CN104620817A - Highland barley biological control disease-resistant and growth promoting method - Google Patents
Highland barley biological control disease-resistant and growth promoting method Download PDFInfo
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- CN104620817A CN104620817A CN201510058085.3A CN201510058085A CN104620817A CN 104620817 A CN104620817 A CN 104620817A CN 201510058085 A CN201510058085 A CN 201510058085A CN 104620817 A CN104620817 A CN 104620817A
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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
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- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention discloses a highland barley biological control disease-resistant and growth promoting method. The highland barley biological control disease-resistant and growth promoting method comprises the steps that bacillus subtilis are made into high density bacterial suspension with the density ranging from 109cfu/mLto 1010cfu/mL; healthy highland barley straws are cut into small sections to compost, the bacillus subtilis high density bacterial suspension is sprayed in the composting process, a biological fertilizer capable of effectively restraining and killing highland barley pathology funguses is obtained after fermentation and is used on highland barley biological control disease-resistant and growth promotion. According to the highland barley biological control disease-resistant and growth promoting method, the bacillus subtilis producing beta dextranase are plant probiotics with a disease-resistant and growth promoting function, the biological fertilizer is added into the compost to effectively strengthening the biological control function on highland barley disease funguses, restraining and killing effects on sheath rot pathogenic bacteria are showed, and the highland barley yield is improved. According to the highland barley biological control disease-resistant and growth promoting method, treatment of the composting process is easy, no waste water is discharged, the investment is small, the energy consumption is low, the operation is easy, industrialized mass production is easy to achieve, the economic benefit is good, and the application prospect is wide.
Description
Technical field
The present invention relates to the preparation field of biological control fertilizer, be specifically related to a kind of disease-resistant growth-promoting method of highland barley biological control.
Background technology
Highland barley (Hordeum vulgare Linn.var.nudum Hook.f.) is a kind of cereal crop of grass family Hordeum, because shell coetonium in it is separated, seed is exposed, therefore also known as naked barley, highland barley, meter great Mai, mainly originating from the ground such as Tibet, China, Qinghai, Sichuan, Yunnan, is Tibetan's main food.Highland barley is planted on Qinghai-Tibet Platean about history in 3500, extends to inspirit culture field among material and culture, Qinghai-Tibet Platean defines rich connotation, is rich in the culture of the highland barley of national characters.Highland barley has extensively medicinal and nutritive value, has released the highland barley products such as highland barley fine dried noodles, highland barley steamed bun, barley nutritional powder.Highland barley is the primary raw material of zanba, roasted qingke barley flour first of the treasured of Tibet four.
Sheath maize ear rot is highland barley Major Diseases, and Phoma macdonaldii is one of pathogen.Plant pathology fungi is mainly present in soil, infection plant in plant growth, causes plant disease, causes production loss.Therefore, fungal diseases of plants to be eliminated from biological control angle, kill or suppress the pathology fungi in soil most important, and use the bio-fertilizer with biological control effect to be the effective way realizing this purpose.Bio-fertilizer development trend should be greatly develop resistance high, can the bacillus of existence steady in a long-term.Particularly bacillus can produce multiple antifungal substance, has remarkable inhibitory action to phytopathogen, and to person poultry harmless, does not also pollute the environment, and will play an important role in control fungal diseases of plants.
In straight or closely straight shaft-like in bacillus form, aerobic or amphimicrobian, Gram-positive, can produce resistance endospore under certain environmental conditions.Bacillus extensively surely grows in plant, is considered to generally acknowledged grade-safe microorganism (generally regarded as safety, GRAS).Bacillus subtilis is a kind of typical bacillus, produce the antibacterial materials such as antibacterial protein, polypeptides matter, lipid material and beta dextranase, particularly hydrolase plant pathology fungi anti-with it ability such as beta dextranase has substantial connection, because the composition of fungal cell wall is glucan.
Summary of the invention
The invention provides the measure of biotic control of the disease-resistant growth-promoting of a kind of highland barley, by being incorporated in highland barley natural flora by the bacillus subtilis (CICC 10339) producing beta dextranase, make the bio-fertilizer that effectively can suppress to kill by biological control approach highland barley pathology fungi.
A kind of disease-resistant growth-promoting method of highland barley biological control, comprises the following steps:
1) bacillus subtilis (CICC 10339) is cultivated, make density 10
9~ 10
10the high density bacteria suspension of cfu/mL;
2) healthy for highland barley stalk is cut into segment and carries out compost, spray step 1 in composting process) in high density bacteria suspension, compost ferments after terminating, after fermentation, obtain the bio-fertilizer that can effectively suppress to kill highland barley pathology fungi, this bio-fertilizer is used in the disease-resistant growth-promoting of highland barley biological control.
In the present invention, healthy for highland barley stalk is cut into trifle compost, and make the flora in prepared bio-fertilizer be healthy highland barley plant naturally flora, this flora and highland barley plant coevolution grow, define mutual beneficial syntaxial system, have certain biological control effect to disease fungi.Further, the bacillus subtilis (CICC 10339) that can produce beta dextranase adds in compost, significantly strengthens the biological control effect to highland barley disease fungi.Bacillus subtilis (CICC 10339), owing to producing beta dextranase, has good decomposition to highland barley plant fiber, by composting fermentation process is necessary, therefore fully can be dissolved in compost flora and breeds stably and lastingly.As the prebiotic effect of lactic acid bacteria for animal, the microorganism killing plant pathology fungi is suppressed to be referred to as plants probiotics.In bio-fertilizer prepared by the present invention, bacillus subtilis (CICC 10339) is namely highland barley probio, has remarkable biological control effect to highland barley pathology fungi.
Step 1) in, bacillus subtilis (CICC 10339) adopts prior art, from Chinese industrial Microbiological Culture Collection administrative center (CICC), and address: No. 24, Road, Jiuxianqiao, Chaoyang District, Beijing City institute No. 6 building; Preservation date is 2007, and bacterial classification keeps being numbered 10339.Bacillus subtilis (CICC 10339) can be bought to Chinese industrial Microbiological Culture Collection administrative center.
Bacillus subtilis (CICC 10339) is cultivated, comprising: the density of setting out of bacillus subtilis (CICC10339) is 10
6~ 10
7individual/mL, being seeded to inoculum concentration in liquid nutrient medium is 1% ~ 5%, 33 DEG C ~ 39 DEG C shaken cultivation 20 ~ 28h, is 10 to the liquid-tight degree of bacterium
9~ 10
10the high density bacteria suspension of cfu/mL.
Further preferably, bacillus subtilis (CICC 10339) is cultivated, comprising: the density of setting out of bacillus subtilis (CICC 10339) is 10
6~ 10
7individual/mL, being seeded to inoculum concentration in liquid nutrient medium is 2% ~ 3%, 36 DEG C ~ 37 DEG C shaken cultivation 22 ~ 26h, is 10 to the liquid-tight degree of bacterium
9~ 10
10the high density bacteria suspension of cfu/mL.
Described liquid nutrient medium, in 1L, comprises following component:
Further preferably, described liquid nutrient medium, in 1L, comprises following component:
Liquid nutrient medium is at 121 DEG C of sterilizing 20min.
Step 2) in, spray step 1 in composting process) in high density bacteria suspension, comprising: when compost 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.
Spray step 1 in composting process) in high density bacteria suspension, comprising: when compost internal temperature reaches 32 DEG C, spray step 1) in high density bacteria suspension, continue compost, when compost internal temperature reaches 43 DEG C, compost terminates.
Step 2) in, after compost terminates, compost is stirred spreading for cooling.Described fermentation adopts nature bottom fermentation.
Compared with prior art, tool of the present invention has the following advantages:
In the present invention, healthy for highland barley stalk is cut into trifle compost, and make the flora in prepared bio-fertilizer be healthy highland barley plant naturally flora, this flora and highland barley plant coevolution grow, define mutual beneficial syntaxial system, have certain biological control effect to disease fungi.
Bacillus subtilis (CICC 10339) produces beta dextranase, and strong destruction pathology fungal cell wall, has pathology fungi and significantly suppress killing action.As the prebiotic effect of lactic acid bacteria for animal, the microorganism killing plant pathology fungi is suppressed to be referred to as plants probiotics.Namely the bacillus subtilis (CICC 10339) of being rich in bio-fertilizer prepared by the present invention is highland barley probio, has remarkable biological control effect to highland barley pathology fungi.
Composting process carries out in its natural state, the stable highland barley plant natural flora of Tiny ecosystem is relied on to ferment, and bacillus subtilis (CICC 10339) is owing to producing beta dextranase, good decomposition is had to highland barley plant fiber, by composting fermentation process is necessary, therefore, bacillus subtilis (CICC10339) fully can be dissolved in compost flora and breed stably and lastingly.Composting process process is easy, non-wastewater discharge, and small investment, energy consumption are low, easy to operate, is easy to industrialization large-scale production, has good economic benefit and wide application prospect.
Embodiment
Embodiment 1
1) produce the High Density Cultivation of the bacillus subtilis (CICC 10339) of beta dextranase, the density of setting out of bacillus subtilis (CICC 10339) is 1.22 × 10
6individual/mL, is seeded to liquid nutrient medium (glucose 14g/L, beef extract 25g/L, potassium dihydrogen phosphate 2.45g/L, groundnut meal 10g/L, pH=7.0,121 DEG C of sterilizing 20min) in, inoculum concentration is 2%, 37 DEG C of shaken cultivation 24h, and the liquid-tight degree of bacterium is 2.46 × 10
9cfu/mL, obtains high density bacteria suspension.
2) the common 100kg of healthy for highland barley stalk is cut into trifle and carries out Natural compost, spray 1 when compost internal temperature reaches 32 DEG C) in the high density bacteria suspension 2L for preparing, continue compost, when compost internal temperature is more than 43 DEG C, compost terminates, stir spreading for cooling, after fermentation under nature, becomes the bio-fertilizer being rich in the bacillus subtilis producing beta dextranase, effectively can suppress the bio-fertilizer killing highland barley pathology fungi.
3) step 1) in the high density bacteria suspension for preparing and step 2) in the bio-fertilizer for preparing significantly there is Beta dextranase vigor, have highland barley pathology fungi and significantly suppress killing action, specifically see the following form 1.
Sheath maize ear rot pathogen Phoma macdonaldii is purchased from Chinese industrial Microbiological Culture Collection administrative center (CICC), and bacterium numbering is 2613.
Beta dextranase vitality test: adopt reducing sugar test method, Beta glucan can be degraded into oligosaccharides and monose by Beta dextranase, the reduction group that it has under boiling water bath condition can with DNS reagent generation chromogenic reaction, the colour developing depth is directly proportional to reducing sugar growing amount, and reducing sugar growing amount is directly proportional to reactant liquor Beta glucanase vigor, thus utilize the light absorption value of colorimetric estimation reactant liquor to calculate reducing sugar growing amount, obtain Beta dextranase vigor.Beta glucanase unit of activity defines: under condition determination, and hydrolysis Beta glucan per minute produces the enzyme amount needed for 1 μm of ol reducing sugar (with glucose meter), is defined as a Ge Meihuo unit (IU).
The antibacterial broadband of dull and stereotyped suppression: adopt dull and stereotyped spray-on process, test strains point is connected on NA flat board, often dull and stereotyped 2 strains, each 3 repetitions, cultivates 24h for 28 DEG C, the filter paper being moistened with chloroform is put into stifling 20min bottom inverted culture dish, repeats once, kill bacterium to be measured; Disease fungus is made into 10
8bacteria suspension, is sprayed on rapidly with aseptic laryngeal spray (0.1ml/ ware) on the flat board being connected to bacterium to be measured, and after 48-60h is cultivated in 28 DEG C of continuation, record forms inhibition zone size.
Dull and stereotyped antagonism: pour NA medium into flat board, becomes triangle to place Oxford cup, adds zymotic fluid 100 μ L in the cup of Oxford on medium after solidifying, 28 DEG C of incubators are cultivated, after disease fungus growth, record 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) produce the High Density Cultivation of the bacillus subtilis (CICC 10339) of beta dextranase, the density of setting out of bacillus subtilis (CICC 10339) is 1.80 × 10
6individual/mL, is seeded to liquid nutrient medium (glucose 14g/L, beef extract 25g/L, potassium dihydrogen phosphate 2.45g/L, groundnut meal 10g/L, pH=7.0,121 DEG C of sterilizing 20min) in, inoculum concentration is 3%, 37 DEG C of shaken cultivation 24h, and the liquid-tight degree of bacterium is 3.34 × 10
9cfu/mL, obtains high density bacteria suspension.
2) the common 100kg of healthy for highland barley stalk is cut into trifle and carries out Natural compost, spray 1 when compost internal temperature reaches 32 DEG C) in the high density bacteria suspension 3L for preparing, continue compost, when compost internal temperature is more than 43 DEG C, compost terminates, stir spreading for cooling, after fermentation under nature, becomes the bio-fertilizer being rich in the bacillus subtilis producing beta dextranase.
3) step 1) in the high density bacteria suspension for preparing and step 2) in the bio-fertilizer for preparing significantly there is Beta dextranase vigor, have highland barley pathology fungi and significantly suppress killing action, specifically see the following form 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) produce the High Density Cultivation of the bacillus subtilis (CICC 10339) of beta dextranase, the density of setting out of bacillus subtilis (CICC 10339) is 1.68 × 10
6individual/mL, is seeded to liquid nutrient medium (glucose 14g/L, beef extract 25g/L, potassium dihydrogen phosphate 2.45g/L, groundnut meal 10g/L, pH=7.0,121 DEG C of sterilizing 20min) in, inoculum concentration is 3%, 37 DEG C of shaken cultivation 24h, and the liquid-tight degree of bacterium is 2.33 × 10
9cfu/mL, obtains high density bacteria suspension.
2) the common 100kg of healthy for highland barley stalk is cut into trifle and carries out Natural compost, spray 1 when compost internal temperature reaches 32 DEG C) in the high density bacteria suspension 3L for preparing, continue compost, when compost internal temperature is more than 43 DEG C, compost terminates, stir spreading for cooling, after fermentation under nature, becomes the bio-fertilizer being rich in the bacillus subtilis producing beta dextranase.
3) step 2) in the bio-fertilizer for preparing to highland barley, there is obvious growth-promoting prebiotic effect, improve single plant yield 8.2% (p<0.05), specifically see the following form 3 and table 4.Examination highland barley kind is supplied to be dark blue 320.
Table 3
Table 4
Claims (9)
1. the disease-resistant growth-promoting method of highland barley biological control, is characterized in that, comprise the following steps:
1) bacillus subtilis CICC 10339 is cultivated, make density 10
9~ 10
10the high density bacteria suspension of cfu/mL;
2) healthy for highland barley stalk is cut into segment and carries out compost, spray step 1 in composting process) in high density bacteria suspension, compost ferments after terminating, after fermentation, obtain the bio-fertilizer that can effectively suppress to kill highland barley pathology fungi, this bio-fertilizer is used in the disease-resistant growth-promoting of highland barley biological control.
2. the disease-resistant growth-promoting method of highland barley biological control according to claim 1, is characterized in that, step 1) in, bacillus subtilis CICC 10339 is cultivated, comprising: the density of setting out of bacillus subtilis CICC 10339 is 10
6~ 10
7individual/mL, being seeded to inoculum concentration in liquid nutrient medium is 1% ~ 5%, 33 DEG C ~ 39 DEG C shaken cultivation 20 ~ 28h, is 10 to the liquid-tight degree of bacterium
9~ 10
10the high density bacteria suspension of cfu/mL.
3. the disease-resistant growth-promoting method of highland barley biological control according to claim 2, is characterized in that, step 1) in, bacillus subtilis CICC 10339 is cultivated, comprising: the density of setting out of bacillus subtilis CICC 10339 is 10
6~ 10
7individual/mL, being seeded to inoculum concentration in liquid nutrient medium is 2% ~ 3%, 36 DEG C ~ 37 DEG C shaken cultivation 22 ~ 26h, is 10 to the liquid-tight degree of bacterium
9~ 10
10the high density bacteria suspension of cfu/mL.
4. the biological control preparation method of the disease-resistant growth-promoting of the highland barley according to Claims 2 or 3, is characterized in that, described liquid nutrient medium, in 1L, comprises following component:
5. the disease-resistant growth-promoting method of highland barley biological control according to claim 4, is characterized in that, described liquid nutrient medium, in 1L, comprises following component:
6. the disease-resistant growth-promoting method of highland barley biological control according to claim 1, it is characterized in that, step 2) in, spray step 1 in composting process) in high density bacteria suspension, comprise: when compost 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.
7. the disease-resistant growth-promoting method of highland barley biological control according to claim 6, it is characterized in that, step 2) in, spray step 1 in composting process) in high density bacteria suspension, comprise: when compost internal temperature reaches 32 DEG C, spray step 1) in high density bacteria suspension, continue compost, when compost internal temperature reaches 43 DEG C, compost terminates.
8. the disease-resistant growth-promoting method of highland barley biological control according to claim 1, is characterized in that, step 2) in, after compost terminates, compost is stirred spreading for cooling.
9. the disease-resistant growth-promoting method of highland barley biological control according to claim 1, is characterized in that, step 2) in, described fermentation adopts nature bottom fermentation.
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| CN109503263A (en) * | 2018-12-06 | 2019-03-22 | 西藏袁氏农业科技发展有限公司 | A kind of formula and preparation method of highland barley organic medical fertilizer |
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