CN102776136B - Composite fungicide for peanuts to resist continuous cropping, as well as preparation method and applications thereof - Google Patents
Composite fungicide for peanuts to resist continuous cropping, as well as preparation method and applications thereof Download PDFInfo
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- CN102776136B CN102776136B CN 201110262196 CN201110262196A CN102776136B CN 102776136 B CN102776136 B CN 102776136B CN 201110262196 CN201110262196 CN 201110262196 CN 201110262196 A CN201110262196 A CN 201110262196A CN 102776136 B CN102776136 B CN 102776136B
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Abstract
The invention provides a composite fungicide for peanuts to resist continuous cropping, as well as a preparation method and applications of the composite fungicide, and specifically provides a composite fungicide for micrococcus lylae O6, leminorella richardii L2, ardisia japonica phyllobacterium C3 and the peanuts to resist continuous cropping. According to the composite fungicide provided by the invention, the allelochemical degrading efficiency is high; and strains existing in natural soils are taken as main materials, the degraded product can be taken as a carbon source by bacteria to be converted into a harmless subject, the carbon source in the soil is increased, the roots of crops can absorb the degraded subjects conveniently, and the environment can not be polluted. According to the composite fungicide, three strains are cultured continuously, the genic mutation can not occur, the degrading properties can be inherited stably; meanwhile, the quantity of beneficial bacteria in the soil can be increased, so that the soil fertility is increased; and furthermore, the preparation method of the composite fungicide provided by the invention is simple, the extraction process is simple, toxic substances can not be added in the preparation process, and the use is simple.
Description
Technical field
The present invention relates to a kind of composite fungus agent of biological field, relate in particular to the anti-continuous cropping composite fungus agent and its production and application of cultivating peanut.
Background technology
Well-known peanut is one of important oil crops, and the Shandong Province is one of Chinese most important peanut producing region and base for export and foreign exchange earning, and main growing area is in zones such as Jiaodong Peninsula, Southern Shandong Province and Midwest, Shandong.Sown area is generally more than 85hm2 throughout the year, ultimate production is more than more than 350 tons, account for 20% and 28% of the whole nation respectively, per unit area yield was up to 4256kg/hm2 in 2006, annual export volume is more than 500,000 tons, account for 70% of national export volume, sown area, gross output, per unit area yield and foreign exchange earning all rank first in the country.In recent years, Shandong Province's peanut cultivation area is big, and concentrate in the producing region, but every year bigger continuous cropping area is arranged all, and 140,000 hm2 are arranged approximately, and indivedual counties, city's peanut continuous cropping area account for 40% of total sown area, and the trend that increases is year by year arranged.Area is big because Shandong Province's peanut is produced, and the producing region is concentrated relatively, and the continuous cropping area accounts for the 1/4-1/3 of the whole province's peanut area throughout the year, is one of principal element that influences Shandong peanut production.Test for many years and investigation result according to people such as Zhang Sisu, the general poor growth of continuous cropping peanut, the underproduction is more than 10%, and high person reaches more than 30%, and along with the increase of the continuous cropping time limit, the underproduction is heavier.The peanut continuous cropping for many years, change has taken place in quantity and the composition of soil microorganisms, bacterial number reduces, fungi quantity increases.
Root exudates has played direct or indirect effect in continuous cropping obstacle.Increasing evidence, continuous cropping obstacle and the allelochemical in the root exudates of crop are closely related.Rice etc. propose on the basis of big quantity research, and the material that causes the crop allelopathy mainly is a secondary metabolite in the plant materials, and allelochemical produces by acetic acid approach or shikimic acid pathway in secondary metabolism.
Yu J.Q and Mat su I isolate 11 kinds of aldehydes matters from the root secretion of cucumber, have 10 kinds of aldehydes matters to have bio-toxicity, and in cucumber continuous cropping planting process, the aldehydes matter that root system discharges runs up to a certain degree, will suppress the growth of second stubble crop.
Hexadecanoic acid, oleic acid etc. are the materials of having been identified its sterilant and having suppressed higher plant growth effect, and phthalic acid also has discovery etc. in the root exudates of soybean, and is listed in allelochemical
Along with being extensive use of of chemical fertilizer, bring an output revolution to plant husbandry, but used chemical fertilizer that the microbial ecological balance is destroyed continuously, the soil compaction sclerosis, the soil-borne disease aggravation, more alarming is that environment for human survival is polluted.Beginning one's study in the world wide solves the improvement problem of soil, the BOM bacterium of the vinelandii that the U.S. is arranged of bio-bacterial manure the earliest, Japan etc., and use is arranged in China, but price is more expensive, function singleness, and popularization is restricted; The bacterial manure of the most manufacturer production of China mostly is single culture greatly, and function singleness, effect are not remarkable, and the anti-continuous cropping special bacteria agent of peanut also seldom has the use of production in China.
Summary of the invention
Purpose of the present invention is intended to overcome above-mentioned the deficiencies in the prior art, the one anti-continuous cropping composite fungus agent and its production and application of cultivating peanut is provided, the invention provides a complex microbial inoculum of cultivating peanut special use, the allelochemical that causes the peanut continuous cropping obstacle of degrading, be based on peanut allelochemical degradation bacteria specifically, and be furnished with the composite fungus agent of the peanut allelochemical of can degrading that multiple bacterial classification makes.The invention provides several bacterial strains can with carbochain long resolve into the short material harmless of carbochain to the deleterious root exudates of peanut root growth to the peanut root growth, particularly generally acknowledged allelochemical hexadecanoic acid, oleic acid, phthalic acid etc. are degraded, and make these innoxious substances in soil, become the carbon source of microorganism, reduce soil acidity, improve soil physical and chemical property, increase soil water-retaining, and can promote the beneficial bacteria growth, regulate the structure of microorganism in the continuous cropping peanut soil, strengthen the soil diseases and insect pests resistance.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
The invention provides Micrococcus lylae O6, its deposit number is: CGMCC 5064; Li Shi Le Minuo Salmonella L2, its deposit number is: CGMCC 5063; Japanese Ardisia Herb leaf bacillus C3, its deposit number is: CGMCC 5065.
The one anti-continuous cropping composite fungus agent of cultivating peanut also is provided, and it contains in Micrococcus lylae O6, Li Shi Le Minuo Salmonella L2, the Japanese Ardisia Herb leaf bacillus C3 bacterial strain one or more composite bacteria suspension.
Further improvement to technical scheme: the living bacteria count of described bacteria suspension is 1 * 10
10～1.4 * 10
10Cfu/ml, described Micrococcus lylae O6: Li Shi Le Minuo Salmonella L2: the bacterium ratio of counting of Japanese Ardisia Herb leaf bacillus C3 is 5: 1: 1.
The present invention also provides the preparation method of the anti-continuous cropping composite fungus agent of peanut, respectively described Micrococcus lylae O6 is inoculated into contain in the oleic substratum, Li Shi Le Minuo Salmonella L2 be inoculated into contain in the phthalic acid substratum, Japanese Ardisia Herb leaf bacillus C3 is inoculated in the substratum that contains hexadecanoic acid, shaking culture, be inoculated in the liquid nutrient medium shaking culture again according to 1% inoculum size.
The present invention also provides the application of the anti-continuous cropping composite fungus agent of peanut in the anti-continuous cropping of peanut.
Further improvement to technical scheme: the described composite fungus agent allelochemical in the peanut of can degrading.
Further improvement to technical scheme: described allelochemical is oleic acid, phthalic acid and hexadecanoic acid.
Further improvement to technical scheme: during described composite bacteria suspension dilution 1000-2000 doubly is manured into soil.
Compared with prior art, advantage of the present invention and positively effect are:
1, composite fungus agent of the present invention is to allelochemical degradation efficiency height.Because the bacterial strain that the present invention uses all screens from the peanut rhizosphere soil, has made full use of the effect of degraded in soil bacterium, the residual allelochemical of rhizosphere continuous cropping of degrading efficiently, degradation rate reaches 75%.
2, composite fungus agent of the present invention is pollution-free.The present invention adopts biotechnology, based on the bacterial classification that exists in the natural soil, the product after its degraded can be converted into innoxious substance as carbon source by bacterium, increases the soil carbon source, help the absorption of crop root, do not exist environment is produced pollution degradation material.
3, strain stability of the present invention is good.These three kinds of bacterial strains can the producer sudden change after can cultured continuously, and its degradation characteristic can genetic stability; Can increase simultaneously the quantity of beneficial bacteria in the soil, increase soil fertility.
4, the preparation method of composite fungus agent of the present invention adopts common method in the biotechnology, and condition is simply gentle, and extraction process is simple, and does not add toxic substance in the preparation process, uses simple.
Description of drawings
Fig. 1 is the gas-chromatography collection of illustrative plates of root exudates among the present invention, wherein 1: laurostearic acid, 2: TETRADECONIC ACID, 3: hexadecanoic acid, 4: stearic acid, 5: oleic acid, 6: Succinic Acid, 7: phthalic acid.
Fig. 2 is the starch degradation test qualification result of phthalic acid degradation bacteria bacterial strain L2 among the present invention.
Fig. 3 is the starch degradation test qualification result of oleic acid degradation bacteria bacterial strain O6 among the present invention.
Fig. 4 is the starch degradation test qualification result of hexadecanoic acid degradation bacteria bacterial strain C3 among the present invention.
Fig. 5 is that the oleic degradation rate of Micrococcus lylae O6 degraded is measured collection of illustrative plates among the present invention.
Fig. 6 be among the present invention Li Shi Le Minuo Salmonella L2 degraded phthalic acid degradation rate measure collection of illustrative plates.
Fig. 7 be among the present invention Japanese Ardisia Herb leaf bacillus C3 degraded hexadecanoic acid degradation rate measure collection of illustrative plates.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is described in further detail.
Embodiment 1
1, bacterial classification source
Soil is collected in the Dong Longwan village, Furukawa town, Laixi City and plants peanut peanut ground for many years continuously, is respectively peanut continuous cropping 1 year, the soil in 2 years, 4 years, 7 years, and soil type is the river moisture soil, and soil texture type is a sand, and peanut varieties is educated No. 16 for flower.The soil sample of sample peanut root is brushed into valve bag with brush, and gather around the root system in the 2mm scope soil, collect altogether about the heavily about 500g of soil, refrigeration is used for the isolation identification of Screening of Bioflocculant-producing Bacteria mensuration and allelochemical.
2, bacterial screening method
Nutrient agar (NA substratum): peptone 5.0g; Beef extract 3.0g; Glucose 2.5g; Yeast powder 1.0g; Agar powder 16.0-18.0g; Distilled water 1000ml; PH7.0-7.2.Other prepares the NA liquid nutrient medium.
Carbon-free basic medium (CM substratum): K
2HPO
47.5g; KH
2PO
42.0g; MgSO
47H
2O0.2g; CaCl
22H
2O 15mg; NaCl 0.5g; (NH
4)
2SO
42.0g; Distilled water 1000ml; PH 7.2-7.6.
The peanut allelochemical: oleic acid, phthalic acid, hexadecanoic acid, these allelochemicals are measured in experiment, and its content ratio is about 5: 1: 1.These allelochemicals are all influential to peanut percentage of germination, plant height, root length and chlorophyll, and 4 years content of peanut continuous cropping promptly reaches higher level, has than significant inhibitory effect.
Screening step: get fresh soil sample 10g and join in the 100ml CM substratum that contains oleic acid 2%, phthalic acid 1 ‰, hexadecanoic acid 2% respectively, place 170r/min, cultivate 72h on 30 ℃ of shaking tables.Succeeding transfer culture three times is drawn 100 μ l nutrient solutions and is inoculated on the NA flat board, cultivates 24h for 30 ℃, then according to the different shape of bacterium colony at random picking list bacterium colony purifying preserve their degradation efficiency to be determined after 3 generations.After gas chromatograph-mass spectrometer (GC-MS) was measured their degradation efficiency, the bacterial strain of choosing degradation efficiency height, inheritance stability carried out strain identification.
The allelochemical hexadecanoic acid, oleic acid, the measuring method of the degradation rate of phthalic acid: NA substratum branch is installed in the test tube of 10ml, add sterilized oleic acid 2% in the test tube separately, phthalic acid 1 ‰, hexadecanoic acid 2%, and the corresponding degradation bacteria strains 1 of inoculation encircles respectively, place 30 ℃ of constant temperature vibrating machines, cultivate 72h under the speed governing 170r/min condition, press 3ml, 3ml, 4ml adds 1: 1 alcohol-ether solvent in proper order respectively for three times, vibration 1min, get upper solution, collect the back in Rotary Evaporators, evaporate to dryness under 40 ℃ of rotating speed 60r/min conditions, use 1ml, 1ml, the 1%H2SO4-methanol solution of 1ml is washed matrass three times, solution is collected in adds a cover the little funnel in curved footpath in the 25ml triangular flask, water bath with thermostatic control 60min in 70 ℃ of thermostat water baths, 3ml is pressed in the cooling back, 3ml, 4ml adds n-hexane extraction in proper order, supernatant liquor is collected in the machine for the treatment of in the 10ml volumetric flask measures with gas chromatograph-mass spectrometer (GC-MS).
GC measures the content of allelochemical in the soil and sees Fig. 1.Determine the relative content of allelochemical in the soil according to peak area.Record the content of various root exudates in the soil by gas chromatogram (Fig. 1), be respectively: hexadecanoic acid 134.03mg/kg, oleic acid 284.12mg/kg, phthalic acid 11.58mg/kg (allelochemical under this content peanut can't normal growth).
Vapor-phase chromatography (GC) condition determination: GC analyzes in centralab of Qingdao Agricultural University and measures, and carries out on Agilent Technologies HP6890N gas chromatograph, adopts fid detector.GC condition: pillar model HP-FFAP, specification 30m * 320 μ m * 0.25 μ m, do not shunt carrier gas He gas (99%), flow 10ml/min, column temperature is from 120-280 ℃, 20 ℃/min of temperature programming, and in the time of 280 ℃, keep 5min, 120 ℃ of injector temperatures, 280 ℃ of detector temperatures, sample size 1 μ l.
3, strain identification
GC measures the degradation rate of allelochemical, and the calculation formula of degradation rate is: degradation rate (%)=(control sample residual quantity-processing sample residual quantity)/control sample residual quantity * 100%.According to experimental result choose higher to the allelochemical degradation capability, growing ability is vigorous and bacterial strain that can genetic stability as aimed strain, do further Test Identification.
With reference to the method for eastern elegant pearl etc. degradation bacteria strains is carried out that conventional morphology is identified and Physiology and biochemistry measure (eastern elegant pearl etc. common bacteria system identification handbook. Beijing, Science Press, 2001:353-379).
Table 1: the morphological specificity of three bacterial strains
Table 2: the Physiology and biochemistry of three bacterial strains is measured
Fig. 2, Fig. 3, Fig. 4 are seen in the evaluation of bacterial strain starch test, and the oleic bacterial strain O6 that degrades is Micrococcus lylae (M.lylae), and degradation rate is 77.12% (degradation rate is measured collection of illustrative plates and seen Fig. 5); The bacterial strain L2 of degraded phthalic acid is a Li Shi Le Minuo Salmonella (L.richardii), degradation rate 68.39% (the degradation rate collection of illustrative plates is seen Fig. 6); The bacterial strain C3 of degraded hexadecanoic acid is a Japanese Ardisia Herb leaf bacillus (P.myrisacearum), and degradation rate is 82.28% (the degradation rate collection of illustrative plates is seen Fig. 7).
The above-mentioned bacterial strains that screens is carried out culture presevation, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on July 22nd, 2011; Deposit number is respectively: Li Shi Le Minuo Salmonella L2 (Leminorella richardii) is numbered CGMCC No.5063; Micrococcus lylae O6 (Micrococcus lylae) is numbered CGMCC No.5064; Japanese Ardisia Herb leaf bacillus C3 (Phyllobacterium myrisacearum) is numbered CGMCC No.5065.
4, spawn culture
With the inoculation of degraded allelochemical to contain respectively oleic acid 2%, phthalic acid 1 ‰, hexadecanoic acid 2% the CM substratum of 100ml in, place 170r/min, 30 ℃ of shaking culture 72h.Draw 100 μ l nutrient solutions respectively and be inoculated in the 100ml NA substratum that adds corresponding allelochemical 30 ℃ of shaking culture 72h.
5, the preparation of composite fungus agent
The main component of composite fungus agent of the present invention is an allelochemical degraded composite bacteria suspension, and living bacteria count is 1 * 10
10～1.4 * 10
10Cfu/ml, wherein O6 degradation bacteria living bacteria count is 7.14 * 10
9～1 * 10
10Cfu/ml; L2 degradation bacteria living bacteria count is 1.43 * 10
9～2 * 10
9Cfu/ml; C3 degradation bacteria living bacteria count is 1.43 * 10
9～2 * 10
9Cfu/ml.The bacterium ratio of counting that wherein preferred allelochemical degraded composite fungus agent is Micrococcus lylae O6, Li Shi Le Minuo Salmonella L2, Japanese Ardisia Herb leaf bacillus C3 is 5: 1: 1 a mixed fermentation liquid.
During described composite bacteria suspension dilution 1000-2000 doubly is manured into soil.
Above embodiment is only in order to illustrating technical scheme of the present invention, but not limits it; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.
Claims (8)
1. a strain oleic acid degradation bacteria strains, its name is called Micrococcus lylae O6(
Micrococcus lylae), deposit number is CGMCC 5064.
2. a strain phthalic acid degradation bacteria strains, its name is called Li Shi Le Minuo Salmonella L2(
Leminorella richardii), deposit number is CGMCC 5063.
3. a strain hexadecanoic acid degradation bacteria strains, its name is called Japanese Ardisia Herb leaf bacillus C3(
Phyllobacterium myrisacearum), deposit number is CGMCC 5065.
4. the anti-continuous cropping composite fungus agent of cultivating peanut, it is characterized in that it is the composite bacteria suspension that contains Micrococcus lylae O6, Li Shi Le Minuo Salmonella L2 and Japanese Ardisia Herb leaf bacillus C3 bacterial strain, described Micrococcus lylae O6 deposit number is CGMCC 5064, described Li Shi Le Minuo Salmonella L2 deposit number is CGMCC 5063, and described Japanese Ardisia Herb leaf bacillus C3 deposit number is CGMCC 5065;
The living bacteria count of described bacteria suspension is 1 * 10
10~ 1.4 * 10
10Cfu/ml, described Micrococcus lylae O6: Li Shi Le Minuo Salmonella L2: the bacterium ratio of counting of Japanese Ardisia Herb leaf bacillus C3 is 5:1:1.
5. the preparation method of the anti-continuous cropping composite fungus agent of peanut according to claim 4, it is characterized in that respectively described Micrococcus lylae O6 being inoculated into contain in the oleic substratum, Li Shi Le Minuo Salmonella L2 be inoculated into contain in the phthalic acid substratum, Japanese Ardisia Herb leaf bacillus C3 is inoculated in the substratum that contains hexadecanoic acid, shaking culture, be inoculated in the liquid nutrient medium shaking culture again according to 1% inoculum size.
6. according to claim 4 one application of anti-continuous cropping composite fungus agent in the anti-continuous cropping of peanut of cultivating peanut.
7. according to claim 6 one application of anti-continuous cropping composite fungus agent in the anti-continuous cropping of peanut of cultivating peanut is characterized in that: the described composite fungus agent allelochemical in the peanut of can degrading, described allelochemical is oleic acid, phthalic acid and hexadecanoic acid.
8. according to claim 7 one application of anti-continuous cropping composite fungus agent in the anti-continuous cropping of peanut of cultivating peanut is characterized in that: during described composite bacteria suspension dilution 1000-2000 doubly is manured into soil.
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| CN104738091B (en) * | 2015-03-14 | 2017-09-22 | 青岛农业大学 | A kind of preparation and application of anti-peanut continuous cropping composite bacteria agent |
| CN104813874A (en) * | 2015-04-28 | 2015-08-05 | 吉林省农业科学院 | Cultivation method for comprehensively preventing peanut disease and pest injury and continuous cropping obstacles |
| CN115997766A (en) * | 2022-11-21 | 2023-04-25 | 云南农业大学 | Mixtures of phthalic acid, palmitic acid and azelaic acid and their use |
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| CN101857500B (en) * | 2010-06-21 | 2011-06-01 | 山东宝源生物有限公司 | Microbe organic fertilizer containing chelated microelements and production method thereof |
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