CN1163446C

CN1163446C - Microbial streptomycete fertilizer and production process

Info

Publication number: CN1163446C
Application number: CNB00124616XA
Authority: CN
Inventors: 伟邓; 邓伟
Original assignee: 邓伟
Current assignee: Beijing jiulongsheng Microbial Resource Development Co., Ltd.
Priority date: 2000-09-26
Filing date: 2000-09-26
Publication date: 2004-08-25
Anticipated expiration: 2020-09-26
Also published as: CN1344699A

Abstract

The present invention relates to streptomycete microbial manure and a production method thereof. The microbial manure is manufactured by adopting specific streptomycete DW999 as production strain, utilizing various agricultural and sideline products as substrates, and selecting a multifunctional full-automatic solid fermentation tank for fermentation. Compared with general bacterium manure, effective viable bacterium amount contained in each gram of products is 10 to 50 times higher; the present invention contains various plant growth regulating substances of which the content is 10 to 100 times of that of the bacterium manure; the present invention generates various active substances for resisting pathogeny bacteria, fungi and viruses. The products have the comprehensive effect that yield increasing effect is conspicuous, stress resistance is strong, crop fruit quality is improved and soil structure is improved. Grain crops increase about 10% in yield, economic crops increase more than 16% in the yield, and vegetables and fruits increase more than 25% in the yield through large numbers of tests.

Description

Microbial streptomycete fertilizer and manufacture method thereof

The present invention relates to a kind of microbial fertilizer and manufacture method thereof.

People know, farm crop, the growth of plants such as garden crop is different with the growth of vegetation, vegetation is generally no longer irrigated after covering ground and applies fertilizer, vegetation self-sow and breeding under the ecological condition of locality, and farm crop, garden crop all are to spend under artificial intervention in whole growing, mode of its artificial control comprise irrigates, applies fertilizer and carry out the prevention and control of plant diseases, pest control etc. crop, in the hope of obtaining high crop yield, fruit quality is good.In order to solve the demand of population growth to output, use chemical fertilizer, agricultural chemicals as the major measure of increasing production in a large number medium-term and long-term dependence of agriculture production, so green manure, farm manure are out in the cold, the amount of application of chemical fertilizer is more and more, agriculture production cost is anxious to be increased, also cause soil compaction simultaneously, fertility descends, because the loss of chemical fertilizer, agricultural chemicals, residual, quality of agricultural product and HUMAN HEALTH have been influenced, cause environmental pollution, also destroyed original microecological balance in the crop rhizosphere soils simultaneously, soil-borne disease is increased the weight of.Soil is the nutrition source of crop growthing development, and it is made up of mineral substance, organism and microorganism three parts.Soil mineral is the main body that soil material is formed, and the matrix that is plant-growth is again the main source of plant inorganic nutrients.Organic matter is fertilizer provision from soil, protects fertile important substance basis, is again the necessary composition that forms soil aggregate and keep soil from packing together.Microorganism is the organism of living in the soil, is to transform the indispensable active substance of soil fertility.Relation between soil mineral, organism and the microorganism three just look like the relation of skeleton, muscle and the blood cell of animal, and microorganism is wherein to enliven most integral part.Important soil fertility such as formation and decomposition, the nutrient that conversion, the soil ulmin of soil material and energy are participated in the activity of soil microorganisms directly discharges, nitrogen is fixing form and growth course.Therefore, in soil, enrich beneficial microorganism quantity, also can strengthen soil microbial activities, increase soil fertility.Particularly the soil microbial activities around the farm crop rhizosphere is even more important to the improvement of root system of plant nutrient environment, and using bacterial manure at rhizosphere can increase useful mushroom quantity and effect in the root layer soil.The basis of agricultural is at soil, microorganism is the factor of enlivening of soil genesis and development, from the development sustainable agriculture and set up the angle of good ecotope, advocating organic and inorganic, balanced the using of microorganism is to make rational use of resources, the measure that applies fertilizers scientifically of maintenance high crop yield, stable yields.

CN89102107.8 discloses a kind of method of utilizing streptomycete to produce the phytokinin mixed preparation, CN99100373.X has done further improvement to this production method, the large increase of cell fission cellulose content, fermentation level improves 4.2 times, the rate of recovery reaches more than 90%, the product water content is low, storage-stable.

Patent such as CN88100023.X, CN95116540.3 all relates to microorganisms such as utilizing vinelandii, phosphorus bacteria, potassium bacterium, adopt fermentation process to obtain fermented liquid, adsorb fermented liquid with adsorbability material again, replenish some nutritive elements then and just produce microbial fertilizer.These microbial fertilizers can activate by the phosphorus of soil fixing, potassium nutrition element, make it can be by plant absorbing; All kinds of plant growth substances that produce in the microbial proliferation process can strengthen plant-growth, can also suppress disease by some pathogenic micro-organism of antagonism; Improve the soil, reduce applying quantity of chemical fertilizer etc.

But CN89102107.8 and CN99100373.X just utilize microorganism to obtain phytokinin, and the microorganism live body does not continue to be utilized; CN88100023.X, CN95116540.3 etc. utilize bacterial micro-organisms such as vinelandii, phosphorus bacteria, potassium bacterium after fermentation, make microbial fertilizer with carrier absorption, although some fertilizer also produce plant growth regulating substance like this, but the content of finding them is not high, the plant growth regulating substance effect is limited, can not give full play to action of microorganisms.This product is that the specific bacterial strain of screening adopts advanced zymotechnique and equipment to make, and is rich in the complex body of beneficial microorganism and multiple meta-bolites (plant growth regulating substance, antibacterial substance), but comprehensive adjustment plant-growth and growth after using.

For this reason, the production method that the purpose of this invention is to provide a kind of microbial streptomycete fertilizer.

Another one purpose of the present invention provides a kind of microbial streptomycete fertilizer (being commonly called as the nine grand microbial fertilizers that rise usually).

The present invention realizes in the following manner:

A kind of manufacture method of strepto-bacterial manure is characterized in that this method undertaken by following step:

(1) slant strains medium preparation and spawn culture: get the cleaned potato 200-400 weight part of peeling, add entry, its potato and water weight ratio are 0.8-1: 1-1.2, boiled 20-30 minute, after filtering, filtrate adds glucose 10-30 weight part, agar 15-30 weight part, add water its total amount is added to the 1000-1200 weight part, heating is melted fully until agar, makes described substratum like this;

The above-mentioned substratum branch that obtains installs in the test tube, sterilization is 0.5-1.0 hour under 121 ℃, 0.1 MPa pressure, inoculating strain streptomycete (Streptomyces sp.) DW999 (Chinese typical culture collection center, the patent culture is accepted registration number CCTCC No.M200022), 28 ℃-32 ℃ one weeks of slant culture, the sorus of giving birth to until gas is white in color;

(2) shake a preparation of bottle bacterium culture medium and a spawn culture: with glucose and/or sucrose, Semen Maydis powder, bean cake powder, MgSO ₄, K ₂HPO ₄, (NH ₄) ₂SO ₄, CaCO ₃Shake a bottle bacterium culture medium, the pH7.5-8.0 of this substratum with water-soluble being prepared into of NaCl;

Above-mentioned substratum branch is installed in the bottle, and sterilization is 0.8-2.0 hour under 121 ℃, 0.1 MPa pressure, and cooling temperature is reduced to 30-35 ℃, inoculates above-mentioned slant culture bacterial classification, on 180 rev/mins of rotary shaking tables, shakes training 18-24 hour in 28-32 ℃;

(3) liquid fermentation tank seed culture medium and enlarged culturing: its substratum is with to shake bottle bacterium culture medium identical, this substratum was 121 ℃, 0.1 MPa pressure sterilization 0.5-1.0 hour, insert above-mentioned shaking behind the cool to room temperature and cultivate son, fermentor tank pressure is the 0.02-0.08 MPa, and ventilation is expressed as 1 with volume/volume: 0.5-0.7;

(4) solid fermentation: the solid fermentation material is selected from following material: one or more in cavings and/or crop stalk, wheat bran, ground phosphate rock, turfy soil, vegetable mould, pond sludge and/or the loam, Semen Maydis powder or starch and plant oil cake, allow sterilization 0.8-2.0 hour under 121 ℃, 0.1 MPa pressure of its material, inoculate the fermentor tank seed liquor behind the cool to room temperature; Cultivate at 28-32 ℃, ventilation is expressed as 1 with volume/volume: 0.3-1, fermentor tank pressure are the 0.02-0.08 MPa, ferment 72-100 hour, add phosphate fertilizer, potash fertilizer and/or micronutrient fertiliser thereafter, and oven dry obtains described product again.

The add-on of described fermentor tank seed liquor is the 5-15% of solid fermentation weight of material by weight.

Shake in bottle bacterium culture medium glucose or sucrose, Semen Maydis powder, bean cake powder, MgSO described ₄, K ₂HPO ₄, (NH ₄) ₂SO ₄, CaCO ₃With the weight percentage of NaCl be respectively 0.5-1.0%, 0.5-2%, 1.5-3%, 0.02-0.3%, 0.02-0.3%, 0.1-0.3%, 0.3-0.5% and 0.1-0.5%.

In the solid fermentation material, cavings and/or crop stalk are one or more 30-50%, plant oil cake 4-8% and Semen Maydis powder or the starch 5-10% in 20-30%, wheat bran 1-3%, ground phosphate rock 5-10%, turfy soil, vegetable mould, pond sludge and/or the loam by weight percentage, the water yield is the 35-50% of solid fermentation weight of material, and pH transfers to 6.5-8.5.

Described plant oil cake is selected from soyabean cake, sesame-send cake, cottonseed cake, rapeseed cake and/or peanut cake; Described starch is selected from W-Gum, rice starch, wheat starch, barley starch, oat starch and/or potato starch; Described crop stalk is selected from Chinese sorghum, corn, wheat, barley, oat, cotton, soybean, paddy rice, broad bean, pea, rape and/or sesame straw.

Described phosphate fertilizer is selected from potassium primary phosphate, normal superphosphate, double superhosphate, fused(calcium magnesium)phosphate, monocalcium phosphate and/or bone meal.

Described potash fertilizer is selected from Repone K, vitriolate of tartar and/or cement kiln ash potassium fertilizer.

Described micronutrient fertiliser is selected from boron fertilizer, zinc fertilizer, molydbenum fertilizer, manganese fertilizer, copper fertilizer and/or iron fertilizer.

Described boron fertilizer is selected from boric acid and/or borax; Described zinc fertilizer is Zinc Sulphate Heptahydrate, zinc chloride and/or zinc oxide; Described molydbenum fertilizer is selected from ammonium molybdate, Sodium orthomolybdate and/or molybdic oxide; Described manganese fertilizer is selected from manganous sulfate, manganese oxide, Manganous chloride tetrahydrate and/or manganous carbonate; Described copper fertilizer is selected from cupric sulfate pentahydrate, cupric chloride, cupric oxide and/or copper mine slag; Described iron fertilizer is selected from iron vitriol and/or iron protocarbonate.

Described solid fermentation carries out in multi-functional full-automatic solid-state fermentation tank, and this solid-state fermentation tank integrates spice, sterilization, inoculation, fermentation and dries.

This strepto-bacterial manure contains the above spore chain mould that lives of 2,000,000,000/gram.

The present invention will be described in detail belows.

The present invention uses streptomycete (Streptomyces sp.) DW999 (Chinese typical culture collection center, the patent culture is accepted registration number CCTCC No.M200022).Its nutrition in No. 4, ISP and No. 5 substratum of ISP (in the base) mycelium is a tawny, ripe aerial mycelium, sorus white.The polynary branch of substrate mycelium, mycelium is wide 1.2 to 1.5 microns, and the single raft branch of aerial mycelium forms tight or loose spiral.On organic substratum, do not generate melanoid.The conidium smooth surface.Produce various plants growth regulatory substances such as zeatin, two hydrogen zeatin, isopentenyl gland purine, indolylacetic acid, Plant hormones regulators,gibberellins, dormin, phytokinin during this bacterium metabolism, also produce the active substance of multiple antibiotics.

Described ISP4 substratum is inorganic salt--the Starch Agar substratum, and its medium preparation method is as follows:

At first prepare solution I, available a small amount of cold distilled water is with 10 gram Zulkovsky starch furnishing pasty states, and adding distil water is added 500 milliliters and obtained solution I again.

Secondly, preparation solution II:

Get 1.0 gram K ₂HPO ₄, 1.0 the gram MgSO ₄7H ₂O, 1.0 gram NaCl, 2.0 gram (NH ₄) ₂SO ₄, 2.0 the gram CaCO ₃Add 500 ml distilled waters, add 1.0 milliliters of micro-salts solutions after the dissolving again and just obtain solution II.This trace element salts solution is with 0.1 gram FeSO ₄7H ₂O, 0.1 gram MnCl ₂4H ₂O, 0.1 gram ZnSO ₄7H ₂O is dissolved in and obtains in 100 ml distilled waters.

The pH of solution II is 7.0-7.4, or its pH regulator is arrived in the described scope.

At last, solution I and solution II are mixed, in mixed solution, add 20 gram agar, melted 15-20 minute packing and sterilization then at 100 ℃.

Described ISP5 substratum is a glycerine--the asparagine nutrient agar, and its medium preparation method is as follows:

With the anhydrous L-asparagine of 1.0 grams, 10.0 gram glycerine, 1.0 gram anhydrous K ₂HPO ₄Dissolve in 1.0 liters of distilled water, add 1.0 milliliters of micro-salts solutions again, obtain a kind of mixing solutions.This trace element salts solution is with 0.1 gram FeSO ₄7H ₂O, 0.1 gram MnCl ₂4H ₂O, 0.1 gram ZnSO ₄7H ₂O is dissolved in and obtains in 100 ml distilled waters.

The pH of this mixing solutions is about 7.0-7.4, or its pH regulator is arrived in the described scope.

At last, in mixing solutions, add 20 gram agar, melted 15-20 minute packing and sterilization then at 100 ℃.

The present preparation method of description chain mould fertilizer in more detail.

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 200-400 weight part, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 0.8-1: 1-1.2, preferably 1: 1, boils 20-30 minute, preferably 0.5 hour, the potato of boiling is filtered with three layers of gauze or other filter plants, removes filter residue, collects filtrate, add glucose 10-30 weight part toward filtrate again, 20 weight parts preferably, agar 15-30 weight part, preferably 18-20 weight part, water replenishes total amount up to the 1000-1200 weight part then, slowly heating is melted fully until agar again, should note during heating stirring, and makes described slant strains substratum like this;

Carrying out slant strains then cultivates.The above-mentioned substratum branch that obtains is installed in 16 * 160mm test tube (every pipe 3-4 milliliter), these test tubes clog with tampon, again these test tubes are placed in the high-pressure sterilizing pot under 121 ℃, 0.1 MPa pressure sterilization 0.5-1.0 hour, thereafter these test tube substratum are put into the inclined-plane.Described high-pressure sterilizing pot is produced by Shanghai Medical Nuclear Instrument Factory, and commodity are called the portable pressure steam sterilizer of electric heating.

The above-mentioned sterilized culture medium inoculated bacterial strain streptomycete DW999 in the slope that puts puts these test tubes in the incubator at 28 ℃ of-32 ℃ of slant culture then, and at least one week of incubation time, the sorus of giving birth to until gas is white in color; Described incubator is produced by Sida Experiment Instrument Factory, Chongqing, and commodity are called electro-heating standing-temperature cultivator.

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with glucose or sucrose 0.5-1.0%, Semen Maydis powder 0.5-2%, bean cake powder 1.5-3%, MgSO ₄0.02-0.3%, K ₂HPO ₄0.02-0.3%, (NH ₄) ₂SO ₄0.1-0.3%, CaCO ₃0.3-0.5%, with NaCl 0.1-0.5% described substratum, the pH7.5-8.0 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch installed in the 500ml triangular flask (generally adorn 100-150 milliliter/bottle), allow sterilization 0.5-1.0 hour under 121 ℃, 0.1 MPa pressure of this substratum, after the sterilization, this substratum cools off, temperature is reduced to 30-35 ℃, inoculates above-mentioned slant culture bacterial classification then, every 1 bottle of liquid nutrient medium of pityrosporion ovale suspension inoculation that the inclined-plane makes with sterilized water, on 180 rev/mins of rotary shaking tables, shake training 18-24 hour in 28-32 ℃; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, sterilization 0.5-1.0 hour under 121 ℃, 0.1 MPa pressure of this substratum, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 1-5%, fermentor tank pressure is the 0.02-0.08 MPa, and air quantity is expressed as 1 with volume/volume: 0.5-0.7; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from one or more following materials: cavings or crop stalk, wheat bran, ground phosphate rock, turfy soil, vegetable mould, in pond sludge and/or the loam one or more, Semen Maydis powder or starch and plant oil cake, in the solid fermentation material, cavings or crop stalk are 20-30% by weight percentage, wheat bran 1-3%, ground phosphate rock 5-10%, turfy soil, vegetable mould, one or more 40-50% in pond sludge and/or the loam, plant oil cake 4-8% and Semen Maydis powder or starch 5-10%, adding the water yield is the 35-50% of described solid fermentation inventory, pH6.5-8.5.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

Described plant oil cake is soyabean cake, sesame-send cake, cottonseed cake, rapeseed cake and/or peanut cake; Described starch is W-Gum, rice starch, wheat starch, barley starch, oat starch and/or potato starch; Described crop stalk is Chinese sorghum, corn, wheat, barley, oat, cotton, soybean, paddy rice, broad bean, pea, rape, castor-oil plant and/or sesame straw.

Materials such as ground phosphate rock, agricultural crop straw, oil cake all need to pulverize, and reach the 80-100 order.Can use any pulverizer to pulverize.Turfy soil, vegetable mould, pond sludge, loam also should suitably be controlled the raw material water content, if water content is too high, then need to dewater in advance.

Allow sterilization 0.8-2.0 hour under 121 ℃, 0.1 MPa pressure of above-mentioned solid fermentation material, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size is counted 5-15% by the solid fermentation weight of material, fully stir the inoculation back, make its material inoculation evenly, cultivate at 28-32 ℃, note ventilating, ventilation is expressed as 1 with volume/volume: 0.3-1, fermentor tank pressure is the 0.02-0.08 MPa, thalli growth situation in the material is observed in sampling in per 8 hours, ferments to stop fermentation in 72-100 hour, if necessary, after fermentation, add phosphate fertilizer, potash fertilizer and/or micronutrient fertiliser, oven dry again, control water-content to reach＜10%, described product obtained like this.

Except above-mentioned key points for operation, other operations, comprising the certain operations condition, all should strictly carry out according to the principle of determining in the biotechnology, these principles should be that those skilled in the art are known, particularly can be with reference to " biotechnology " (Huadong Chemical College press) Yu Juntang, Tang Xiaoxuan chief editor, the content described in December, 1991 first version.

Adopt the colony counting method of the agricultural industry criteria NY22-94 of the People's Republic of China (PRC) " microbial fertilizer " regulation to detect, the strepto-bacterial manure that aforesaid method makes can reach the above spore chain mould that lives of 2,000,000,000/gram, can reach 10,000,000,000/gram spore chain mould alive, these strepto-bacterial manure can also contain phosphate fertilizer, potash fertilizer and trace element fertilizer and great number of organic matters, therefore, this fertilizer integrates organism, inorganics and microorganism.

This fertilizer can be used as that crop seeds is handled, is stained with root, is grown seedlings, base manure and foliage-spray.Method for treating seeds comprises seed dressing, vexed kind, seed soaking.Seed dressing method is earlier with the seed water-soaked, mixes thoroughly with this fertilizer again, and the back of drying in the shade is sowed.The weight proportion of crop seed and fertilizer of the present invention is 20: 1.The weight proportion of vegetable seed and fertilizer of the present invention is 1: 1.Every mu of consumption is this fertilizer of 0.5-1 kilogram.

Vexed kind of method is to add 4 kg water ratios according to every kilogram of fertilizer, and fertilizer of the present invention was soaked 30 minutes, pours seed then into and mixes thoroughly, and the seed add-on is that every kilogram of fertilizer can add 10-15 kilogram seed.After the vexed 12-20 in lucifuge place hour, spread the sowing of drying in the shade out again.

Seed-soaking method adds 20 kilograms of seeds and soaked 10-12 hour after to be fertilizer of the present invention according to every kilograms of product convert 20 kilograms in water.

After being stained with method for root and being fertilizer of the present invention and converting 20 kilograms in water according to every kilograms of product, its solution is made for to be stained with root when thing is transplanted and to use.

Method for culturing seedlings be with every kilogram of fertilizer of the present invention with after 50 kilograms of nutrition soil (fertilizer that becomes thoroughly decomposed) mix, make seedbed or feeding block seedlings raising and use.

The base applying method be every mu with spreading manuer in holes or ditch spread behind 2-3 kilogram fertilizer of the present invention and about 40 kilograms of fertilizers (preferably the adding 10-15 kilogram cake fertilizer again) mixing, also can be by the use of topdressing of this proportioning mixing post-trenching.

Spraying method is that fertilizer of the present invention mixes immersion after 30 minutes with water according to weight ratio 1: 20-50, gets supernatant liquor and sprays.

Streptomycete quasi-microorganism fertilizer has following positively effect:

(1) this microbial fertilizer adopts the specific chains mould DW999 that obtains through mutagenesis screening for producing bacterial classification, can utilize multiple agricultural byproducts to carry out growth and breeding for matrix, and every gram product living bacteria count than the high 10-50 of present bacterial fertilizer doubly.

(2) data according to modern study shows, plant growth regulating substance is naturally occurring a series of organic compound in the plant materials, its content is very low, the various physiological activities of plant (are planted embryogeny, seed germination, cauline leaf is grown, is yielded positive results etc.) interact closely related with different types of plant growth regulating substance, the various plants growth substance that utilizes specific beneficial microorganism in the breeding metabolic process, to be produced, stimulate and regulate plant growth, have pure natural, cost low, act on comprehensive characteristics.This microbial fertilizer meta-bolites contains the various plants growth regulatory substance, and as zeatin, two hydrogen zeatin, isopentenyl gland purine, indolylacetic acid, Plant hormones regulators,gibberellins, dormin etc., its content are 10-100 times of general bacterial fertilizer.

Strepto-bacterial manure of the present invention adopts high pressure liquid chromatographic analysis through analytic centre of China Forestry Science Research Institute, and it the results are shown in following table 1.

Table 1 streptomycete fertilizer high pressure liquid chromatographic analysis result

The sample title	Content, microgram/gram
	Content, microgram/gram			Z	GA ₃	IAA
	Microbial fertilizer (005 crowd of streptomycete DW999)	4.4	29.8	Z	GA ₃	IAA	0.8
Microbial fertilizer (008 crowd of streptomycete DW999)	Microbial fertilizer (005 crowd of streptomycete DW999)	4.4	29.8	4.5	34.7	0.8	0.8
Microbial fertilizer (008 crowd of streptomycete DW999)	Microbial fertilizer (010 crowd of streptomycete DW999)	4.5	19.8	4.5	34.7	0.8	0.8
Microbial fertilizer (031 crowd of streptomycete DW999)	Microbial fertilizer (010 crowd of streptomycete DW999)	4.5	19.8	4.5	29.3	1.6	0.8
Microbial fertilizer (031 crowd of streptomycete DW999)	Microbial fertilizer (066 crowd of streptomycete DW999)	3.0	43.6	4.5	29.3	1.6	0.8
Microbial fertilizer (086 crowd of streptomycete DW999)	Microbial fertilizer (066 crowd of streptomycete DW999)	3.0	43.6	3.1	24.8	0.8	0.8

Z represents zeatin

GA ₃Represent Plant hormones regulators,gibberellins

IAA represents indolylacetic acid

(3) after this microbial fertilizer is concentrated and is applied to farm crop root district, the spore of dormancy brings back to life breeding rapidly, they are seized by the position, the active substance of the multiple disease-resistant indigenous bacteria of the competition of nutritive substance, oxygen and generation, fungi and virus, form a kind of biological barrier at crop rhizosphere, stop harmful bacterium decide grow, invade, and the growth of inhibition root pathogens, thereby the minimizing soil-borne disease has strengthened the resistance against diseases of crop.Once carried out following test: elder generation is difference inoculating two kinds pathogenic bacteria bacteria suspension in culture dish bottom substratum, inoculate 4 DW999 bacterium pieces, around the bacterium piece, tangible inhibition zone occurs after cultivating for some time, thereby can illustrate that product of the present invention has the disease resistance effect.Is example with the DW999 bacterial strain to the restraining effect (antagonistic effect) of phytopathogen, its experiment operational condition is: inoculate the pathogenic bacteria bacteria suspension respectively earlier in culture dish bottom substratum, inoculate 4 DW999 bacterium pieces, around the bacterium piece, tangible inhibition zone occurs after cultivating for some time, thereby verified the field prophylaxis effect of this fertilizer.

(4) streptomycete fertilizer of the present invention inserts soil, after a large amount of breedings, and N in the soil organic matter, P, the conversion capability of K can improve 10-25%.

(5) this product has obvious effect of increasing production, and strong stress resistance improves the crop and fruit quality, the net effect of the structure of improving the soil.Through a large amount of tests, statistical study determines, it is about 10% that food crop are increased production, and cash crop are increased production more than 16%, and vegetable and fruit increases production more than 25%.

Illustrate further the present invention by following embodiment, and these embodiment do not limit protection scope of the present invention.

Accompanying drawing of the present invention is restraining effect (antagonistic effect) diagram of DW999 bacterial strain to phytopathogen.

Embodiment 1

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 200 weight parts, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 0.8: 1, boils 0.5 hour, and the potato of boiling is with three layers of filtered through gauze, remove filter residue, collect filtrate, add glucose 10 weight parts, agar 15 weight parts toward filtrate again, water replenishes total amount up to 1000 weight parts then, slowly heating is melted fully until agar again, slowly stirs during heating, makes described slant strains substratum like this;

Carrying out slant strains then cultivates.The above-mentioned substratum branch that obtains is installed in 16 * 160mm test tube (every pipe 3-4 milliliter), these test tubes clog with tampon, again these test tubes are placed in the high-pressure sterilizing pot under 121 ℃, 0.1 MPa pressure sterilization 0.5 hour, thereafter these test tube substratum are put into the inclined-plane.Described high-pressure sterilizing pot is produced by Shanghai Medical Nuclear Instrument Factory, and commodity are called the portable pressure steam sterilizer of electric heating.

The above-mentioned sterilized culture medium inoculated bacterial strain streptomycete DW999 in the slope that puts puts these test tubes in the incubator at 28 ℃ of slant culture then, and in one week of incubation time, the sorus of giving birth to until gas is white in color; Described incubator is produced by Sida Experiment Instrument Factory, Chongqing, and commodity are called electro-heating standing-temperature cultivator.

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with glucose 0.5%, Semen Maydis powder 0.5%, bean cake powder 1.5%, MgSO ₄0.05%, K ₂HPO ₄0.05%, (NH ₄) ₂SO ₄0.1%, CaCO ₃0.3%, with NaCl 0.2% described substratum, the pH7.5 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch is installed in the 500ml triangular flask, allow the sterilization 0.5 hour under 121 ℃, 0.1 MPa pressure of this substratum, after the sterilization, this substratum cools off, temperature is reduced to 30 ℃, inoculates above-mentioned slant culture bacterial classification then, 1 bottle of liquid nutrient medium of every slant pore bacterial suspension inoculation, on 180 rev/mins of rotary shaking tables, shake training 18 hours in 28 ℃; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, this substratum was 121 ℃, 0.1 MPa pressure sterilization 0.5 hour, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 1%, fermentor tank pressure is 0.05 MPa, and air quantity is expressed as 1: 0.5 with volume/volume; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from following material: cavings, wheat bran, ground phosphate rock, soyabean cake and W-Gum, in the solid fermentation material, by weight percentage cavings be 26%, wheat bran 2%, ground phosphate rock 8%, turfy soil 48%, soyabean cake 8% and W-Gum 8%, adding the water yield is 35% of described solid fermentation weight of material, and pH transfers to 8.5.

Ground phosphate rock, turfy soil, soybean cake material all need to pulverize, and reach the 80-100 order.

Allow sterilization 0.8-2 hour under 121 ℃, 0.1 MPa pressure of above-mentioned solid fermentation material, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size counts 5% by the solid fermentation material, and fully stir the inoculation back, makes its material inoculation evenly, cultivate at 28 ℃, note ventilating, ventilation is expressed as 1: 0.3 with volume/volume, and fermentor tank pressure is 0.05 MPa, thalli growth situation in the material is observed in sampling in per 8 hours, fermented 72 hours, and stopped fermentation, again oven dry, control water-content to reach＜10%, obtain described product like this.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

The fertilizer that this embodiment makes has carried out green pepper pot experiment in seedling stage, uses commercially available bacterial micro-organism fertilizer to carry out simultaneous test simultaneously.This test is to adopt common test method to carry out.Its test-results is listed in table 2.

The investigation of table 2 green pepper growing way in seedling stage

Handle	The leaf look	Leaf long (cm)	Leaf wide (cm)	Plant height (cm)	Stem thick (cm)	Radical (bar)	Strain fresh weight (g)	Main root long (cm)	Lateral root long (cm)
Handle	The leaf look	Leaf long (cm)	Leaf wide (cm)	Plant height (cm)	Stem thick (cm)	Radical (bar)	Strain fresh weight (g)	Main root long (cm)	Lateral root long (cm)	Microbial streptomycete fertilizer of the present invention	Dark green	4.6	2.7	6.8	0.2	22	1.0	6.2	3.6
Bacterial micro-organism fertilizer (CK)	Light green	3.5	2.4	5.1	0.15	18	0.6	3.6	3.0	Microbial streptomycete fertilizer of the present invention	Dark green	4.6	2.7	6.8	0.2	22	1.0	6.2	3.6
Bacterial micro-organism fertilizer (CK)	Light green	3.5	2.4	5.1	0.15	18	0.6	3.6	3.0	Than CK+/-		+1.1	+0.3	+1.7	+0.0 5	+4	+0.4	+2.6	+0.6

Can see that by last table green pepper shines and has very remarkable advantages in radical, rectangular comparison of main root length and lateral root seedling stage.

Embodiment 2

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 300 weight parts, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 1: 1, boils 0.5 hour, and the potato of boiling is with three layers of gauze, remove filter residue, collect filtrate, add glucose 20 weight parts, agar 18 weight parts toward filtrate again, water replenishes total amount up to 1000 weight parts then, slowly heating is melted fully until agar again, stirs during heating, makes described slant strains substratum like this;

Carrying out slant strains then cultivates.The above-mentioned substratum branch that obtains is installed in 16 * 160mm test tube (4 milliliters of every pipes), these test tubes clog with tampon, again these test tubes are placed in the high-pressure sterilizing pot under 121 ℃, 0.1 MPa pressure sterilization 0.5 hour, thereafter these test tube substratum are put into the inclined-plane.Described high-pressure sterilizing pot is produced by Shanghai Medical Nuclear Instrument Factory, and commodity are called the portable pressure steam sterilizer of electric heating.

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with glucose 0.6%, Semen Maydis powder 0.5%, bean cake powder 1.8%, MgSO ₄0.2%, K ₂HPO ₄0.1%, (NH ₄) ₂SO ₄0.1%, CaCO ₃0.3%, with NaCl 0.1% described substratum, the pH7.5 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch is installed in the 500ml triangular flask, allow the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, after the sterilization, this substratum cools off, temperature is reduced to 32 ℃, inoculates above-mentioned slant culture bacterial classification then, every 1 bottle of liquid nutrient medium of pityrosporion ovale suspension inoculation that the inclined-plane makes with sterilized water, on 180 rev/mins of rotary shaking tables, shake training 20 hours in 32 ℃; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 2%, fermentor tank pressure is 0.05 MPa, and air quantity is expressed as 1: 0.5 with volume/volume; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from following material: cavings, wheat bran, ground phosphate rock, vegetable mould, Semen Maydis powder and peanut cake, in the solid fermentation material, by weight percentage cavings be 26%, wheat bran 2%, ground phosphate rock 8%, vegetable mould 48%, peanut cake 8% and Semen Maydis powder 8%, adding the water yield is 38% of described solid fermentation weight of material, and pH transfers to 7.5.

Materials such as ground phosphate rock, vegetable mould, peanut cake all need to pulverize, and reach the 80-100 order.

Allow sterilization 0.8-2 hour under 121 ℃, 0.1 MPa pressure pressure of above-mentioned solid fermentation material, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size counts 6% by the solid fermentation material, fully stir the inoculation back, makes its material inoculation evenly, cultivates at 32 ℃, note ventilating, ventilation is expressed as 1: 0.3 with volume/volume, and fermentor tank pressure is 0.06 MPa, and thalli growth situation in the material is observed in sampling in per 8 hours, ferment and stopped fermentation in 80 hours, oven dry again, control water-content to reach＜10%, described product obtained like this.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

The fertilizer that uses this embodiment to make has carried out the test of cigarette strain disease, uses commercially available bacterial micro-organism fertilizer to carry out simultaneous test simultaneously.This test is to adopt common test method to carry out.These test-results are listed in table 3.

Table 3 cigarette strain morbidity survey

Handle	Virus disease (mosaic disease)			Blade face disease (comprising prairie fire, gas spot, red-star like disease etc.)
	Virus disease (mosaic disease)						Sickness rate (%)	Disease refers to (%)	Prevention effect (%)	Sickness rate (%)	Disease refers to (%)	Prevention effect (%)
	Bacterial micro-organism fertilizer (CK)	37.5	56.7	0	25.5	40.1	Sickness rate (%)	Disease refers to (%)	Prevention effect (%)	Sickness rate (%)	Disease refers to (%)	Prevention effect (%)	0
Microbial fertilizer * of the present invention	Bacterial micro-organism fertilizer (CK)	37.5	56.7	0	25.5	40.1	18.8	30.8	45.7	10.6	32.2	19.7	0
Microbial fertilizer * of the present invention	Microbial fertilizer * * of the present invention	15.5	21.6	61.9	9.8	27.5	18.8	30.8	45.7	10.6	32.2	19.7	31.4

* every mu is used 1 kilogram of microbial fertilizer of the present invention.

* uses 2 kilograms of microbial fertilizers of the present invention for every mu.

As can be seen from the above table, compare with existing commercially available bacterial micro-organism fertilizer, microbial streptomycete fertilizer of the present invention has fabulous result for control tobacco leaf virus disease and blade face disease.

Embodiment 3

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 300 weight parts, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 1: 1, boils 0.6 hour, and the potato of boiling is with three layers of filtered through gauze, remove filter residue, collect filtrate, add glucose 15 weight parts, agar 15 weight parts toward filtrate again, water replenishes total amount up to 1000 weight parts then, slowly heating is melted fully until agar again, slowly stirs during heating, makes described slant strains substratum like this;

The above-mentioned sterilized culture medium inoculated bacterial strain streptomycete DW999 in the slope that puts puts these test tubes in the incubator at 30 ℃ of slant culture then, and in one week of incubation time, the sorus of giving birth to until gas is white in color; Described incubator is produced by Sida Experiment Instrument Factory, Chongqing, and commodity are called electro-heating standing-temperature cultivator.

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with glucose 0.6%, Semen Maydis powder 0.8%, bean cake powder 2%, MgSO ₄0.1%, K ₂HPO ₄0.1%, (NH ₄) ₂SO ₄0.2%, CaCO ₃0.4%, with NaCl 0.2% described substratum, the pH7.6 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch is installed in the 500ml triangular flask, allow the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure pressure of this substratum, after the sterilization, this substratum cools off, temperature is reduced to 30 ℃, inoculates above-mentioned slant culture bacterial classification then, every 1 bottle of liquid nutrient medium of pityrosporion ovale suspension inoculation that the inclined-plane makes with sterilized water, on 180 rev/mins of rotary shaking tables, shake training 20 hours in 30 ℃; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure pressure of this substratum, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 2%, fermentor tank pressure is 0.06 MPa, and air quantity is expressed as 1: 0.6 with volume/volume; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from following material: cavings, wheat bran, ground phosphate rock, wheat stalk, cottonseed cake and soybean starch, pond sludge, in the solid fermentation material, by weight percentage cavings be 26%, wheat bran 2%, ground phosphate rock 8%, wheat stalk 8%, cottonseed oil cake 8% and soybean starch 8%, pond sludge 40%, adding the water yield is 40% of described solid fermentation weight of material, and pH transfers to 8.0.

Materials such as ground phosphate rock, wheat stalk, cottonseed oil cake, pond sludge all need to pulverize, and reach the 80-100 order.

Allow above-mentioned solid fermentation material 121 ℃, 0.1 MPa pressure sterilization 0.8-2 hour, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size counts 5% by the solid fermentation material, and fully stir the inoculation back, makes its material inoculation evenly, cultivate at 28 ℃, note ventilating, ventilation is expressed as 1: 0.3 with volume/volume, and fermentor tank pressure is 0.06 MPa, thalli growth situation in the material is observed in sampling in per 8 hours, fermented 72 hours, and stopped fermentation, again oven dry, control water-content to reach＜10%, obtain described product like this.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

The fertilizer that this embodiment makes has carried out wheat seed soaking field test, uses commercially available bacterial micro-organism fertilizer to carry out simultaneous test simultaneously.This test is to adopt common test method to carry out.Its test-results is listed in table 4-6.

Each leaf emergence questionnaire of table 4 wheat

Each handles survey product summary sheet table 5 wheat

Each handles anti-Powdery Mildew cartogram table 6 wheat

4-5 can see that compare with existing bacterial micro-organism fertilizer, fertilizer of the present invention has remarkable contribution to the wheat seedling rate by table, and wheat has tangible effect of increasing production.Can see that by table 6 fertilizer of the present invention has very significant effect to the control wheat powdery mildew.

Embodiment 4

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 400 weight parts, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 1: 1, boils 0.6 hour, and the potato of boiling is with three layers of filtered through gauze, remove filter residue, collect filtrate, add glucose 25 weight parts, agar 25 weight parts toward filtrate again, water replenishes total amount up to 1000 weight parts then, slowly heating is melted fully until agar again, slowly stirs during heating, makes described slant strains substratum like this;

Carrying out slant strains then cultivates.The above-mentioned substratum branch that obtains is installed in 16 * 160mm test tube (every pipe 3-4 milliliter), these test tubes clog with tampon, again these test tubes are placed in the high-pressure sterilizing pot under 121 ℃, 0.1 MPa pressure sterilization 0.6 hour, thereafter these test tube substratum are put into the inclined-plane.Described high-pressure sterilizing pot is produced by Shanghai Medical Nuclear Instrument Factory, and commodity are called the portable pressure steam sterilizer of electric heating.

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with glucose 0.7%, Semen Maydis powder 1.2%, bean cake powder 2%, MgSO ₄0.1%, K ₂HPO ₄0.2%, (NH ₄) ₂SO ₄0.2%, CaCO ₃0.4%, with NaCl 0.3% described substratum, the pH7.6 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch is installed in the 500ml triangular flask, allow this substratum sterilization under 121 ℃, 0.1 MPa pressure 0.6 hour, after the sterilization, this substratum cools off, and temperature is reduced to 30 ℃, inoculates above-mentioned slant culture bacterial classification then, every 1 bottle of liquid nutrient medium of pityrosporion ovale suspension inoculation that the inclined-plane makes with sterilized water, on 180 rev/mins of rotary shaking tables, shake training 20 hours in 30 ℃, note during cultivation stirring; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 2%, fermentor tank pressure is 0.06 MPa, and air quantity is expressed as 1: 0.6 with volume/volume; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from following material: cavings, wheat bran, ground phosphate rock, vegetable mould, turfy soil, cottonseed cake and Semen Maydis powder, in the solid fermentation material, by weight percentage cavings be 26%, wheat bran 2%, ground phosphate rock 8%, vegetable mould 35%, turfy soil 13%, cottonseed cake 8% and Semen Maydis powder 8%, adding the water yield is 42% of described solid fermentation weight of material, and pH transfers to 8.5.

Ground phosphate rock, vegetable mould, turfy soil, cottonseed cake material all need to pulverize, and reach the 80-100 order.

Allow sterilization 0.8-2 hour under 121 ℃, 0.1 MPa pressure of above-mentioned solid fermentation material, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size counts 8% by the solid fermentation material, and fully stir the inoculation back, makes its material inoculation evenly, cultivate at 30 ℃, note ventilating, ventilation is expressed as 1: 0.5 with volume/volume, and fermentor tank pressure is 0.06 MPa, thalli growth situation in the material is observed in sampling in per 8 hours, fermented 80 hours, and stopped fermentation, again oven dry, control water-content to reach＜10%, obtain described product like this.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

The fertilizer that this embodiment makes has carried out green pepper, eggplant field test, uses commercially available bacterial micro-organism fertilizer to carry out simultaneous test simultaneously.This test is to adopt common test method to carry out.Its test-results is listed in table 7.

Embodiment 5

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 300 weight parts, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 1: 1, boils 0.6 hour, and the potato of boiling is with three layers of filtered through gauze, remove filter residue, collect filtrate, add glucose 25 weight parts, agar 25 weight parts toward filtrate again, water replenishes total amount up to 1000 weight parts then, slowly heating is melted fully until agar again, slowly stirs during heating, makes described slant strains substratum like this;

Carrying out slant strains then cultivates.The above-mentioned substratum branch that obtains is installed in 16 * 160mm test tube (every pipe 3-4 milliliter), these test tubes clog with tampon, again these test tubes are placed in the high-pressure sterilizing pot under 121 ℃, 0.1 MPa pressure sterilization 0.5 hour, thereafter the substratum in these test tubes are put into the inclined-plane.Described high-pressure sterilizing pot is produced by Shanghai Medical Nuclear Instrument Factory, and commodity are called the portable pressure steam sterilizer of electric heating.

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with glucose 0.8%, Semen Maydis powder 1.5%, bean cake powder 2%, MgSO ₄0.1%, K ₂HPO ₄0.1%, (NH ₄) ₂SO ₄0.2%, CaCO ₃0.4%, with NaCl 0.2% described substratum, the pH7.6 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch is installed in the 500ml triangular flask, allow the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, after the sterilization, this substratum cools off, and temperature is reduced to 30 ℃, inoculates above-mentioned slant culture bacterial classification then, every 1 bottle of liquid nutrient medium of pityrosporion ovale suspension inoculation that the inclined-plane makes with sterilized water, on 180 rev/mins of rotary shaking tables, shake training 24 hours in 30 ℃, note during cultivation stirring; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, this substratum is 121 ℃, 0.1 MPa pressure sterilization 0.6 hour, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 2%, fermentor tank pressure is 0.06 MPa, and air quantity is expressed as 1: 0.6 with volume/volume; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from following material: cavings, wheat bran, ground phosphate rock, soyabean cake, peanut cake, loam and barley starch, in the solid fermentation material, by weight percentage cavings be 26%, wheat bran 2%, ground phosphate rock 8%, loam 48%, soyabean cake 4%, peanut cake 4% and barley starch 8%, adding the water yield is 42% of described solid fermentation weight of material, and pH transfers to 8.5.

Ground phosphate rock, loam, soyabean cake, peanut cake material all need to pulverize, and reach the 80-100 order.

Allow above-mentioned solid fermentation material 121 ℃, 0.1 MPa pressure sterilization 0.6 hour, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size counts 8% by the solid fermentation material, and fully stir the inoculation back, makes its material inoculation evenly, cultivate at 28 ℃, note ventilating, ventilation is expressed as 1: 0.5 with volume/volume, and fermentor tank pressure is 0.06 MPa, thalli growth situation in the material is observed in sampling in per 8 hours, fermented 85 hours, and stopped fermentation, again oven dry, control water-content to reach＜10%, obtain described product like this.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

The fertilizer that this embodiment makes has carried out the kidney bean field test, uses commercially available bacterial micro-organism fertilizer to carry out simultaneous test simultaneously.This test is to adopt common test method to carry out.Its test-results is listed in table 7.

Embodiment 6

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 400 weight parts, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 1: 1, boils 0.6 hour, and the potato of boiling is with three layers of filtered through gauze, remove filter residue, collect filtrate, add glucose 25 weight parts, agar 25 weight parts toward filtrate again, water replenishes total amount up to 1200 weight parts then, slowly heating is melted fully until agar again, slowly stirs during heating, makes described slant strains substratum like this;

The above-mentioned sterilized culture medium inoculated streptomycete bacterial strain DW999 in the slope that puts puts these test tubes in the incubator at 30 ℃ of slant culture then, and in one week of incubation time, the sorus of giving birth to until gas is white in color; Described incubator is produced by Sida Experiment Instrument Factory, Chongqing, and commodity are called electro-heating standing-temperature cultivator.

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with sucrose 0.8%, Semen Maydis powder 1.5%, bean cake powder 2%, MgSO ₄0.1%, K ₂HPO ₄0.1%, (NH ₄) ₂SO ₄0.2%, CaCO ₃0.4%, with NaCl 0.2% described substratum, the pH7.6 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch is installed in the 500ml triangular flask, allow the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, after the sterilization, this substratum cools off, temperature is reduced to 30 ℃, inoculates above-mentioned slant culture bacterial classification then, every 1 bottle of liquid nutrient medium of pityrosporion ovale suspension inoculation that the inclined-plane makes with sterilized water, on 180 rev/mins of rotary shaking tables, shake training 20 hours in 30 ℃; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 4%, fermentor tank pressure is 0.06 MPa, and air quantity is expressed as 1: 0.6 with volume/volume; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from following material: cavings, wheat bran, ground phosphate rock, vegetable mould, cottonseed cake and Semen Maydis powder, in the solid fermentation material, by weight percentage cavings be 26%, wheat bran 3%, ground phosphate rock 8%, vegetable mould 47%, cottonseed cake 8% and Semen Maydis powder 8%, adding the water yield is 40% of described solid fermentation weight of material, and pH transfers to 8.0.

Ground phosphate rock, vegetable mould, cottonseed cake material all need to pulverize, and reach the 80-100 order.Allow sterilization 0.8-2 hour under 121 ℃, 0.1 MPa pressure of above-mentioned solid fermentation material, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size counts 10% by the solid fermentation material, fully stir the inoculation back, make its material inoculation evenly, cultivate at 30 ℃, note ventilating, ventilation is expressed as 1: 0.6 with volume/volume, fermentor tank pressure is 0.05 MPa, thalli growth situation in the material is observed in sampling in per 8 hours, fermented 80 hours, stop fermentation, oven dry again, control water-content to reach＜10%, add then in solid fermentation weight of material 5% potassium primary phosphate, 4% vitriolate of tartar and 0.1% trace compound mixture, this trace compound mixture is by zinc sulfate, copper sulfate, ammonium molybdate and manganous sulfate be according to weight ratio 1: 1: 0.5: 0.8 mixes and obtains, and just obtains described product after fully mixed.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

The fertilizer that this embodiment makes has carried out the wild cabbage field test, uses commercially available bacterial micro-organism fertilizer to carry out simultaneous test simultaneously.This test is to adopt common test method to carry out.Its test-results is listed in table 7.

Embodiment 7

At first prepare the slant strains substratum.Use potato-glucose agar medium.This medium preparation method is as follows: potato is cleaned, remove foreign material, peel, be cut into small pieces, get its potato 300 weight parts, put into 800 ml beakers, add entry, amount of water is potato and water weight ratio 1: 1, boils 0.6 hour, and the potato of boiling is with three layers of filtered through gauze, remove filter residue, collect filtrate, add glucose 25 weight parts, agar 25 weight parts toward filtrate again, water replenishes total amount up to 1200 weight parts then, slowly heating is melted fully until agar again, slowly stirs during heating, makes described slant strains substratum like this;

Secondly, a bottle bacterium culture medium is shaken in preparation.According to following weight percent with sucrose 0.8%, Semen Maydis powder 1.6%, bean cake powder 2%, MgSO ₄0.1%, K ₂HPO ₄0.1%, (NH ₄) ₂SO ₄0.2%, CaCO ₃0.5%, with NaCl 0.5% described substratum, the pH7.7 of its solution of obtaining soluble in water.

Carrying out shaking table then cultivates.Above-mentioned substratum branch is installed in the 500ml triangular flask, allow the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, after the sterilization, this substratum cools off, and temperature is reduced to 30 ℃, inoculates above-mentioned slant culture bacterial classification then, every 1 bottle of liquid nutrient medium of pityrosporion ovale suspension inoculation that the inclined-plane makes with sterilized water, on 180 rev/mins of rotary shaking tables, shake training 20 hours in 30 ℃, note during cultivation stirring; Described rotary shaking table is produced by Harbin Donglian Electronic ﹠ Technology Development Co., Ltd., and commodity are called the shaking culture case.

The 3rd step, preparation liquid fermentation tank seed culture medium and enlarged culturing: the substratum that seed tank culture is used and aforementioned to shake bottle bacterium culture medium identical.Enlarged culturing is carried out according to following step: 300 liters of substratum of preparation in 500 liters of fermentor tanks, the sterilization 0.6 hour under 121 ℃, 0.1 MPa pressure of this substratum, insert above-mentioned shaking behind the cool to room temperature and cultivate son, inoculum size is in liquid fermentation tank seed culture medium 4%, fermentor tank pressure is 0.06 MPa, and air quantity is expressed as 1: 0.8 with volume/volume; Described liquid fermentation tank is produced by the biochemical suite of equipment in Huifeng factory, and commodity are called liquid fermentation tank.

The 4th step, solid fermentation.The solid fermentation material is selected from following material: cavings, wheat bran, ground phosphate rock, turfy soil, sesame oil cake and W-Gum, in the solid fermentation material, by weight percentage cavings be 26%, wheat bran 2%, ground phosphate rock 8%, turfy soil 48%, sesame oil cake 8% and W-Gum 8%, adding the water yield is 40% of described solid fermentation weight of material, and pH transfers to 8.5.

Ground phosphate rock, turfy soil, sesame oil cake material all need to pulverize, and reach the 80-100 order.

Allow sterilization 0.8-2 hour under 121 ℃, 0.1 MPa pressure of above-mentioned solid fermentation material, inoculate the fermentor tank seed liquor behind the cool to room temperature; Fermentor tank seed liquor inoculum size counts 12% by the solid fermentation material, fully stir the inoculation back, make its material inoculation evenly, cultivate at 28 ℃, note ventilating, ventilation is expressed as 1: 0.8 with volume/volume, fermentor tank pressure is 0.05 MPa, thalli growth situation in the material is observed in sampling in per 8 hours, fermented 80 hours, stop fermentation, oven dry again, control water-content to reach＜10%, add then in solid fermentation weight of material 4% fused(calcium magnesium)phosphate, 4% vitriolate of tartar and 0.1% trace compound mixture, this trace compound mixture is by zinc sulfate, copper sulfate, ammonium molybdate and manganous sulfate be according to weight ratio 1: 1: 0.5: 0.8 mixes and obtains, and just obtains described product after fully mixed.Described solid-state fermentation tank is that commodity are called multi-functional full-automatic solid-state fermentation tank by Beijing Jiulongsheng micro-organisms source development Co., Ltd's development.

The fertilizer that this embodiment makes has carried out the Radix Dauci Sativae field test, uses commercially available bacterial micro-organism fertilizer to carry out simultaneous test simultaneously.This test is to adopt common test method to carry out.Its test-results is listed in table 7.

The effect of increasing production of table 7 on Different Crop

Crop varieties	Handle	Cell production, kilogram					The comparison photograph+,-
		Cell production, kilogram					The comparison photograph+,-		I	II	III	On average	The folding per mu yield	Kg/ mu	％
		Green pepper	Microbial streptomycete fertilizer of the present invention	28	23.3	16.3	22.53	2253	I	II	III	On average	The folding per mu yield	Kg/ mu	％	+420	+22.9 1
Bacterial micro-organism fertilizer (CK)	24		Microbial streptomycete fertilizer of the present invention	28	23.3	16.3	22.53	2253	17	14	18.33	1833				+420	+22.9 1
Bacterial micro-organism fertilizer (CK)	24		Eggplant	Microbial streptomycete fertilizer of the present invention	36.7	33.4	43.4	37.83	17	14	18.33	1833			3783	+556	+17.2 3
Bacterial micro-organism fertilizer (CK)	33.4	26.7		Microbial streptomycete fertilizer of the present invention	36.7	33.4	43.4	37.83	36.7	32.27	3227				3783	+556	+17.2 3
Bacterial micro-organism fertilizer (CK)	33.4	26.7		Kidney bean	Microbial streptomycete fertilizer of the present invention	26	21	22	36.7	32.27	3227			23	2300	+333	+16.9 6
Bacterial micro-organism fertilizer (CK)	23	17	19		Microbial streptomycete fertilizer of the present invention	26	21	22	19.67	1967				23	2300	+333	+16.9 6
Bacterial micro-organism fertilizer (CK)	23	17	19		Broccoli	Microbial streptomycete fertilizer of the present invention	34	31	19.67	1967			31	32	3200	+440	+15.9 0
Bacterial micro-organism fertilizer (CK)	28	26	29	27.6		Microbial streptomycete fertilizer of the present invention	34	31	2760				31	32	3200	+440	+15.9 0
Bacterial micro-organism fertilizer (CK)	28	26	29	27.6		Radix Dauci Sativae	Microbial streptomycete fertilizer of the present invention	40	2760			34.4	32.8	35.73	3573	+960	+36.7 0
Bacterial micro-organism fertilizer (CK)	30.4	25.6	22.4	26.13	2613		Microbial streptomycete fertilizer of the present invention	40				34.4	32.8	35.73	3573	+960	+36.7 0

Above-mentioned table 4 result clearly illustrates that microbial streptomycete fertilizer effect of increasing production of the present invention is higher than the effect of increasing production of bacterial micro-organism fertilizer in the prior art very significantly.

Claims

1, a kind of manufacture method of strepto-bacterial manure is characterized in that this method undertaken by following step:

(1) slant strains medium preparation and spawn culture: get the cleaned potato 200-400 weight part of peeling, add entry, its potato and water weight ratio are 0.8-1: 1-1.2, boiled 20-30 minute, after filtering, filtrate adds glucose 10-30 weight part, agar 15-30 weight part, add water total amount is added to the 1000-1200 weight part, heating is melted fully until agar, makes described substratum like this;

(2) shake a preparation of bottle bacterium culture medium and a spawn culture: with glucose or sucrose, Semen Maydis powder, bean cake powder, MgSO ₄, K ₂HPO ₄, (NH ₄) ₂SO ₄, CaCO ₃Shake a bottle bacterium culture medium, the pH7.5-8.0 of this substratum with water-soluble being prepared into of NaCl;

Above-mentioned substratum branch is installed in the triangular flask, and sterilization is 0.5-1.0 hour under 121 ℃, 0.1 MPa pressure, and the 30-35 ℃ of above-mentioned slant culture bacterial classification of inoculation reduced in cooling then, on 180 rev/mins of rotary shaking tables, shakes training 18-24 hour in 28-32 ℃;

(3) liquid fermentation tank seed culture medium and enlarged culturing: its substratum is with to shake bottle bacterium culture medium identical, sterilization 0.5-1.0 hour under 121 ℃, 0.1 MPa pressure of this substratum, insert above-mentioned shaking behind the cool to room temperature and cultivate son, fermentor tank pressure is the 0.02-0.08 MPa, and ventilation is expressed as 1 with volume/volume: 0.5-0.7;

(4) solid fermentation: the solid fermentation material is selected from one or more following materials: (a) one or more in cavings and/or crop stalk, (b) wheat bran, (c) ground phosphate rock, (d) turfy soil, vegetable mould, pond sludge and/or the loam, (e) Semen Maydis powder or starch and (f) plant oil cake, transfer sterilization 0.5-1.0 hour under 121 ℃, 0.1 MPa pressure of its material of pH relief, inoculate the fermentor tank seed liquor behind the cool to room temperature; Cultivate at 28-32 ℃, ventilation is expressed as 1 with volume/volume: 0.3-1, fermentor tank pressure are the 0.02-0.08 MPa, ferment 72-100 hour, the fermentation back adds phosphate fertilizer, potash fertilizer and/or micronutrient fertiliser, obtains described product 35-40 ℃ of oven dry again.

2, the manufacture method of strepto-bacterial manure according to claim 1 is characterized in that shaking in bottle bacterium culture medium glucose or sucrose, Semen Maydis powder, bean cake powder, MgSO described ₄, K ₂HPO ₄, (NH ₄) ₂SO ₄, CaCO ₃With the NaCl weight percentage be respectively 0.5-1.0%, 0.5-2%, 1.5-3%, 0.02-0.3%, 0.02-0.3%, 0.1-0.3%, 0.3-0.5% and 0.1-0.5%.

3, the manufacture method of strepto-bacterial manure according to claim 1, it is characterized in that in the solid fermentation material, one or more 40-50%, plant oil cake 4-8% in cavings and/or crop stalk 20-30%, wheat bran 1-3%, ground phosphate rock 5-10%, turfy soil, vegetable mould, pond sludge and/or the loam and Semen Maydis powder or starch 5-10%, solid fermentation substratum in this ratio preparation adds water 35-50%, pH6.5-8.5.

4, the manufacture method of strepto-bacterial manure according to claim 1 is characterized in that described plant oil cake is soyabean cake, sesame-send cake, cottonseed cake, rapeseed cake and/or peanut cake; Described starch is W-Gum, rice starch, wheat starch, barley starch, oat starch, soybean starch and/or potato starch; Described crop stalk is Chinese sorghum, corn, wheat, barley, oat, cotton, soybean, paddy rice, broad bean, pea, rape and/or sesame straw.

5, the manufacture method of strepto-bacterial manure according to claim 1 is characterized in that described phosphate fertilizer is potassium primary phosphate, normal superphosphate, double superhosphate, fused(calcium magnesium)phosphate, monocalcium phosphate and/or bone meal.

6, the manufacture method of strepto-bacterial manure according to claim 1 is characterized in that described potash fertilizer is Repone K, vitriolate of tartar and/or cement kiln ash potassium fertilizer.

7, the manufacture method of strepto-bacterial manure according to claim 1 is characterized in that described micronutrient fertiliser is boron fertilizer, zinc fertilizer, molydbenum fertilizer, manganese fertilizer, copper fertilizer and/or iron fertilizer.

8, the manufacture method of strepto-bacterial manure according to claim 7 is characterized in that described boron fertilizer is boric acid and/or borax; Described zinc fertilizer is Zinc Sulphate Heptahydrate, zinc chloride and/or zinc oxide; Described molydbenum fertilizer is ammonium molybdate, Sodium orthomolybdate and/or molybdic oxide; Described manganese fertilizer is manganous sulfate, manganese oxide, Manganous chloride tetrahydrate and/or manganous carbonate; Described copper fertilizer is cupric sulfate pentahydrate, cupric chloride, cupric oxide and/or copper mine slag; Described iron fertilizer is iron vitriol and/or iron protocarbonate.

9, the manufacture method of strepto-bacterial manure according to claim 1, the add-on that it is characterized in that described fermentor tank seed liquor are the 5-15% of solid fermentation weight of material.

10, the manufacture method of strepto-bacterial manure according to claim 1 is characterized in that described solid fermentation carries out in multi-functional full-automatic solid-state fermentation tank, this solid-state fermentation tank integrates spice, sterilization, inoculation, fermentation and dries.

11, the strepto-bacterial manure of making according to the described method of arbitrary claim among the claim 1-10 is characterized in that this strepto-bacterial manure contains the above spore chain mould that lives of 2,000,000,000/gram.