Summary of the invention
The objective of the invention is to the problems referred to above; Provide a kind of have diseases prevention, short give birth to and the rhizosphere probiotic strain-brown streptomycete of wine laterite of multiple functions such as detoxifcation as microbial inoculum effective constituent, and provide under the cover simulation soil natural envrionment conditions and be suitable for the brown streptomycete growth of wine laterite zymotechnique.
The technical scheme that the present invention provides is: the brown drappus microbial inoculum of this wine laterite is to be that the viable bacteria culture of the brown streptomycete viable bacteria of wine laterite bacterial classification through the preparation of liquid submerged fermentation or solid fermentation of CGMCCNo.2664 is that effective ingredient is formed with the deposit number.The brown streptomycete of streptomyces wine laterite (Streptomycesvinaceusdrappus), tentative S506 by name has submitted Chinese common micro-organisms preservation center preservation on September 11st, 2008, and the tomato rhizosphere soil separates acquisition to this bacterial classification from the Luancheng County, Hebei province.
The brown streptomyces species determined dna sequence of wine laterite result is in the brown drappus microbial inoculum of described wine laterite:
GGCGTGCTTAACACATGCAAGTCGAACGATGAACCACTTCGGTGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGATCCTCGCAGGCATCTGCGAGGTTCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACC?GGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAACCCTGGAGACAGGGTCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCTTGTGGTGCTGGGACTCACGGGAGACCGCCGGGTCACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGGAGCTGTCG。
The brown streptomycete viable bacteria of described wine laterite culture can also add peat composed of rotten mosses powder as carrier or auxiliary material.
The preparation method of the brown drappus microbial inoculum of described wine laterite is characterized in that step comprises:
A, actication of culture: on the brown streptomycete inoculation of wine laterite Zhi Gaoshi synthetic medium flat board, 28-35 ℃ of cultivation transferred 2-3 time repeatedly, cultivates for 1 week, makes actication of culture;
The liquid seeds preparation of B, the brown streptomycete of wine laterite
The seed culture material is that wheat bran or bran powder, 2-4% Semen Maydis powder, 0.5-2% soybean cake powder, 0.5-2% glycerine, 0.1% SODIUMNITRATE, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water of 2-8% is formed by mass ratio; Nature pH; Be sub-packed in triangular flask 121-130 ℃ autoclaving 30-60 minute; Be cooled to 25-35 ℃; The brown streptomyces species of wine laterite that inoculation step A obtains, under 25-30 ℃, shake-flask culture 48-72 hour;
The liquid submerged fermentation of C, the brown streptomycete of wine laterite
Culture with step B is a seed; Ratio with 3-10% is inoculated in the liquid culture material of liquid fermentation tank; The liquid culture material is identical with step B with culture condition, and aeration-agitation was cultivated 72 hours, and the peat composed of rotten mosses powder absorption that adds two volumes makes drappus microbial inoculum.
The preparation method of the brown drappus microbial inoculum of described wine laterite, it is that 2% bran powder, 2% Semen Maydis powder, 1% soybean cake powder, 0.5% glycerine, 0.1% SODIUMNITRATE, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio that the liquid seeds of the brown streptomycete of the described wine laterite of step B and step C is cultivated material.
The preparation method of the brown drappus microbial inoculum of described wine laterite, it is that 4% bran powder, 4% Semen Maydis powder, 2% soybean cake powder, 0.5% glycerine, 0.1% saltpetre, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio that the liquid seeds of the brown streptomycete of the described wine laterite of step B and step C is cultivated material.
The preparation method of the brown drappus microbial inoculum of described wine laterite, described step comprises:
A, actication of culture: on the brown streptomycete inoculation of wine laterite Zhi Gaoshi synthetic medium flat board, 28-35 cultivates, and transfers 2-3 time repeatedly, cultivates for 1 week, makes actication of culture;
The liquid seeds preparation of B, the brown streptomycete of wine laterite
The seed culture material is that wheat bran or bran powder, 2-4% Semen Maydis powder, 0.5-2% soybean cake powder, 0.5-2% glycerine, 0.1% SODIUMNITRATE, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water of 2-8% is formed by mass ratio; Nature pH; Be sub-packed in triangular flask 121-130 ℃ autoclaving 30-60 minute; Be cooled to 25-35 ℃; The brown streptomyces species of wine laterite that inoculation step A obtains, under 25-30 ℃, shake-flask culture 48-72 hour;
The solid fermentation of C, the brown streptomycete of wine laterite
The solid fermentation material is wheat bran or the rice bran of 35-50% by mass ratio, medical stone powder, shale powder or the zeolite powder of 5-10%, the soybean cake powder of 1-2%; 0.05% saltpetre, 0.15% potassium hydrogenphosphate, 0.15% sodium-chlor, 0.4% lime carbonate and remaining water are formed, and 121-130 ℃ of autoclaving is cooled to 25-35 ℃; Insert the liquid spawn of step B in the ratio of 3-10%, stir, be sub-packed in aseptic koji tray; Under 30 ± 5 ℃ of conditions; Be interrupted aerobic culture 72 hours, 35-50 ℃ of low temperature air-dry to material moisture below 12%, make drappus microbial inoculum.
The preparation method of the brown drappus microbial inoculum of described wine laterite; The solid fermentation material of the brown streptomycete of the described wine laterite of step C is 35% wheat bran, 10% medical stone powder, 1% soybean cake powder by mass ratio, and 0.1% saltpetre, 0.15% potassium hydrogenphosphate, 0.15% sodium-chlor, 0.4% lime carbonate and remaining water are formed.
The preparation method of the brown drappus microbial inoculum of described wine laterite; The solid fermentation material of the brown streptomycete of the described wine laterite of step C is 50% wheat bran, 5% shale powder, 2% soybean cake powder by mass ratio, and 0.1% saltpetre, 0.15% potassium hydrogenphosphate, 0.15% sodium-chlor, 0.4% lime carbonate and remaining water are formed.
The preparation method of the brown drappus microbial inoculum of described wine laterite; The solid fermentation material of the brown streptomycete of the described wine laterite of step C is 50% wheat bran, 5% zeolite powder, 1% soybean cake powder by mass ratio, and 0.1% saltpetre, 0.15% potassium hydrogenphosphate, 0.15% sodium-chlor, 0.4% lime carbonate and remaining water are formed.
The application of the brown drappus microbial inoculum of wine laterite of the present invention comprises: its viable bacteria culture is used to prepare the soil organisms renovation agent or the amendment of control cultivation continuous cropping obstacle, disease prevention growth-promoting and function of detoxification.
The substantive distinguishing features that the present invention obtained is with significant technical progress: with northern facilities vegetable production status is background; On the basis of the present situation of further verifying continuous cropping obstacle, formation and the origin cause of formation; Having proposed to realize the research thinking of efficient stable control through the comprehensive regulation to the little ecology of rhizosphere and root system, is that effective constituent is prepared the brown drappus microbial inoculum of wine laterite with the brown streptomycete S506 of the rhizosphere probiotic strain wine laterite bacterial classification with multiple functions such as diseases prevention, short living and detoxifcations; A kind of " the liquid production of hybrid seeds; solid fermentation " novel process that is suitable for the brown streptomycete of wine laterite is provided, has realized that living bacteria count and spore production rate are high, living contaminants is few; The technical requirements that fermentation period is short is suitable for suitability for industrialized production.The viable bacteria body of this bacterium is applied the vegetables rhizosphere; Can effectively prevent and treat various vegetables root diseases such as blight, root rot, root knot nematode disease; And having significant promotion root system development and degraded root system from poisonous substance matter function, a continuous cropping obstacle difficult problem that generally takes place at effectively control vegetables, fruit tree, medicinal material etc. has broad prospect of application.
Embodiment
The bacterial classification that the brown drappus microbial inoculum of wine laterite of the present invention provides is: the brown streptomycete of streptomyces wine laterite (Streptomyces vinaceusdrappus); Tentative S506 by name; Submit Chinese common micro-organisms preservation center preservation on September 11st, 2008, deposit number is CGMCCNo.2664.The tomato rhizosphere soil separates acquisition to this bacterial classification from the Luancheng County, Hebei province.
The brown drappus microbial inoculum of wine laterite of the present invention (Streptomyces vinaceusdrappus) S506 mainly is to utilize its viable bacteria culture, produces soil organisms renovation agent or amendment with disease prevention growth-promoting and function of detoxification, is used for the control of continuous cropping obstacle.
But the preparation of the brown drappus microbial inoculum viable bacteria of wine laterite culture gets final product also solid fermentation of liquid submerged fermentation; The substratum material there is not particular requirement; The carbon source, nitrogenous source, phosphorus source and the inorganic salt that satisfy strain growth get final product; Wheat bran, rice bran, starch, all kinds of residual oil grouts and phosphoric acid salt, salt etc. all can be used as the cultivation material, and to the no particular requirement of pH value, culturing process is suitably ventilated or stirred.Higher viable bacteria density is to enhance productivity, and improves effect, reduces the key of production and use cost.Only under the simulated soil natural environmental condition, this bacterial strain just can be bred by normal growth, and produces a large amount of conidiums, and therefore, the brown streptomycete S506 of preparation wine laterite microbial inoculum adopts solid fermentation process more promising than liquid fermentation process.
The brown streptomycete S506 of embodiment 1 wine laterite liquid fermentation agent
A, actication of culture: on the brown streptomycete inoculation of wine laterite Zhi Gaoshi synthetic medium flat board, 28-35 ℃ of cultivation transferred 2-3 time repeatedly, cultivates for 1 week, makes actication of culture; Take out the growth conditions of observing thalline, with electron microscope scanning, mycelia and conidium photo are as shown in Figure 1.Visible by Fig. 1, the bacterium colony of the brown streptomycete S506 of wine laterite of the present invention is rounded, and substrate mycelium does not have tabula, does not rupture; Aerial hyphae is luxuriant, multi-branched; The fibrillae of spores volution, spore oval, smooth surface.
The liquid seeds preparation of B, the brown streptomycete of wine laterite
The seed culture material is that 8% wheat bran or bran powder, 4% Semen Maydis powder, 2% soybean cake powder, 2% glycerine, 0.1% SODIUMNITRATE, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio; Nature pH; Be sub-packed in triangular flask 121-130 ℃ autoclaving 30-60 minute, be cooled to 25-35 ℃, the brown streptomyces species of wine laterite that inoculation step A obtains; Under 25-30 ℃, shake-flask culture 48-72 hour;
The liquid submerged fermentation of C, the brown streptomycete of wine laterite
Culture with step B is a seed; Ratio with 3-10% is inoculated in the liquid culture material of liquid fermentation tank; The liquid culture material is identical with step B with culture condition, and aeration-agitation was cultivated 72 hours, and the peat composed of rotten mosses powder absorption that adds two volumes makes drappus microbial inoculum.
The brown streptomycete S506 of embodiment 2 wine laterite liquid fermentation agent
A, steps A are with embodiment 1;
The seed culture material of B, C, the brown streptomycete S506 of wine laterite is that 2% bran powder, 2% Semen Maydis powder, 1% soybean cake powder, 0.5% glycerine, 0.1% SODIUMNITRATE, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio, and fermentation and preparation condition are with embodiment 1.The liquid fermentation agent of the brown streptomycete S506 of embodiment 3 wine laterite
A, steps A are with embodiment 1;
It is that 4% bran powder, 4% Semen Maydis powder, 2% soybean cake powder, 0.5% glycerine, 0.1% saltpetre, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio that the liquid seeds of B, C, the brown streptomycete of wine laterite is cultivated material.
The seed culture material is that 4% bran powder, 4% Semen Maydis powder, 2% soybean cake powder, 0.5% glycerine, 0.1% saltpetre, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio, and fermentation and preparation condition are with embodiment 1.
The solid fermentation microbial inoculum of the brown streptomycete S506 of embodiment 4 wine laterite
A, steps A are with embodiment 1;
The liquid seeds preparation of B, the brown streptomycete of wine laterite
The seed culture material is that 8% wheat bran or bran powder, 2% Semen Maydis powder, 0.5% soybean cake powder, 0.5% glycerine, 0.1% SODIUMNITRATE, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio; Nature pH; Be sub-packed in triangular flask 121-130 ℃ autoclaving 30-60 minute; Be cooled to 25-35 ℃; The brown streptomyces species of wine laterite that inoculation step A obtains, under 25-30 ℃, shake-flask culture 48-72 hour;
The solid fermentation of C, the brown streptomycete of wine laterite
The solid fermentation material is 45% wheat bran or rice bran by mass ratio, 8% medical stone powder, 1.5% soybean cake powder; 0.05% saltpetre, 0.15% potassium hydrogenphosphate, 0.15% sodium-chlor, 0.4% lime carbonate and remaining water are formed, and 121-130 ℃ of autoclaving is cooled to 25-35 ℃; Insert the liquid spawn of step B in the ratio of 3-10%, stir, be sub-packed in aseptic koji tray; Under 30 ± 5 ℃ of conditions; Be interrupted aerobic culture 72 hours, 35-50 ℃ of low temperature air-dry to material moisture below 12%, make drappus microbial inoculum: viable bacteria content 2 * 10
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The solid fermentation microbial inoculum of the brown streptomycete S506 of embodiment 5 wine laterite
A, steps A are with embodiment 4;
B, liquid seed culture medium are that 4% bran powder, 4% Semen Maydis powder, 2% soybean cake powder, 0.5% glycerine, 0.1% SODIUMNITRATE, 0.4% sodium-chlor, 0.3% lime carbonate, 0.3% potassium hydrogenphosphate, 0.05% sal epsom, 0.05% ferrous sulfate and remaining water are formed by mass ratio, and fermentation condition is with embodiment 4;
The solid fermentation material of C, the brown streptomycete of wine laterite is 35% wheat bran, 10% shale powder, 1% soybean cake powder by mass ratio; 0.1% saltpetre, 0.15% potassium hydrogenphosphate, 0.15% sodium-chlor, 0.4% lime carbonate and remaining water are formed, and fermentation and preparation condition are with embodiment 4.
The solid fermentation microbial inoculum of the brown streptomycete S506 of embodiment 6 wine laterite
A, B, with embodiment 4;
The solid fermentation material of C, the brown streptomycete of wine laterite is 50% wheat bran, 5% zeolite powder, 1% soybean cake powder by mass ratio; 0.1% saltpetre, 0.15% potassium hydrogenphosphate, 0.15% sodium-chlor, 0.4% lime carbonate and remaining water are formed, and fermentation and preparation condition are with embodiment 4.
The brown drappus microbial inoculum applying examples 1 of wine laterite (using) at nursery stage
In vegetable seedling substrate, add the brown drappus microbial inoculum of wine laterite by 1% mass ratio, after mixing, spread seed and grow seedlings.
The brown drappus microbial inoculum applying examples 2 of wine laterite (using) at setting date
When vegetable permanent planting, the brown drappus microbial inoculum of wine laterite is mixed with 5 times thin field soil or the fertilizer that fully becomes thoroughly decomposed, stir, then mixture is evenly executed in people's planting pit.Final singling afterwards, ridging, same routine operation waters.
More than use and to rhizosphere microflora, root system is shown that from effect test-results such as the releasing of poisonous substance matter and field application the brown streptomycete S506 of wine laterite has following comprehensive function:
(1) significantly promotes root system development, improve the anti-adversity of plant, promoted the effective utilization of plant, for the good quality and high output of the middle and later periods of growing is laid a good foundation to soil nutrient to non-irrigated, saline and alkaline, soil-borne disease etc.;
(2) significantly improve rhizosphere viable bacteria total amount, and improve the rhizosphere microflora structure, bacterium, actinomycetes quantity are improved, filamentous fungus, pathogenic bacteria quantity descend;
(3) effectively control pathogens such as sickle-like bacteria, rhizoctonia, root knot nematode to the infecting of root system, preventive effect and common chemical sterilant pentachloro-nitro this quite, the control of nematode has been surpassed Avrmectin;
(4) degraded phenolic acids root system is significantly alleviated root system secretion and first crop root system and is rotted to discharge the injury of toxin to root system from poisonous substance matter.
Use in continuous cropping vegetables old liberated area for many years, can significantly improve the quality of breeding seedling, disease is few, and well developed root system shortens the transplanting seedling time after transplanting, and improves the later stage resistance against diseases.In the growth middle and later periods, can effectively control the generation of root disease and overground part disease, reduce medication 40~50%, comprehensive output increases more than 25%.In recent years, on the various vegetables on ground such as Hebei, Shandong, Henan, Fujian, Shanghai and fruit tree, used, all shown same disease-resistant, raising the output and quality improving effect, all surpassed similar technology both domestic and external and product.Testing data is seen " application test report " in detail.
Above-mentioned description about brown drappus microbial inoculum of wine laterite and application only proposes as several kinds of technical schemes of the present invention, and conduct is not to its single restricted condition.