At leavening temperature is that 25 ± 0.5 ℃, stirring velocity are that 160r/min, fermentor tank internal gas pressure are 0.5 atmospheric condition bottom fermentation 24～48h; Get bacterium and count the bacterium liquid that concentration reaches 20～4,000,000,000/milliliter, promptly accomplish the preparation of the bacillus subtilis microbial agent of control herbal medicine soil-borne disease; Wherein in the step 3 when beginning fermentation air flow be 0.8VVM, behind the fermentation beginning 7h to the fermentation ends air flow be 1VVM.
The present invention prevents and treats the subtilis X1 of herbal medicine soil-borne disease, is the isolating biocontrol strain that genseng root rot, sclerotium disease, damping-off, blight, flat shellfish sclerotium disease and soil-borne diseases such as shizandra berry root-rot, base rot disease are had higher preventive effect from genseng root soil sample.
The present invention prevents and treats the microbial inoculum of the subtilis of herbal medicine soil-borne disease; Authentic medicinal herbs seed such as genseng, Ping Bei and shizandra berry and plantation soil are handled; Make beneficial microbe colony in seed-coat, Gen Biao and rhizosphere soil, grow surely, grow; Utilize competition and antagonistic action to suppress growth of pathogenic bacteria and breeding, control the generation of soil-borne disease and spread; Simultaneously through improving the active plant resistance that strengthens of root defensive ferment; The microbial inoculum biological prevention and control agent is to people, animal safety, and is free from environmental pollution, can not injure non-target organism, can not bring out the resistance of pathogenic bacteria; Improvement Chinese medicinal materials quality, the control of control herbal medicine soil-borne disease such as genseng root rot, damping-off, blight and sclerotium disease and flat shellfish sclerotium disease and diseases such as shizandra berry root-rot, base rot disease reaches 60%～80%, improves Chinese medicinal materials output; The preparation low production cost; Simple to operate, easy to implement, can produce in batches and be applicable; Significant to production of our province healthful Chinese crud drug and sustainable agriculture development, have certain development and application values.
Embodiment
Embodiment one: the subtilis of this embodiment control herbal medicine soil-borne disease; It is the subtilis X1 of control herbal medicine soil-borne disease; For subtilis (Bacillus subtilis), belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; Deposit number is CGMCC No.4037, and preservation date is on July 26th, 2010; It is gram-positive microorganism, and is shaft-like, and length is 1.2 μ m～1.4 μ m, and wide is 0.6 μ m～0.8 μ m, give birth in the gemma, and ellipse, atrichia does not produce pigment; On beef-protein medium, form the oyster white bacterium colony, gauffer, the edge is irregular, and the bacterium colony umbo is carinate.
The subtilis of control herbal medicine soil-borne disease is according to " the outstanding Bacteria Identification handbook preliminary judgement of uncle is a bacillus, is accredited as bacillus (Bacillus) through the SHERLOCK microflora then in this embodiment.
The genseng canopy soil sample separation of ferreous forestry bureau deer ring farm obtains the subtilis X1 of control herbal medicine soil-borne disease from the Heilongjiang Province in this embodiment, and its separation method is following:
1, adopt dilution-plate method to separate soil bacteria; Take by weighing the genseng root soil sample 10g that picks up from the ferreous forestry bureau in Heilongjiang Province deer ring farm genseng canopy different zones respectively; Put into the triangular flask that fills the 90mL sterilized water; Place the 10min that fully vibrates on the shaking table, the bacterium in the soil is well-dispersed in the diluent, process the soil suspension-s of 1: 10 concentration;
2, treat soil suspension-s post precipitation, with aseptic pipette, extract 1mL supernatant, immigration fills in the test tube of 9mL sterilized water, and fully concussion shakes up, and processes 1: 100 soil suspension-s; By that analogy, process concentration and be respectively 10
-3, 10
-4, 10
-5Doubly dilute suspension, select 10
-3, 10
-4, 10
-5Each 100 μ L of the diluent of three concentration coat in the NYDA solid medium, and each concentration is done 3, is inverted in then in 37 ℃ of constant incubators and cultivates 18h; Add up the colony count that grows on each flat board; Picking list bacterium colony, the purifying of on the NYDA solid medium, ruling repeatedly is till obtaining being sheerly bacterial strain; To be sheerly bacterial strain list colony inoculation in the NYD liquid nutrient medium, and cultivate 18～20h in 37 ℃ of incubators, and get bacterium liquid 700 μ l and join in the Eppendorf pipe, and add 50% glycerine, 300 μ l then, mixing is placed on-70 ℃ of refrigerator and cooled and freezes preservation.
Embodiment two: this embodiment and embodiment one are different is that the subtilis X1 starch hydrolysis of control herbal medicine soil-borne disease is positive; Fat hydrolysis is negative; Gelatin hydrolysis is positive; The litmus milk test is positive; Hydrolysis of urea is negative; Hydrogen sulfide production test is negative; Indole test is positive; Methyl red test is negative; The Fu Pu test is positive; Citrate test is positive; Peroxidase test is positive; Oxidase test is negative; Can utilize dextrose plus saccharose to produce not aerogenesis of acid; The hippurate test is positive; Nitrate reduction test is positive; Good health is long; Growth pH value is 6.0～8.0; Growth temperature is 20～40 ℃.Other is identical with embodiment one.
Embodiment three: the bacterial preparation process of the subtilis of this embodiment control herbal medicine soil-borne disease carries out according to the following steps: the bacterium liquid of one, getting the subtilis X1 of the control herbal medicine soil-borne disease that-70 ℃ of glycerine preserve; Streak inoculation is on the NYDA solid medium; Cultivate 18～20h in 30 ℃ of thermostat containers; Picking list colony inoculation is in the NYD of 5ml liquid nutrient medium, and 30 ℃, frequency are that 18～20h is cultivated in concussion under the 150r/min condition, obtain activation bacterium liquid; Two, get 0.1ml activation bacterium liquid and coat on the eggplant bottle of dress NYDA substratum, in 30 ℃ of thermostat containers, cultivate 18～20h, get activated spawn; Three, activated spawn is inoculated in the fermentor tank that the NYD liquid nutrient medium is housed; At leavening temperature is that 25 ± 0.5 ℃, stirring velocity are that 160r/min, fermentor tank internal gas pressure are 0.5 atmospheric condition bottom fermentation 24～48h; Get bacterium and count the bacterium liquid that concentration reaches 20～4,000,000,000/milliliter, promptly accomplish the preparation of the bacillus subtilis microbial agent of control herbal medicine soil-borne disease; Wherein in the step 3 when beginning fermentation air flow be 0.8VVM, behind the fermentation beginning 7h to the fermentation ends air flow be 1VVM.
The bacillus subtilis microbial agent thin up that this embodiment prepares gained control herbal medicine soil-borne disease is used for genseng seed soaking, filling root or sprinkling for 50 times; Its control that prevents and treats genseng soil-borne disease genseng root rot and sclerotium disease is tested as follows:
1, supply examination cause of disease microbial inoculum: Fusarium oxysporum and sclerotium disease pathogenic bacteria, on the wheat substratum, cultivate, treat that mycelia is covered with after, grind for use;
2, chemical agent supplies the reagent agent: 50% carbendazol wettable powder, chemical industry ltd of Nanjing Novartis;
3, method
3.1 the ginseng seed screening, screening is full ginseng seed evenly;
3.2 inoculation pathogenic bacteria preparation
The ground pathogenic bacteria is used after the dilution proportion by 1: 5 with fine sand, remove and put ginseng seed after the every basin of blank group evenly sprinkles one deck germ, spread one deck soil again and cover.
3.3 experimental design: do 7 processing altogether, program request is to whole good furrow face after the seed treatment, and the cave distance is 10cm * 10cm, 2 in every cave, and earthing waters sufficient water, performs mark, and each handles 3m
2, repeat 3 times;
Handle 1: the blank group, directly ginseng seed is not inoculated pathogenic bacteria;
Handle 2: the pathogenic bacteria contrast, directly sow ginseng seed, the inoculation Fusarium oxysporum;
Handle 3: chemical agent contrast, the ginseng seed of sowing usefulness, inoculation Fusarium oxysporum;
Handle 4: the ginseng seed that the bacillus subtilis microbial agent of sowing control herbal medicine soil-borne disease soaked, inoculation Fusarium oxysporum;
Handle 5: the pathogenic bacteria contrast, directly sow ginseng seed, the inoculation sclerotium bacteria;
Handle 6: the ginseng seed that the bacillus subtilis microbial agent of sowing control herbal medicine soil-borne disease soaked, inoculation sclerotium bacteria;
Handle 7: chemical agent contrast, the ginseng seed of sowing usefulness, inoculation sclerotium bacteria;
4, the bacillus subtilis microbial agent method of use of control herbal medicine soil-borne disease
1) seed soaking: respectively soaked 30 minutes the back sowing of drying in the shade with 50 times of diluents of genus bacillus X1 microbial inoculum;
2) irritate root: seedling stage and transplanting phase, respectively irritate root once with 50 times of diluents of genus bacillus X1 microbial inoculum, 100 milliliters of every strain consumptions;
3) spray: 50 times of diluents of genus bacillus X1 microbial inoculum respectively sprayed once in seedling stage and early flowering season;
5, pilot survey:
Observe about 20 days and emerge, the record seedling rate; Root rot of investigation in per 5 days, sclerotium disease incidence after genseng is emerged; Calculate sickness rate; Experimental result is as shown in table 1, and is visible, and the bacillus subtilis microbial agent of gained control herbal medicine soil-borne disease reaches 76.2% and 87.3% to the control of genseng root rot and sclerotium disease in this enforcement.
Table 1
Adopt aforesaid method that genseng damping-off, blight, flat shellfish sclerotium disease and shizandra berry root-rot, base rot disease are experimentized, the result prevents and treats the herbal medicine soil-borne disease for gained in this enforcements bacillus subtilis microbial agent reaches 60%～80% to genseng damping-off, blight, the control of putting down shellfish sclerotium disease and shizandra berry root-rot, base rot disease.
Embodiment four: this embodiment and embodiment three are different is that the NYDA solid medium is added zero(ppm) water and is settled to 1000mL by the agar powder of the glucose of the Carnis Bovis seu Bubali cream of the yeast extract paste of 5g, 8g, 10g and 15g and forms in the step 1.Other step and parameter are identical with embodiment three.
Embodiment five: this embodiment and embodiment four are different is that the NYD liquid nutrient medium is added zero(ppm) water and is settled to 1000mL by the glucose of the Carnis Bovis seu Bubali cream of the yeast extract paste of 5g, 8g and 10g and forms in the step 1.Other step and parameter are identical with embodiment four.
Embodiment six: the bacterial preparation process of the subtilis of this embodiment control herbal medicine soil-borne disease carries out according to the following steps: the bacterium liquid of one, getting the subtilis X1 of the control herbal medicine soil-borne disease that-70 ℃ of glycerine preserve; Streak inoculation is on the NYDA solid medium; Cultivate 18～20h in 30 ℃ of thermostat containers; Picking list colony inoculation is in the NYD of 5ml liquid nutrient medium, and 30 ℃, frequency are that 18～20h is cultivated in concussion under the 150r/min condition, obtain activation bacterium liquid; Two, get 0.1ml activation bacterium liquid and coat on the eggplant bottle of dress NYDA substratum, in 30 ℃ of thermostat containers, cultivate 18～20h, get activated spawn; Three, activated spawn is inoculated in the fermentor tank that 10～15L NYD liquid nutrient medium is housed; At leavening temperature is that 25 ± 0.5 ℃, stirring velocity are that 160r/min, fermentor tank internal gas pressure are 0.5 atmospheric condition bottom fermentation 24～48h; Get bacterium and count the bacterium liquid that concentration reaches 20～4,000,000,000/milliliter, promptly accomplish the preparation of the bacillus subtilis microbial agent of control herbal medicine soil-borne disease; Wherein the inoculum size of activated spawn is 1% of the interior NYD liquid nutrient medium volume of fermentor tank in the step 3; Air flow is 0.8VVM when beginning fermentation in the step 3, behind the fermentation beginning 7h to the fermentation ends air flow be 1VVM.
Claims (5)
1. subtilis that prevents and treats the herbal medicine soil-borne disease; It is characterized in that it is the subtilis X1 of control herbal medicine soil-borne disease; For subtilis (Bacillus subtilis), belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; Deposit number is CGMCC No.4037, and preservation date is on July 26th, 2010; It is gram-positive microorganism, and is shaft-like, and length is 1.2 μ m～1.4 μ m, and wide is 0.6 μ m～0.8 μ m, give birth in the gemma, and ellipse, atrichia does not produce pigment; On beef-protein medium, form the oyster white bacterium colony, gauffer, the edge is irregular, and the bacterium colony umbo is carinate.
2. the subtilis of control herbal medicine soil-borne disease according to claim 1 is characterized in that the subtilis X1 starch hydrolysis that prevents and treats the herbal medicine soil-borne disease is positive, fat hydrolysis is negative, gelatin hydrolysis is positive, the litmus milk test is positive, hydrolysis of urea is negative, hydrogen sulfide production test is negative, indole test is positive, methyl red test is negative, the Fu Pu test is positive, citrate test is positive, peroxidase test is positive, oxidase test is negative, aerogenesis, hippurate test are not positive, nitrate reduction test is positive, aerobic growth, growth pH value are 6.0～8.0, growth temperature is 20～40 ℃ to utilize dextrose plus saccharose to produce acid.
3. the bacterial preparation process that comprises the subtilis of the herbal medicine of control described in the claim 1 soil-borne disease; The bacterial preparation process that it is characterized in that preventing and treating the subtilis of herbal medicine soil-borne disease carries out according to the following steps: the bacterium liquid of one, getting the subtilis X1 of the control herbal medicine soil-borne disease that-70 ℃ of glycerine preserve; Streak inoculation is on the NYDA solid medium; Cultivate 18～20h in 30 ℃ of thermostat containers; Picking list colony inoculation is in the NYD of 5ml liquid nutrient medium, and 30 ℃, frequency are that 18～20h is cultivated in concussion under the 150r/min condition, obtain activation bacterium liquid; Two, get 0.1ml activation bacterium liquid and coat on the eggplant bottle of dress NYDA substratum, in 30 ℃ of thermostat containers, cultivate 18～20h, get activated spawn; Three, activated spawn is inoculated in the fermentor tank that the NYD liquid nutrient medium is housed; At leavening temperature is that 25 ± 0.5 ℃, stirring velocity are that 160r/min, fermentor tank internal gas pressure are 0.5 atmospheric condition bottom fermentation 24～48h; Get bacterium and count the bacterium liquid that concentration reaches 20～4,000,000,000/milliliter, promptly accomplish the preparation of the bacillus subtilis microbial agent of control herbal medicine soil-borne disease; Wherein in the step 3 when beginning fermentation air flow be 0.8VVM, behind the fermentation beginning 7h to the fermentation ends air flow be 1VVM.
4. according to the bacterial preparation process of the subtilis of the said control herbal medicine of claim 3 soil-borne disease, it is characterized in that NYDA solid medium in the step 1 is added zero(ppm) water and is settled to 1000mL by the agar powder of the glucose of the Carnis Bovis seu Bubali cream of the yeast extract paste of 5g, 8g, 10g and 15g to form.
5. according to the bacterial preparation process of the subtilis of the said control herbal medicine of claim 3 soil-borne disease, it is characterized in that NYD liquid nutrient medium in the step 1 is added zero(ppm) water and is settled to 1000mL by the glucose of the Carnis Bovis seu Bubali cream of the yeast extract paste of 5g, 8g and 10g to form.
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| CN102391965B (en) * | 2011-11-17 | 2013-01-30 | 吉林省集韩生物肥有限公司 | Preparation method of biological agent for preventing and treating ginseng diseases |
| CN105087449A (en) * | 2015-09-18 | 2015-11-25 | 黑龙江省科学院微生物研究所 | Pilot-scale fermentation process of biocontrol strain X1 |
| CN105039230A (en) * | 2015-09-18 | 2015-11-11 | 黑龙江省科学院微生物研究所 | Biocontrol strain X1 fermentation medium and small-scale fermentation technology |
| CN108990639A (en) * | 2018-08-22 | 2018-12-14 | 四川省农业科学院经济作物育种栽培研究所 | A kind of preventing control method of Rhizoma Chuanxiong soil-borne disease |
| CN111362751B (en) * | 2020-04-24 | 2022-07-12 | 山东省现代中药研究院有限公司 | Special compound microbial disease-resistant growth-promoting bacterial fertilizer for ginseng and preparation method thereof |
| CN113403218B (en) * | 2021-05-13 | 2022-07-01 | 南京本源生态农业科技有限公司 | Biocontrol strain and application thereof in preventing and treating soft rot of thunberg fritillary bulb and promoting growth of thunberg fritillary bulb |
| CN113603525A (en) * | 2021-07-21 | 2021-11-05 | 山东京青农业科技有限公司 | Special organic bacterial fertilizer for araliaceae panax plants and preparation method thereof |
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