CN106396862A - Disease controlling microbial fertilizer special for tuberous crops and preparation method thereof - Google Patents

Disease controlling microbial fertilizer special for tuberous crops and preparation method thereof Download PDF

Info

Publication number
CN106396862A
CN106396862A CN201610746263.6A CN201610746263A CN106396862A CN 106396862 A CN106396862 A CN 106396862A CN 201610746263 A CN201610746263 A CN 201610746263A CN 106396862 A CN106396862 A CN 106396862A
Authority
CN
China
Prior art keywords
mycopowder
streptomycete
medium
series bacillus
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610746263.6A
Other languages
Chinese (zh)
Inventor
蒋常德
张国睿
胡艳晖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Foshan Yanhui Biotechnology Co Ltd
Original Assignee
Foshan Yanhui Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Foshan Yanhui Biotechnology Co Ltd filed Critical Foshan Yanhui Biotechnology Co Ltd
Priority to CN201610746263.6A priority Critical patent/CN106396862A/en
Publication of CN106396862A publication Critical patent/CN106396862A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00Other organic fertilisers
    • C05F11/02Other organic fertilisers from peat, brown coal, and similar vegetable deposits
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05GMIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05GMIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/80Soil conditioners
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor

Abstract

The invention discloses a disease controlling microbial fertilizer special for tuberous crops and a preparation method thereof, in particular to a microbial fertilizer special for controlling sweet potato black rot and a preparation method thereof. The microbial fertilizer is prepared by mixing of the following raw materials: antimycoin streptomyces powder, wet streptomyces powder, paenibacillus pabuli powder, paenibacillus jamilae powder, and potassium fulvate raw powder. The strains involved in the invention can utilize the antibiosis, competition, hyperparasitism and bacteriolysis effects between or within microbial species, or utilize secondary metabolites to induce disease resistance of sweet potatoes, and can produce a variety of antibiotics like endomycin compound, non-polyene antifungal antibiotics, dihydrostreptomycin, humidin and cobalamin to inhibit sweet potato black rot pathogenic bacteria and strengthen the disease prevention effect. The microbial fertilizer provided by the invention is safe to human and livestock, and is environment-friendly. Application of the microbial fertilizer provided by the invention to control of sweet potato black rot is not easy to produce drug resistance. The microbial fertilizer has the advantages of simple preparation method, low cost and easy use.

Description

A kind of potato class microbial bacterial manure special of controlling disease and preparation method thereof
Technical field
The invention belongs to microbial manure technical field is and in particular to a kind of potato class microbial bacterial manure special of controlling disease And preparation method thereof.
Background technology
Rhizoma Dioscoreae esculentae is a kind of stable high yield, nutritious, broad-spectrum crops, is important cereal crops, is again Good forage crop, is also insutrial crop.In recent years, both at home and abroad the nutrition health-care functions of Rhizoma Dioscoreae esculentae have been carried out deep Research, the height that it has increasingly caused people as a kind of novel nourishing health functional food is focused on.Rhizoma Dioscoreae esculentae is producing, was storing Pollution-free in journey, nuisanceless, it is " 21 century optimal food " that relevant experts evaluate it, and sweet potato food is tied in human foods In structure, shared proportion will be more and more big, and its impact will be further extensive, and the industrialization prospect of Rhizoma Dioscoreae esculentae is very wide.China Rhizoma Dioscoreae esculentae Pest and disease damage harm is serious it has been found that reports has kind more than 30, and wherein sweet potato black rot is that generation is tighter than wide, comparison of causing harm The fungal disease of weight.Melasma can occur in Rhizoma Dioscoreae esculentae seedling stage, field growth period and storage period, causes rotten kind, dead seedling, growth Hypoevolutism etc. endangers.The preventing and treating of Rhizoma Dioscoreae esculentae disease is mainly carried out including from disease-resistant variety, physical control, cultural control, biology The comprehensive preventive health measuress such as preventing and treating and chemical prevention.It is a kind of most economical effective and safe ready preventing and treating side from disease-resistant variety Method, but due to the suitable difficulty of Rhizoma Dioscoreae esculentae breeding for disease resistance, selects that a yield height, quality be excellent, disease resistance is good (and resists multiple diseases Evil) sweet potato variety be not easy very much;Time length that physical control and cultural control measure need, slowly effect, time-consuming;Change The pollution of soil degradation, pesticide residues, water source and environment that medicine of learning to farm is led to, biological chain interruption, disruption of ecological balance, disease produce A series of problems, such as resistance, increasingly shows especially.The method of Biological control is more and more paid attention to, and will prevent and treat but without correlation Rhizoma Dioscoreae esculentae disease carries out disclosure to the related invention that fertilizer combines.
Content of the invention
Problem to be solved by this invention is to develop a kind of potato class microbial bacterial manure special of controlling disease, prepares this micro- The raw material of bio-bacterial manure is by weight:Antimycoin streptomycete mycopowder 20-40 part, wet streptomycete mycopowder 10-30 part, feedstuff class Bacillus cereuss mycopowder 10-30 part, Jia meter Li series bacillus mycopowder 20-40 part, potassium fulvate former powder 20-40 part;
In described microbial-bacterial fertilizer, antimycoin streptomycete, wet streptomycete, feedstuff series bacillus, Jia meter Li class spore bar Bacterium, four kinds of bacterium are the energy symbiotic co-existences that screening obtains from more than 400 plants of microbial strains, can produce multiple antibiotics such as endomycin Complex, non-polyenoid antifungal antibiotic, dihydrostreptomycin, humidin and cobalamins class are suppressing sweet potato black rot pathogen There is especially powerful effect, thus reaching the effect preventing and treating sweet potato black rot;On the other hand, the strain of the present invention can be comprehensive Antibiosis between using microbial species or in kind, competition, superparasitism, bacteriolysiss, or Rhizoma Dioscoreae esculentae is induced by secondary metabolite Produce disease resistance, strengthen its protection effect;Again, 4 plants of bacterial strains of this used in the present invention are all directly can to divide from soil From obtaining, there is fixed nitrogen, phosphorus decomposing, potassium decomposing, and there are root mark growth-promoting functions, can be numerous in crop surface, plant inside or soil While reproductive growth, and orient plant rhizosphere and secrete some secondary metabolites, it is possible to increase plant is to the absorption of nutrient, stimulation Plant strain growth plays the effect of fertilizer;Potassium fulvate in the present invention also can provide potash fertilizer and organic matter by crop simultaneously.
Another purpose, there is provided a kind of preparation method of the potato class microbial bacterial manure special of controlling disease, it is specifically made Preparation Method, comprises the following steps:
Step one, the preparation of antimycoin streptomycete mycopowder
Take out antimycoin streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7 My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as antimycoin streptomycete seed liquor;
Fermentation:The antimycoin streptomycete seed liquor of above-mentioned preparation is seeded to the antimycoin of sterilizing with the inoculum concentration of 1%-3% In strepto- bacteria solid fermentation culture medium, after strain is mixed homogeneously with antimycoin streptomycete solid medium, the anti-of kind will be connected Moldin strepto- bacteria solid fermentation culture medium is spread out on fermentation tank, and height of materials is 3-6cm, 28-32 DEG C of temperature control, first day air Humidity remains 60%-75%, and air humidity remains 40%-50% later, ferments 3-4 days, detects that its spore content is not less than 20 Hundred million CFU/g, you can low-temperature air-drying to moisture is less than 8%, pulverized 40 mesh sieves, that is, obtains antimycoin streptomycete mycopowder;
Wherein, described Gause I solid medium:Potassium nitrate:1 gram, soluble starch:20 grams, dipotassium hydrogen phosphate:0.5, Magnesium sulfate:0.5, sodium chloride:0.5, ferrous sulfate:0.01 gram, agar:20 grams, distilled water supplies 1000ml, PH7.2~7.4;
Wherein, described antimycoin strepto- bacteria solid fermentation culture medium:Maize alcohol stillage 80%, rapeseed cake 2%, maize cob meal 1%, stone Powder 10%, wheat bran 7%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%- 60%.The condition of each medium sterilization is:0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Step 2, the preparation of wet streptomycete mycopowder
Take out wet streptomyces species preservation pipe, draw flat board with PDA solid medium and recovered, cultivate 7 days for 30 DEG C.Under flat board Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of PDA solid mediums, cultivates 6-8 for 30 DEG C in incubator My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense Spend for 0.1 hundred million cfu/ml, as wet streptomycete seed liquor;
Fermentation:The wet streptomycete seed liquor of above-mentioned preparation is seeded to the wet strepto- bacteria solid fermentation training of sterilizing with 5% inoculum concentration On foster base, after strain is mixed homogeneously with wet streptomycete solid medium, the wet strepto- bacteria solid fermentation culture medium stand planted will be connected On fermentation tank, height of materials is 5-8cm, 28-32 DEG C of temperature control, and a few days ago air humidity remains 60%-75%, and air is wet later Degree remains 40%-50%, ferments 4-6 days, detects that its spore content is not less than 1,000,000,000 CFU/g, you can low-temperature air-drying moisture is less than 8%, pulverize 40 mesh sieves, that is, obtain wet streptomycete mycopowder;
Wherein, described PDA culture medium:Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000mL, agar 20g;
Wherein, described wet strepto- bacteria solid fermentation culture medium:Powdered rice hulls 80%, cottonseed meal 2%, Semen Maydis powder 1%, stone powder 2%, wheat bran 15%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%.Each culture Base sterilizing condition be:0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Step 3, the preparation of Jia meter Li series bacillus mycopowder
Take out Jia meter Li series bacillus preservation pipe, draw flat board respectively with nitrogen-free solid medium and recovered, 30 DEG C of cultures 72 Hour.Under flat board, picking single bacterium colony is seeded to equipped with the Fructus Solani melongenae bottle of nitrogen-free solid medium, trains in 30 DEG C of incubators Support 72 hours, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, be seeded to equipped with 300L Jia meter Li class spore bar In the 500L fermentation tank of bacterium seed culture medium, open stirring 120r/min, ventilation is 100L/min, after 8-24 hour within first 8 hours Ventilation is 200L/min, and after 24 hours, ventilation is 300L/min, 30 DEG C of culture 32-48 hours, treats total bacteria count content not Less than 2,500,000,000 cfu/ml, you can as Jia meter Li series bacillus seed liquor;
Fermentation:The Jia meter Li series bacillus seed liquor of above-mentioned preparation is seeded to Jia meter Li class spore with the ratio of 10%-20% On bacillus solid medium, mix, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature to be 30-40 DEG C, sends out Ferment 36-48 hour, treats that spore content is not less than 6,000,000,000 cfu/g, you can dry, treats that moisture is less than 10%, pulverized 100 mesh Sieve, that is, obtain Jia meter Li series bacillus mycopowder;
Wherein, described nitrogen-free solid medium:Sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid Calcium 0.1g, Calcium Carbonate 5g, agar 18g, distilled water 1000mL, pH7.2;
Wherein, described Jia meter Li series bacillus seed culture medium:Molasses 20g/L, tapioca starch 10 g/L, rapeseed cake powder 40 g/L, Magnesium sulfate 0.4 g/L, manganese sulfate 0.6g/L, potassium dihydrogen phosphate 0.6 g/L, Calcium Carbonate 10 g/L, pH7.0.Each medium sterilization Condition be:0.10-0.15MPa, 121 DEG C sterilize 30 minutes;
Wherein, Jia meter Li series bacillus solid medium:Maize alcohol stillage 57%, wheat bran 35%, cottonseed meal 5%, stone powder 1%, thick Semen Maydiss Skin 2%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.The condition of each medium sterilization is: 0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Step 4, the preparation of feedstuff series bacillus mycopowder
Take out feedstuff series bacillus preservation pipe, draw flat board respectively with nutrient solid medium and recovered, 30 DEG C of cultures 48 hours.Under flat board, picking single bacterium colony is seeded to equipped with the Fructus Solani melongenae bottle of nutrient solid medium, cultivates at 30 DEG C Cultivate 48 hours in case, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, be seeded to equipped with 300L liquid seeds In the 500L fermentation tank of culture medium, open stirring 120r/min, ventilation is 200L/min within first 10 hours, and after 10 hours, ventilation is 320L/min, 30 DEG C of culture 16-24 hours, treat that total bacterial content is not less than 3,000,000,000 cfu/ml, you can as feedstuff series bacillus Seed liquor;
Fermentation:The feedstuff series bacillus seed liquor of above-mentioned preparation is added to feedstuff with the inoculum concentration of the 10%-20% of total inoculum concentration In series bacillus solid fermentation culture medium, mix, in tray top fermentation, windrow thickness is 5-10cm, control fermentation temperature is 30-40 DEG C, 36-48 hour of fermenting, treat that spore content is not less than 8,000,000,000 cfu/g, you can dry, treat that moisture is less than 10%, powder Broken mistake 100 mesh sieve, that is, obtain feedstuff series bacillus mycopowder;
Wherein, described nutrient broth solid medium:Peptone 10.0 g/L, Carnis Bovis seu Bubali cream 3.0 g/L, sodium chloride 5.0 g/L, Agar 20 g/L, pH 7.2 ± 0.2;
Wherein, described liquid seed culture medium:Semen Maydis powder 35 g/L, bean cake powder 20 g/L, fish flour 5 g/L, magnesium sulfate 0.6g/ L, manganese sulfate 0.5 g/L, potassium dihydrogen phosphate 0.3 g/L, Calcium Carbonate 5.0 g/L, pH7.0;
Wherein, feedstuff series bacillus solid fermentation culture medium:Wheat bran 60%, DDGS(Maize alcohol stillage)32%, cottonseed meal 5%, stone powder 3%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.The condition of each medium sterilization For:0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Step 5, by the various mycopowder made above by antimycoin streptomycete mycopowder 20-40 part, wet streptomycete mycopowder 10-30 part, feedstuff series bacillus mycopowder 10-30 part, Jia meter Li series bacillus mycopowder 20-40 part, potassium fulvate former powder 20- 40 parts;The potato class microbial bacterial manure special of mix homogeneously, as controlling disease.
Wherein, the application process of the potato class microbial bacterial manure special of described controlling disease is bottom application microbial-bacterial fertilizer 5- 10kg/ mu.
The present invention has the advantage that and beneficial effect:, the antimycoin streptomycete that comprises in the present invention, wet streptomycete, Feedstuff series bacillus, four kinds of bacterium of Jia meter Li series bacillus are the energy symbiosis that screening obtains from more than 400 plants of microbial strains Coexist, can be in the root of plant, stem, in leaf and soil can growth and breeding, multiple antibiotics can be produced and can produce multiple antibacterials The complex of element such as endomycin, non-polyenoid antifungal antibiotic, dihydrostreptomycin, humidin and cobalamins class are suppressing Rhizoma Dioscoreae esculentae black Pinta pathogen has especially powerful effect, thus reaching the effect preventing and treating sweet potato black rot, its drug effect to sweet potato black rot Height, average preventive effect is more than 87.62% and with strong points;, the present invention strain can comprehensively utilize microbial species between or Antibiosis in kind, competition, superparasitism, bacteriolysiss, or induce Rhizoma Dioscoreae esculentae to produce disease resistance by secondary metabolite, strengthen it Protection effect;, this four plants of bacterial strains used in the present invention be all can be directly isolated to obtain from soil, there is fixed nitrogen, solution Phosphorus, potassium decomposing, all there are root mark growth-promoting functions, in crop surface, plant inside or soil while flourish, and can orient Plant rhizosphere secretes some secondary metabolites, it is possible to increase plant plays fertilizer to the absorption of nutrient, stimulation plant strain growth Effect;(4), composite microbic bacterial fertilizer of the present invention, to people, animal safety, belongs to environmentally friendly, using the inventive method preventing and treating kind Solanum cinerea is not likely to produce Drug resistance, and preparation method of the present invention is simple, strain is many, and extensively, fertilizer efficiency is notable for the antagonistic substance of generation, Low cost, use are simple.
Specific embodiment
Embodiment 1
A kind of potato class microbial bacterial manure special of controlling disease is it is characterised in that prepare the raw material of this microbial-bacterial fertilizer by weight Part it is:30 parts of antimycoin streptomycete mycopowder, 20 parts of wet streptomycete mycopowder, 20 parts of feedstuff series bacillus mycopowder, Jia meter Li class 30 parts of bacillus cereuss mycopowder, 30 parts of the former powder of potassium fulvate;Its concrete preparation method, comprises the following steps:
Step one, the preparation of antimycoin streptomycete mycopowder
Take out antimycoin streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7 My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as antimycoin streptomycete seed liquor;
Fermentation:The antimycoin streptomycete seed liquor of above-mentioned preparation is seeded to the antimycoin of sterilizing with the inoculum concentration of 1%-3% In strepto- bacteria solid fermentation culture medium, after strain is mixed homogeneously with antimycoin streptomycete solid medium, the anti-of kind will be connected Moldin strepto- bacteria solid fermentation culture medium is spread out on fermentation tank, and height of materials is 3-6cm, 28-32 DEG C of temperature control, first day air Humidity remains 60%-75%, and air humidity remains 40%-50% later, ferments 3 days, detects that its spore content is 2,500,000,000 CFU/ G, low-temperature air-drying to moisture is less than 8%, pulverized 40 mesh sieves, that is, obtains antimycoin streptomycete mycopowder;
Wherein, described Gause I solid medium:Potassium nitrate:1 gram, soluble starch:20 grams, dipotassium hydrogen phosphate:0.5, Magnesium sulfate:0.5, sodium chloride:0.5, ferrous sulfate:0.01 gram, agar:20 grams, distilled water supplies 1000ml, PH7.2~7.4;
Wherein, described antimycoin strepto- bacteria solid fermentation culture medium:Maize alcohol stillage 80%, rapeseed cake 2%, maize cob meal 1%, stone Powder 10%, wheat bran 7%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%- 60%.The condition of each medium sterilization is:0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Step 2, the preparation of wet streptomycete mycopowder
Take out wet streptomyces species preservation pipe, draw flat board with PDA solid medium and recovered, cultivate 7 days for 30 DEG C.Under flat board Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of PDA solid mediums, cultivates 6-8 for 30 DEG C in incubator My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense Spend for 0.1 hundred million cfu/ml, as wet streptomycete seed liquor;
Fermentation:The wet streptomycete seed liquor of above-mentioned preparation is seeded to the wet strepto- bacteria solid fermentation training of sterilizing with 5% inoculum concentration On foster base, after strain is mixed homogeneously with wet streptomycete solid medium, the wet strepto- bacteria solid fermentation culture medium stand planted will be connected On fermentation tank, height of materials is 5-8cm, 28-32 DEG C of temperature control, and a few days ago air humidity remains 60%-75%, and air is wet later Degree remains 40%-50%, ferments 5 days, detects that its spore content is 1,200,000,000 CFU/g, you can low-temperature air-drying moisture is less than 8%, pulverizes Cross 40 mesh sieves, that is, obtain wet streptomycete mycopowder;
Wherein, described PDA culture medium:Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000mL, agar 20g;
Wherein, described wet strepto- bacteria solid fermentation culture medium:Powdered rice hulls 80%, cottonseed meal 2%, Semen Maydis powder 1%, stone powder 2%, wheat bran 15%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%.Each culture Base sterilizing condition be:0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Step 3, the preparation of Jia meter Li series bacillus mycopowder
Take out Jia meter Li series bacillus preservation pipe, draw flat board respectively with nitrogen-free solid medium and recovered, 30 DEG C of cultures 72 Hour.Under flat board, picking single bacterium colony is seeded to equipped with the Fructus Solani melongenae bottle of nitrogen-free solid medium, trains in 30 DEG C of incubators Support 72 hours, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, be seeded to equipped with 300L Jia meter Li class spore bar In the 500L fermentation tank of bacterium seed culture medium, open stirring 120r/min, ventilation is 100L/min, after 8-24 hour within first 8 hours Ventilation is 200L/min, and after 24 hours, ventilation is 300L/min, 30 DEG C of culture 32-48 hours, treats total bacteria count content not Less than 2,500,000,000 cfu/ml, you can as Jia meter Li series bacillus seed liquor;
Fermentation:The Jia meter Li series bacillus seed liquor of above-mentioned preparation is seeded to Jia meter Li series bacillus with 15% ratio On solid medium, mix, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature to be 30-40 DEG C, fermentation 40 Hour, detection spore content is 6,500,000,000 cfu/g, dries, treats that moisture is less than 10%, pulverized 100 mesh sieves, that is, obtains with rice In series bacillus mycopowder;
Wherein, described nitrogen-free solid medium:Sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid Calcium 0.1g, Calcium Carbonate 5g, agar 18g, distilled water 1000mL, pH7.2;
Wherein, described Jia meter Li series bacillus seed culture medium:Molasses 20g/L, tapioca starch 10 g/L, rapeseed cake powder 40 g/L, Magnesium sulfate 0.4 g/L, manganese sulfate 0.6g/L, potassium dihydrogen phosphate 0.6 g/L, Calcium Carbonate 10 g/L, pH7.0.Each medium sterilization Condition be:0.10-0.15MPa, 121 DEG C sterilize 30 minutes;
Jia meter Li series bacillus solid medium:Maize alcohol stillage 57%, wheat bran 35%, cottonseed meal 5%, stone powder 1%, thick skin of Semen Maydis 2%, Dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.The condition of each medium sterilization is:0.10- 0.15MPa, 121 DEG C sterilize 30 minutes.
Step 4, the preparation of feedstuff series bacillus mycopowder
Take out feedstuff series bacillus preservation pipe, draw flat board respectively with nutrient solid medium and recovered, 30 DEG C of cultures 48 hours.Under flat board, picking single bacterium colony is seeded to equipped with nutrient solid medium, cultivates 48 in 30 DEG C of incubators Hour, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, it is seeded to equipped with 300L liquid seed culture medium In 500L fermentation tank, open stirring 120r/min, ventilation is 200L/min within first 10 hours, after 10 hours, ventilation is 320L/min , 30 DEG C of culture 16-24 hours, treat that total bacterial content is not less than 3,000,000,000 cfu/ml, you can as feedstuff series bacillus seed liquor;
Fermentation:The feedstuff series bacillus seed liquor of above-mentioned preparation is added to feedstuff with the inoculum concentration of the 10%-20% of total inoculum concentration In series bacillus solid fermentation culture medium, mix, in tray top fermentation, windrow thickness is 5-10cm, control fermentation temperature is 30-40 DEG C, ferment 42 hours, detect that its spore content is 8,400,000,000 cfu/g, you can dry, treat that moisture is less than 10%, pulverize Cross 100 mesh sieves, that is, obtain feedstuff series bacillus mycopowder;
Wherein, described nutrient broth solid medium:Peptone 10.0 g/L, Carnis Bovis seu Bubali cream 3.0 g/L, sodium chloride 5.0 g/L, Agar 20 g/L, pH 7.2 ± 0.2;
Wherein, described liquid seed culture medium:Semen Maydis powder 35 g/L, bean cake powder 20 g/L, fish flour 5 g/L, magnesium sulfate 0.6g/ L, manganese sulfate 0.5 g/L, potassium dihydrogen phosphate 0.3 g/L, Calcium Carbonate 5.0 g/L, pH7.0;
Wherein, feedstuff series bacillus solid fermentation culture medium:Wheat bran 60%, DDGS(Maize alcohol stillage)32%, cottonseed meal 5%, stone powder 3%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.The condition of each medium sterilization For:0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Step 5, by the various mycopowder made above by 30 parts of antimycoin streptomycete mycopowder, 20 parts of wet streptomycete mycopowder, 20 parts of feedstuff series bacillus mycopowder, 30 parts of Jia meter Li series bacillus mycopowder, 30 parts of the former powder of potassium fulvate;Mix homogeneously, that is, Potato class microbial bacterial manure special for controlling disease.
Embodiment 2 microbial-bacterial fertilizer is in the application effect of the big Tanaka of Rhizoma Dioscoreae esculentae
1st, test and Selection is carried out in Sweet Potato For High. yield Production area;
2nd, test address choice is on Zhanjiang, Guangdong Province Xuwen County farm;There is the soil of obvious sweet potato black rot in the previous year Test and compare;
3rd, medicament usage and consumption:
Test group:The microbial-bacterial fertilizer 5kg/ mu that bottom application embodiment 1 produces, compound fertilizer(15-15-15), 10 kg/ mus;
Chemical fertilizer group:Compound fertilizer(15-15-15), 40 kg/ mus;
Fertilizer group:Farm manure 600 kg/ mu;Other control measures are identical;
4th, result of the test:As it can be seen from table 1 the microbial-bacterial fertilizer of the present invention applies chemically composited fertile and fertilizer with respect to complete Group has obvious volume increase advantage in field experiment, and the average yield per mu using the microbial-bacterial fertilizer of the present invention can reach 2432kg/ mu, melasma refers to only 3.12, and entirely also has 2088kg using the yield of chemical fertilizer group, and melasma refers to up to 25.21; And the yield of manure use only has 1876kg, melasma also has an obvious reduction, only 12.45, the microbial bacteria of the present invention Fertilizer can increase production 29.6% with respect to fertilizer group on land for growing field crops, and high to the preventive effect of melasma with chemical fertilizer group with respect to complete Reach 87.62%, there is significant effect.
Table 1 microbial-bacterial fertilizer is in the application effect of the big Tanaka of Rhizoma Dioscoreae esculentae
Average yield per mu(kg) Rate of growth (%) Melasma disease refers to Relative control effect(%)
Test group 2432 29.6 3.12 87.62
Chemical fertilizer group 2088 11.3 25.21
Fertilizer group 1876 12.45 50.61
Below the present invention is described in detail, the above, only the preferred embodiments of the invention, when can not limit The scope of the present invention, that is, all according to the made impartial change of the application scope and modification, all should still belong in covering scope of the present invention.

Claims (2)

1. a kind of potato class microbial bacterial manure special of controlling disease is it is characterised in that prepare the raw material of this microbial-bacterial fertilizer by weight Measuring part is:Antimycoin streptomycete mycopowder 20-40 part, wet streptomycete mycopowder 10-30 part, feedstuff series bacillus mycopowder 10-30 Part, Jia meter Li series bacillus mycopowder 20-40 part, potassium fulvate former powder 20-40 part;Its concrete preparation method, walks including following Suddenly:
Step one, the preparation of antimycoin streptomycete mycopowder
Take out antimycoin streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7 My god, picking single bacterium colony streak inoculation under flat board, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as antimycoin streptomycete seed liquor;
Fermentation:The antimycoin streptomycete seed liquor of above-mentioned preparation is seeded to the antimycoin of sterilizing with the inoculum concentration of 1%-3% In strepto- bacteria solid fermentation culture medium, after strain is mixed homogeneously with antimycoin streptomycete solid medium, the anti-of kind will be connected Moldin strepto- bacteria solid fermentation culture medium is spread out on fermentation tank, and height of materials is 3-6cm, 28-32 DEG C of temperature control, first day air Humidity remains 60%-75%, and air humidity remains 40%-50% later, ferments 3-4 days, detects that its spore content is not less than 20 Hundred million CFU/g, you can low-temperature air-drying to moisture is less than 8%, pulverized 40 mesh sieves, that is, obtains antimycoin streptomycete mycopowder;
Wherein, described Gause I solid medium:Potassium nitrate:1 gram, soluble starch:20 grams, dipotassium hydrogen phosphate:0.5, Magnesium sulfate:0.5, sodium chloride:0.5, ferrous sulfate:0.01 gram, agar:20 grams, distilled water supplies 1000ml, PH7.2~7.4;
Wherein, described antimycoin strepto- bacteria solid fermentation culture medium:Maize alcohol stillage 80%, rapeseed cake 2%, maize cob meal 1%, stone Powder 10%, wheat bran 7%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%- 60%;The condition of each medium sterilization is:0.10-0.15MPa, 121 DEG C sterilize 30 minutes;
Step 2, the preparation of wet streptomycete mycopowder
Take out wet streptomyces species preservation pipe, draw flat board with PDA solid medium and recovered, cultivate 7 days, under flat board for 30 DEG C Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of PDA solid mediums, cultivates 6-8 for 30 DEG C in incubator My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense Spend for 0.1 hundred million cfu/ml, as wet streptomycete seed liquor;
Fermentation:The wet streptomycete seed liquor of above-mentioned preparation is seeded to the wet strepto- bacteria solid fermentation training of sterilizing with 5% inoculum concentration On foster base, after strain is mixed homogeneously with wet streptomycete solid medium, the wet strepto- bacteria solid fermentation culture medium stand planted will be connected On fermentation tank, height of materials is 5-8cm, 28-32 DEG C of temperature control, and a few days ago air humidity remains 60%-75%, and air is wet later Degree remains 40%-50%, ferments 4-6 days, detects that its spore content is not less than 1,000,000,000 CFU/g, you can low-temperature air-drying moisture is less than 8%, pulverize 40 mesh sieves, that is, obtain wet streptomycete mycopowder;
Wherein, described PDA culture medium:Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000mL, agar 20g;
Wherein, described wet strepto- bacteria solid fermentation culture medium:Powdered rice hulls 80%, cottonseed meal 2%, Semen Maydis powder 1%, stone powder 2%, wheat bran 15%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%;Each culture Base sterilizing condition be:0.10-0.15MPa, 121 DEG C sterilize 30 minutes;
Step 3, the preparation of Jia meter Li series bacillus mycopowder
Take out Jia meter Li series bacillus preservation pipe, draw flat board respectively with nitrogen-free solid medium and recovered, 30 DEG C of cultures 72 Hour, under flat board, picking single bacterium colony is seeded to equipped with the Fructus Solani melongenae bottle of nitrogen-free solid medium, trains in 30 DEG C of incubators Support 72 hours, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, be seeded to equipped with 300L Jia meter Li class spore bar In the 500L fermentation tank of bacterium seed culture medium, open stirring 120r/min, ventilation is 100L/min, after 8-24 hour within first 8 hours Ventilation is 200L/min, and after 24 hours, ventilation is 300L/min, 30 DEG C of culture 32-48 hours, treats total bacteria count content not Less than 2,500,000,000 cfu/ml, you can as Jia meter Li series bacillus seed liquor;
Fermentation:The Jia meter Li series bacillus seed liquor of above-mentioned preparation is seeded to Jia meter Li class spore with the ratio of 10%-20% On bacillus solid medium, mix, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature to be 30-40 DEG C, sends out Ferment 36-48 hour, treats that spore content is not less than 6,000,000,000 cfu/g, you can dry, treats that moisture is less than 10%, pulverized 100 mesh Sieve, that is, obtain Jia meter Li series bacillus mycopowder;
Wherein, described nitrogen-free solid medium:Sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid Calcium 0.1g, Calcium Carbonate 5g, agar 18g, distilled water 1000mL, pH7.2;
Wherein, described Jia meter Li series bacillus seed culture medium:Molasses 20g/L, tapioca starch 10 g/L, rapeseed cake powder 40 g/L, Magnesium sulfate 0.4 g/L, manganese sulfate 0.6g/L, potassium dihydrogen phosphate 0.6 g/L, Calcium Carbonate 10 g/L, pH7.0;
Wherein, described Jia meter Li series bacillus solid medium:Maize alcohol stillage 57%, wheat bran 35%, cottonseed meal 5%, stone powder 1%, Thick skin of Semen Maydis 2%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%;The condition of each medium sterilization For:0.10-0.15MPa, 121 DEG C sterilize 30 minutes;
Step 4, the preparation of feedstuff series bacillus mycopowder
Take out feedstuff series bacillus preservation pipe, draw flat board respectively with nutrient solid medium and recovered, 30 DEG C of cultures 48 hours, under flat board, picking single bacterium colony was seeded to equipped with the Fructus Solani melongenae bottle of nutrient solid medium, cultivates at 30 DEG C Cultivate 48 hours in case, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, be seeded to equipped with 300L liquid seeds In the 500L fermentation tank of culture medium, open stirring 120r/min, ventilation is 200L/min within first 10 hours, and after 10 hours, ventilation is 320L/min, 30 DEG C of culture 16-24 hours, treat that total bacterial content is not less than 3,000,000,000 cfu/ml, you can as feedstuff series bacillus Seed liquor;
Fermentation:The feedstuff series bacillus seed liquor of above-mentioned preparation is added to feedstuff with the inoculum concentration of the 10%-20% of total inoculum concentration In series bacillus solid fermentation culture medium, mix, in tray top fermentation, windrow thickness is 5-10cm, control fermentation temperature is 30-40 DEG C, 36-48 hour of fermenting, treat that spore content is not less than 8,000,000,000 cfu/g, you can dry, treat that moisture is less than 10%, powder Broken mistake 100 mesh sieve, that is, obtain feedstuff series bacillus mycopowder;
Wherein, described nutrient broth solid medium:Peptone 10.0 g/L, Carnis Bovis seu Bubali cream 3.0 g/L, sodium chloride 5.0 g/L, Agar 20 g/L, pH 7.2 ± 0.2;
Wherein, described liquid seed culture medium:Semen Maydis powder 35 g/L, bean cake powder 20 g/L, fish flour 5 g/L, magnesium sulfate 0.6g/ L, manganese sulfate 0.5 g/L, potassium dihydrogen phosphate 0.3 g/L, Calcium Carbonate 5.0 g/L, pH7.0;
Wherein, feedstuff series bacillus solid fermentation culture medium:Wheat bran 60%, DDGS(Maize alcohol stillage)32%, cottonseed meal 5%, stone powder 3%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%;The condition of each medium sterilization For:0.10-0.15MPa, 121 DEG C sterilize 30 minutes;
Step 5, by the various mycopowder made above by antimycoin streptomycete mycopowder 20-40 part, wet streptomycete mycopowder 10-30 Part, feedstuff series bacillus mycopowder 10-30 part, Jia meter Li series bacillus mycopowder 20-40 part, potassium fulvate former powder 20-40 part; The potato class microbial bacterial manure special of mix homogeneously, as controlling disease.
2. a kind of potato class microbial bacterial manure special of controlling disease according to claim 1 is it is characterised in that described is anti- The application process controlling the potato class microbial bacterial manure special of disease is bottom application microbial-bacterial fertilizer 5-10kg/ mu.
CN201610746263.6A 2016-08-29 2016-08-29 Disease controlling microbial fertilizer special for tuberous crops and preparation method thereof Pending CN106396862A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610746263.6A CN106396862A (en) 2016-08-29 2016-08-29 Disease controlling microbial fertilizer special for tuberous crops and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610746263.6A CN106396862A (en) 2016-08-29 2016-08-29 Disease controlling microbial fertilizer special for tuberous crops and preparation method thereof

Publications (1)

Publication Number Publication Date
CN106396862A true CN106396862A (en) 2017-02-15

Family

ID=58003501

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610746263.6A Pending CN106396862A (en) 2016-08-29 2016-08-29 Disease controlling microbial fertilizer special for tuberous crops and preparation method thereof

Country Status (1)

Country Link
CN (1) CN106396862A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109251879A (en) * 2018-11-22 2019-01-22 青岛中达农业科技有限公司 A kind of Jie meter La series bacillus fermentation process in high density
CN109266589A (en) * 2018-11-22 2019-01-25 青岛中达农业科技有限公司 Jie meter La series bacillus and its application of one plant of inhibition anthrax-bacilus and sickle-like bacteria
CN109369269A (en) * 2018-11-26 2019-02-22 中国热带农业科学院环境与植物保护研究所 A kind of charcoal composite microbic bacterial fertilizer and preparation method thereof inhibiting soil-borne disease

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102690151A (en) * 2012-06-28 2012-09-26 赵鹏 Composite microbial inoculum organic multielement compound fertilizer and production method thereof
CN102816726A (en) * 2012-09-10 2012-12-12 临沂中磷生物科技有限公司 Novel compound microbial agent and applications thereof
CN102910988B (en) * 2012-11-18 2014-08-20 北京市波普克生态科技有限公司 Compound microorganism liquid bacterial fertilizer for preventing plant diseases and insect pests and preparation method of compound microorganism liquid bacterial fertilizer
CN105432667A (en) * 2015-12-19 2016-03-30 佛山市艳晖生物科技有限公司 Preparation method of bacillus subtilis wettable powder

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102690151A (en) * 2012-06-28 2012-09-26 赵鹏 Composite microbial inoculum organic multielement compound fertilizer and production method thereof
CN102816726A (en) * 2012-09-10 2012-12-12 临沂中磷生物科技有限公司 Novel compound microbial agent and applications thereof
CN102910988B (en) * 2012-11-18 2014-08-20 北京市波普克生态科技有限公司 Compound microorganism liquid bacterial fertilizer for preventing plant diseases and insect pests and preparation method of compound microorganism liquid bacterial fertilizer
CN105432667A (en) * 2015-12-19 2016-03-30 佛山市艳晖生物科技有限公司 Preparation method of bacillus subtilis wettable powder

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109251879A (en) * 2018-11-22 2019-01-22 青岛中达农业科技有限公司 A kind of Jie meter La series bacillus fermentation process in high density
CN109266589A (en) * 2018-11-22 2019-01-25 青岛中达农业科技有限公司 Jie meter La series bacillus and its application of one plant of inhibition anthrax-bacilus and sickle-like bacteria
CN109266589B (en) * 2018-11-22 2020-09-18 青岛中达农业科技有限公司 Paenibacillus jimirabilis for inhibiting anthrax and fusarium and application thereof
CN109369269A (en) * 2018-11-26 2019-02-22 中国热带农业科学院环境与植物保护研究所 A kind of charcoal composite microbic bacterial fertilizer and preparation method thereof inhibiting soil-borne disease

Similar Documents

Publication Publication Date Title
EP2479253B1 (en) Antagonistic bacteria for preventing and treating panama wilt disease of continuously planted banana and microorganism organic fertilizer thereof
CN101696390B (en) Biological preventing and controlling strain of continuous cropping cucumber and watermelon blight and microbe organic fertilizer thereof
CN101759494B (en) Diseases-prevention organic fertilizer prepared by edible fungi residue
CN105218254A (en) A kind of Microbial and ecological organic fertilizers material and preparation method thereof
CN104531533B (en) A kind of composite bacteria agent for preventing and treating plant continuous cropping disease and preparation method thereof, application
CN106386163A (en) Method for cultivating edible fungi by using crop straw
CN103819267A (en) Bacterial manure for peanut planting and preparation method of bacterial manure
CN102491802A (en) Tea seed cake biological organic fertilizer and production method thereof
CN101948780B (en) Antagonist bacterium for preventing and treating continuous cropping hot pepper epidemic disease and microbial organic fertilizer thereof
CN101974428A (en) Complex microbial preparation capable of resisting replant obstacle resistance and preparation method thereof
CN104962490B (en) A kind of Enteromorpha microbial bacterial agent and preparation method thereof
CN104016745B (en) microbial organic fertilizer and preparation method thereof
CN102199049A (en) Microbial organic manure and preparation method thereof
CN103588522B (en) A kind of preparation method of quick-effective mixed fertilizer of organic active liquid
CN103966148A (en) Vegetable waste composting bacterial agent, using method thereof and organic substrate prepared from vegetable waste composting bacterial agent
CN101886055B (en) Antagonistic bacteria NJL-14 for preventing and controlling continuous-cropping tobacco bacterial wilt
CN106591193A (en) Bacillus amyloliquefaciens with broad spectrum growth-promoting and stress-resisting effects
CN105462878A (en) Livestock excrement efficient decomposition agent and fermentation method thereof
CN105439725A (en) Paenibacillus polymyxa pesticide-fertilizer for farm onsite fermentation and applications thereof
CN103194405A (en) Growth-promoting bacteria for promoting ginger growth and preventing and controlling continuous cropping ginger soil-borne wilt and microorganism organic fertilizer produced from growth-promoting bacteria
CN103387428A (en) Preparation method for organic material decomposition agent
CN109020727A (en) A kind of bio-organic fertilizer and preparation method thereof preventing Asparagus Stem Blight
CN106396862A (en) Disease controlling microbial fertilizer special for tuberous crops and preparation method thereof
CN106336329A (en) Microbial bacterial fertilizer special for honeysuckles and preparation method thereof
CN108863550A (en) A kind of dedicated disease prevention growth-promoting composite microbic bacterial fertilizer of tea tree and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
C06 Publication
SE01 Entry into force of request for substantive examination
C10 Entry into substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20170215

WD01 Invention patent application deemed withdrawn after publication