CN104620817B - A kind of Semen avenae nudae Biological control disease-resistant growth-promoting method - Google Patents
A kind of Semen avenae nudae Biological control disease-resistant growth-promoting method Download PDFInfo
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- CN104620817B CN104620817B CN201510058085.3A CN201510058085A CN104620817B CN 104620817 B CN104620817 B CN 104620817B CN 201510058085 A CN201510058085 A CN 201510058085A CN 104620817 B CN104620817 B CN 104620817B
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- 239000002361 compost Substances 0.000 claims abstract description 45
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- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 27
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 27
- 239000000725 suspension Substances 0.000 claims abstract description 27
- 239000003337 fertilizer Substances 0.000 claims abstract description 20
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- 238000001816 cooling Methods 0.000 claims description 5
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- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 abstract description 3
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 240000005979 Hordeum vulgare Species 0.000 description 4
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- 239000008103 glucose Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 235000017060 Arachis glabrata Nutrition 0.000 description 3
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- 238000003756 stirring Methods 0.000 description 3
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- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000209219 Hordeum Species 0.000 description 1
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- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000013040 bath agent Substances 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- General Health & Medical Sciences (AREA)
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- Biotechnology (AREA)
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Abstract
The invention discloses a kind of Semen avenae nudae Biological control disease-resistant growth-promoting method, including: bacillus subtilis is made density 109～1010The high density bacteria suspension of cfu/mL；Semen avenae nudae health straw is cut into segment and carries out compost, composting process sprays bacillus subtilis high density bacteria suspension, fermented after obtain the bio-fertilizer that can effectively suppress to kill Semen avenae nudae pathology fungus, this bio-fertilizer is used in the disease-resistant growth-promoting of Semen avenae nudae Biological control.In the present invention, the bacillus subtilis producing beta glucanase is the plants probiotics that Semen avenae nudae rises disease-resistant growth-promoting functions, is added in compost, significantly strengthens the Biological control effect to Semen avenae nudae disease fungus, display has suppression killing action to sheath maize ear rot pathogen, and improves Semen avenae nudae yield.Composting process processes simplicity, non-wastewater discharge, and small investment, energy consumption are low, easily operate, it is easy to industrialization large-scale production, has good economic benefit and wide application prospect.
Description
Technical field
The present invention relates to the preparation field that Biological control is fertile, be specifically related to a kind of disease-resistant rush of Semen avenae nudae Biological control
Generation method.
Background technology
Semen avenae nudae (Hordeum vulgare Linn.var.nudum Hook.f.) is the one of grass family Hordeum
Cereal crop, because in it, coetonium shell separates, seed is exposed, therefore also known as hull-less barley, Semen avenae nudae, meter great Mai,
Mainly originate from the ground such as Tibet, China, Qinghai, Sichuan, Yunnan, be Tibetan's main food.Semen avenae nudae is in Qinghai-Tibet
On plateau, plantation there are about history in 3500, extends to spirit culture field, green grass or young crops among material and culture
The Semen avenae nudae culture hide and define rich connotation on plateau, being rich in national characters.Semen avenae nudae has the most medicinal and battalion
Support and be worth, released the Semen avenae nudae products such as highland barley fine dried noodles, Semen avenae nudae steamed bread, barley nutritional powder.Semen avenae nudae is Tibet
The primary raw material of Zanba first of four treasured.
Sheath maize ear rot is Semen avenae nudae Major Diseases, and Phoma macdonaldii is one of pathogen.Plant pathology is true
Bacterium is primarily present in soil, infection plant in plant growing, causes plant disease, causes production to damage
Lose.Therefore, fungal diseases of plants to be eliminated from Biological control angle, kill or suppress the pathology in soil
Fungus is most important, and using the bio-fertilizer with Biological control effect is the effective way realizing this purpose
Footpath.Bio-fertilizer development trend should be to greatly develop the bacillus cereus that resistance is high, can survive steadily in the long term.
Particularly bacillus cereus can produce multiple antifungal substance, and phytopathogen is significantly inhibited effect,
And to person poultry harmless, do not pollute the environment yet, will play an important role in preventing and treating fungal diseases of plants.
In straight or the most straight shaft-like in bacillus cereus form, aerobic or amphimicrobian, Gram-positive,
Resistance endospore can be produced under certain environmental conditions.Bacillus cereus is extensively grown in plant surely, quilt
It is considered generally acknowledged grade-safe microorganism (generally regarded as safety, GRAS).Hay
Bud pole bacterium is a kind of typical bacillus cereus, produce antibacterial protein, polypeptides matter, lipid material and
The antibiotic substance such as beta glucanase, the hydrolase plant pathology anti-with it such as particularly beta glucanase
Fungus ability has substantial connection, because the composition of fungal cell wall is glucosan.
Summary of the invention
The invention provides the measure of biotic control of the disease-resistant growth-promoting of a kind of Semen avenae nudae, by beta glucanase will be produced
Bacillus subtilis (CICC 10339) incorporate in Semen avenae nudae natural flora, be made to by biology prevent
Approach of controlling effectively suppresses to kill the bio-fertilizer of Semen avenae nudae pathology fungus.
A kind of Semen avenae nudae Biological control disease-resistant growth-promoting method, comprises the following steps:
1) bacillus subtilis (CICC 10339) is cultivated, make density 109～1010cfu/mL
High density bacteria suspension；
2) Semen avenae nudae health straw is cut into segment and carries out compost, composting process sprays step 1) in height
Density bacteria suspension, compost ferments after terminating, fermented after obtain effectively suppressing to kill Semen avenae nudae pathology
The bio-fertilizer of fungus, is used in this bio-fertilizer in the disease-resistant growth-promoting of Semen avenae nudae Biological control.
In the present invention, Semen avenae nudae health straw is cut into trifle compost, makes the flora in prepared bio-fertilizer i.e.
For healthy Semen avenae nudae plant naturally flora, this flora grows with Semen avenae nudae plant coevolution, defines mutually
Benefit syntaxial system, has certain Biological control effect to disease fungus.Further, beta can be produced
The bacillus subtilis (CICC 10339) of glucanase adds in compost, significantly strengthens Semen avenae nudae disease
The Biological control effect of fungus.Bacillus subtilis (CICC 10339) due to produce beta glucanase,
Semen avenae nudae plant fiber there is is good decomposition, necessary for composting fermentation process, therefore can fully incorporate
Breed stably and lastingly in compost flora.As lactic acid bacteria is killed for the prebiotic effect of animal, suppression
The microorganism of plant pathology fungus can be referred to as plants probiotics.In bio-fertilizer prepared by the present invention, withered
Grass bacillus cereus (CICC 10339) is i.e. Semen avenae nudae probiotic bacteria, has Semen avenae nudae pathology fungus notable biological anti-
Control effect.
Step 1) in, bacillus subtilis (CICC 10339) uses prior art, from China's work
Industry Microbiological Culture Collection administrative center (CICC), address: Road, Jiuxianqiao, Chaoyang District, Beijing City 24
Number No. 6 building of institute；Preservation date is 2007, and strain keeps numbered 10339.Bacillus subtilis
(CICC 10339) can buy to Chinese industrial Microbiological Culture Collection administrative center.
Bacillus subtilis (CICC 10339) is cultivated, including: bacillus subtilis (CICC
10339) density of setting out is 106～107Individual/mL, being seeded to inoculum concentration in fluid medium is 1%～5%,
33 DEG C～39 DEG C of shaken cultivation 20～28h, be 10 to bacterium solution density9～1010The high density bacteria suspension of cfu/mL.
Further preferably, bacillus subtilis (CICC 10339) is cultivated, including: hay bud
The density of setting out of spore bacillus (CICC 10339) is 106～107Individual/mL, is seeded in fluid medium
Inoculum concentration is 2%～3%, and 36 DEG C～37 DEG C of shaken cultivation 22～26h, be 10 to bacterium solution density9～1010cfu/mL
High density bacteria suspension.
Described fluid medium, in terms of 1L, including following components:
Further preferably, described fluid medium, in terms of 1L, including following components:
Fluid medium is at 121 DEG C of sterilizing 20min.
Step 2) in, composting process sprays step 1) in high density bacteria suspension, including: at heap
When fertile internal temperature reaches 30 DEG C～34 DEG C, spray step 1) in high density bacteria suspension, continue compost,
When compost internal temperature reaches 41 DEG C～45 DEG C, compost terminates.
Composting process sprays step 1) in high density bacteria suspension, including: reach at compost internal temperature
During to 32 DEG C, spray step 1) in high density bacteria suspension, continue compost, treat that compost internal temperature reaches
Terminate to compost when 43 DEG C.
Step 2) in, after compost terminates, compost is stirred spreading for cooling.Described fermentation uses naturalness
Bottom fermentation.
Compared with prior art, present invention have the advantage that
In the present invention, Semen avenae nudae health straw is cut into trifle compost, makes the flora in prepared bio-fertilizer i.e.
For healthy Semen avenae nudae plant naturally flora, this flora grows with Semen avenae nudae plant coevolution, defines mutually
Benefit syntaxial system, has certain Biological control effect to disease fungus.
Bacillus subtilis (CICC 10339) produces beta glucanase, strong destruction pathology fungal cell
Wall, significantly inhibits killing action to pathology fungus.As lactic acid bacteria is for the prebiotic effect of animal, press down
System kills the microorganism of plant pathology fungus can be referred to as plants probiotics.Bio-fertilizer prepared by the present invention
In rich in bacillus subtilis (CICC 10339) be i.e. Semen avenae nudae probiotic bacteria, Semen avenae nudae pathology fungus is had
Notable Biological control effect.
Composting process is carried out in its natural state, and the Semen avenae nudae plant natural flora relying on Tiny ecosystem stable is carried out
Fermentation, and bacillus subtilis (CICC 10339) is owing to producing beta glucanase, fine to Semen avenae nudae plant
Dimension has good decomposition, necessary for composting fermentation process, therefore, and bacillus subtilis (CICC
10339) can fully be dissolved in compost flora and breed stably and lastingly.Composting process processes simplicity,
Non-wastewater discharge, small investment, energy consumption are low, easily operate, it is easy to industrialization large-scale production, have good
Economic benefit and wide application prospect.
Detailed description of the invention
Embodiment 1
1) High Density Cultivation of the bacillus subtilis (CICC 10339) of beta glucanase is produced, withered
The density of setting out of grass bacillus cereus (CICC 10339) is 1.22 × 106Individual/mL, is seeded to liquid culture
Base (glucose 14g/L, Carnis Bovis seu Bubali cream 25g/L, potassium dihydrogen phosphate 2.45g/L, peanut cake powder 10g/L,
PH=7.0,121 DEG C of sterilizing 20min) in, inoculum concentration is 2%, and 37 DEG C of shaken cultivation 24h, bacterium solution is close
Degree is 2.46 × 109Cfu/mL, obtains high density bacteria suspension.
2) Semen avenae nudae health straw 100kg altogether is cut into trifle and carries out Natural compost, at compost internal temperature
Reach to spray 1 when 32 DEG C) in the high density bacteria suspension 2L for preparing, continue compost, in treating compost
When portion's temperature is more than 43 DEG C, compost terminates, and stirs spreading for cooling, after fermentation under naturalness, becomes rich in product
The bio-fertilizer of the bacillus subtilis of beta glucanase, can effectively suppress to kill Semen avenae nudae pathology fungus
Bio-fertilizer.
3) step 1) in the high density bacteria suspension for preparing and step 2) in the bio-fertilizer for preparing
Significantly there is Beta glucanase vigor, Semen avenae nudae pathology fungus is had and significantly inhibits killing action, tool
Body see table 1.
During sheath maize ear rot pathogen Phoma macdonaldii manages purchased from Chinese industrial Microbiological Culture Collection
The heart (CICC), bacterium numbering is 2613.
Beta glucanase vitality test: using reducing sugar test method, Beta glucanase can be by Beta
Glucosan is degraded into oligosaccharide and monosaccharide, and the reduction group that it has can try with DNS under the conditions of boiling water bath
Agent generation chromogenic reaction, colour developing the depth be directly proportional to reducing sugar growing amount, and reducing sugar growing amount with instead
Answer liquid Beta glucanase vigor to be directly proportional, thus utilize the light absorption value of colorimetric determination reactant liquor to calculate reduction
Sugar growing amount, obtains Beta glucanase vigor.Beta glucanase unit of activity defines: measuring bar
Under part, hydrolysis Beta glucosan per minute produces the enzyme needed for 1 μm ol reducing sugar (with glucose meter)
Amount, is defined as enzyme unit (IU) alive.
Flat board suppresses antibacterial broadband: using flat board nebulization, test strains point is connected on NA flat board, often puts down
Plate 2 strain, each 3 repetitions, cultivate 24h, the filter paper being moistened with chloroform is put into inverted cultivation for 28 DEG C
Bottom ware, stifling 20min, is repeated once, kills bacterium to be measured；Pathogenic fungi is made into 108Bacteria suspension,
It is sprayed on rapidly with aseptic laryngeal spray on the flat board being connected to bacterium to be measured (0.1ml/ ware), 28 DEG C of continuation
After cultivating 48-60h, record forms inhibition zone size.
Flat board antagonism: pour NA culture medium into flat board, becomes triangle to place Oxford in culture medium after solidification
Cup, adds fermentation liquid 100 μ L in the cup of Oxford, and 28 DEG C of incubators are cultivated, record after pathogenic fungi grows
Inhibition zone size.
Table 1
Note: antibacterial broadband refers to the difference of antibacterial radius and colony radius to be measured ,-: indicate without antibacterial band；+: represent
Antibacterial broadband < 5mm；++ represent antibacterial broadband > 5mm.
Embodiment 2
1) High Density Cultivation of the bacillus subtilis (CICC 10339) of beta glucanase is produced, withered
The density of setting out of grass bacillus cereus (CICC 10339) is 1.80 × 106Individual/mL, is seeded to liquid culture
Base (glucose 14g/L, Carnis Bovis seu Bubali cream 25g/L, potassium dihydrogen phosphate 2.45g/L, peanut cake powder 10g/L,
PH=7.0,121 DEG C of sterilizing 20min) in, inoculum concentration is 3%, and 37 DEG C of shaken cultivation 24h, bacterium solution is close
Degree is 3.34 × 109Cfu/mL, obtains high density bacteria suspension.
2) Semen avenae nudae health straw 100kg altogether is cut into trifle and carries out Natural compost, at compost internal temperature
Reach to spray 1 when 32 DEG C) in the high density bacteria suspension 3L for preparing, continue compost, treat inside compost
When temperature is more than 43 DEG C, compost terminates, and stirs spreading for cooling, after fermentation under naturalness, becomes rich in producing beta
The bio-fertilizer of the bacillus subtilis of glucanase.
3) step 1) in the high density bacteria suspension for preparing and step 2) in the bio-fertilizer for preparing
Significantly there is Beta glucanase vigor, Semen avenae nudae pathology fungus is had and significantly inhibits killing action, tool
Body see table 2.
Table 2
Note: antibacterial broadband refers to the difference of antibacterial radius and colony radius to be measured ,-: indicate without antibacterial band；+: represent
Antibacterial broadband < 5mm；++ represent antibacterial broadband > 5mm.
Embodiment 3
1) High Density Cultivation of the bacillus subtilis (CICC 10339) of beta glucanase is produced, withered
The density of setting out of grass bacillus cereus (CICC 10339) is 1.68 × 106Individual/mL, is seeded to liquid culture
Base (glucose 14g/L, Carnis Bovis seu Bubali cream 25g/L, potassium dihydrogen phosphate 2.45g/L, peanut cake powder 10g/L,
PH=7.0,121 DEG C of sterilizing 20min) in, inoculum concentration is 3%, and 37 DEG C of shaken cultivation 24h, bacterium solution is close
Degree is 2.33 × 109Cfu/mL, obtains high density bacteria suspension.
2) Semen avenae nudae health straw 100kg altogether is cut into trifle and carries out Natural compost, at compost internal temperature
Reach to spray 1 when 32 DEG C) in the high density bacteria suspension 3L for preparing, continue compost, treat inside compost
When temperature is more than 43 DEG C, compost terminates, and stirs spreading for cooling, after fermentation under naturalness, becomes rich in producing beta
The bio-fertilizer of the bacillus subtilis of glucanase.
3) step 2) in the bio-fertilizer for preparing Semen avenae nudae is had obvious growth-promoting prebiotic effect, improve single
Strain yield 8.2% (p < 0.05), is specifically shown in table 3 below and table 4.It is dark blue 320 for examination Semen avenae nudae kind.
Table 3
Table 4
Claims (9)
1. a Semen avenae nudae Biological control disease-resistant growth-promoting method, it is characterised in that comprise the following steps:
1) bacillus subtilis CICC 10339 is cultivated, make density 109～1010Cfu/mL's
High density bacteria suspension；
2) Semen avenae nudae health straw is cut into segment and carries out compost, composting process sprays step 1) in height
Density bacteria suspension, compost ferments after terminating, fermented after obtain effectively suppressing to kill Semen avenae nudae pathology
The bio-fertilizer of fungus, is used in this bio-fertilizer in the disease-resistant growth-promoting of Semen avenae nudae Biological control.
Semen avenae nudae Biological control the most according to claim 1 disease-resistant growth-promoting method, it is characterised in that
Step 1) in, bacillus subtilis CICC 10339 is cultivated, including: bacillus subtilis
The density of setting out of CICC 10339 is 106～107Individual/mL, being seeded to inoculum concentration in fluid medium is
1%～5%, 33 DEG C～39 DEG C of shaken cultivation 20～28h, be 10 to bacterium solution density9～1010Cfu/mL's is highly dense
Degree bacteria suspension.
Semen avenae nudae Biological control the most according to claim 2 disease-resistant growth-promoting method, it is characterised in that
Step 1) in, bacillus subtilis CICC 10339 is cultivated, including: bacillus subtilis
The density of setting out of CICC 10339 is 106～107Individual/mL, being seeded to inoculum concentration in fluid medium is
2%～3%, 36 DEG C～37 DEG C of shaken cultivation 22～26h, be 10 to bacterium solution density9～1010Cfu/mL's is highly dense
Degree bacteria suspension.
4., according to the Semen avenae nudae Biological control disease-resistant growth-promoting method described in Claims 2 or 3, its feature exists
In, described fluid medium, in terms of 1L, including following components:
Regulation pH=6.5～7.5.
Semen avenae nudae Biological control the most according to claim 4 disease-resistant growth-promoting method, it is characterised in that
Described fluid medium, in terms of 1L, including following components:
Regulation pH=7.0.
Semen avenae nudae Biological control the most according to claim 1 disease-resistant growth-promoting method, it is characterised in that
Step 2) in, composting process sprays step 1) in high density bacteria suspension, including: in compost
When portion's temperature reaches 30 DEG C～34 DEG C, spray step 1) in high density bacteria suspension, continue compost, treat
When compost internal temperature reaches 41 DEG C～45 DEG C, compost terminates.
Semen avenae nudae Biological control the most according to claim 6 disease-resistant growth-promoting method, it is characterised in that
Step 2) in, composting process sprays step 1) in high density bacteria suspension, including: in compost
When portion's temperature reaches 32 DEG C, spray step 1) in high density bacteria suspension, continue compost, in treating compost
When portion's temperature reaches 43 DEG C, compost terminates.
Semen avenae nudae Biological control the most according to claim 1 disease-resistant growth-promoting method, it is characterised in that
Step 2) in, after compost terminates, compost is stirred spreading for cooling.
Semen avenae nudae Biological control the most according to claim 1 disease-resistant growth-promoting method, it is characterised in that
Step 2) in, described fermentation uses naturalness bottom fermentation.
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