CN103122330A - Alpine grassland pasture endogenous serratia plymuthica strain GH010 and application thereof as well as microbial agent prepared from same and preparation method - Google Patents
Alpine grassland pasture endogenous serratia plymuthica strain GH010 and application thereof as well as microbial agent prepared from same and preparation method Download PDFInfo
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- CN103122330A CN103122330A CN2012105904709A CN201210590470A CN103122330A CN 103122330 A CN103122330 A CN 103122330A CN 2012105904709 A CN2012105904709 A CN 2012105904709A CN 201210590470 A CN201210590470 A CN 201210590470A CN 103122330 A CN103122330 A CN 103122330A
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- 241000221696 Sclerotinia sclerotiorum Species 0.000 claims abstract description 10
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Abstract
The invention discloses an alpine grassland pasture endogenous serratia plymuthica strain GH010 capable of inhibiting various pathogenic bacteria and discloses a microbial agent prepared from the alpine grassland pasture endogenous serratia plymuthica strain GH010 and a preparation method thereof. The alpine grassland pasture endogenous serratia plymuthica strain GH010 and the microbial agent prepared from the same disclosed by the invention have the beneficial effects of having good inhibition effects on the various pathogenic bacteria; the inhibition effect is 67.5% of chili rhizoctonia solani and 69.5% of rape sclerotinia sclerotiorum; the bacteriostasis rates of cotton rhizoctonia solani, eggplant sclerotinia sclerotiorum, tomato botrytis cinerea, fusarium wilt of cucumber, exserohilum turcicum, bipolaris maydis or bipolaristriticola are more than or equal to 57.5%; and the strain has growth-promoting effects on crops including alfalfa and the like, and the environmental protection properties of a microbial pesticide and a microbial fertilizer prepared from the strain are good, the resistance to drugs is unlikely to generate and the safety is good.
Description
Technical field
The present invention relates to the biological and ecological methods to prevent plant disease, pests, and erosion field, be specifically related to that Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 and its are used and with microbiobacterial agent and the preparation method of its preparation.
Background technology
Chemical bactericide can be residual in environment and agricultural-food, and cause of disease produces resistance gradually to chemical bactericide in addition, and dosage is increased gradually, more increased the weight of residual quantity, and to the murders by poisoning of nontarget organism such as people, domestic animal, beneficial insect and wildlife etc.In China, agricultural product security is subject to people's concern, the particularly Practice for Pesticide Residue in Agricultural Products problem that exceeds standard in recent years, and it is having a strong impact on the whole people's physical and mental health, simultaneously influence ecological environment and agricultural trade.
Along with the raising of people's living standard and the enhancing of environmental consciousness, in International or National market, vegetable products is carried out access system, the vegetables that come into the market must be pollution-free vegetables.The vegetable pesticide residue amount exceeds standard, and faces " Green Trade Barrier ", can't enter the world market under the overall situation of WTO.This will ask for help pesticide species and performance are adjusted, to the public nuisance-free agricultural chemicals future development.Biological pesticide is to produce the good option of preventing and treating disease in the non-polluted farm product process.
Microbial pesticide is " public nuisance-free agricultural chemicals " of generally acknowledging, can prevent and treat disease by the direct or indirect mode of action, and its prevention effect is better, and is nontoxic to people and animals, free from environmental pollution, noresidue; The selectivity that tool is stronger does not injure beneficial organism and natural enemy, does not destroy the eubiosis, is difficult for developing immunity to drugs; Raw materials for production and effective constituent are natural product, and easily degraded, can go back to nature, and guarantee Sustainable development; Also available biotechnology and engineered method are transformed microorganism, improve constantly Performance and quality, bring into play better the feature of environmental protection of biological pesticide.Along with carrying out of Law on Environmental Protection, and participate in competition in the international market, microbial pesticide will as a kind of new and high technology, in protection of the environment, assurance human health, in agricultural sustainable development, be brought into play more and more important effect.
Chemical fertilizer location test result shows, using of phosphorus, potassium fertilizer can cause heavy metal to be accumulated in the soil body, natural resources and energy resources consumes in a large number, environmental pollution; The nitrogen phosphorus fertilizer utilization ratio that applies is low, the nitrogenous fertilizer that is not absorbed and used runs off in the farmland or volatilizees, underground water resources, soil and air have been caused severe contamination, the toxic substance of agricultural and animal products is exceeded standard, bring great harm to human health, simultaneously chemical fertilizer use in a large number for a long time the spoiled soil crumb structure, cause soil compaction, fertility descends.
Microbial fertilizer is as a kind of new-type fertilizer, after being manured into soil, Fast-propagation by its specific bacterial strain, can be fixedly phosphorus, the potassium element of stationary state in nitrogen, release soil in atmosphere, make the nutrient potentiality of environment be given full play to, and build a good soil microorganisms environment for plant growth, the aspects such as fertilizer amount, reduction environmental pollution, raising crops quality are significant reducing.Especially integrate the research and development of the composite microbiological fertilizer of fixed nitrogen, phosphorus decomposing, potassium decomposing and plant growth element, very important effect is arranged in agricultural sustainable development.
At present, although both at home and abroad microbial pesticide and microbial fertilizer aspect have all been dropped into huge manpower and material resources, quantity chemical pesticide and fertilizer are far away more than biological pesticide and fertilizer; In addition, biological pesticide and fertilizer having relatively high expectations to environment.Therefore, develop the microbial pesticide and the fertilizer that meet the local environment condition particularly important, promote the development of local green agriculture, competition, protection of the environment and the assurance human health of the international market for farm products of participation just to seem particularly important.
Summary of the invention
Purpose of the present invention is exactly for above-mentioned defective of the prior art, and the Alpine Grasslands herbage Nei Shengpu city Serratia strain that can suppress multiple pathogenic bacteria GH010 is provided.
To achieve these goals, technical scheme provided by the invention is: Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010, bacterial strain GH010 be serratia (
Serratia plymuthica), being preserved in Chinese common micro-organisms culture presevation administrative center on 08 13rd, 2012, the address is No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, culture presevation is numbered CGMCC NO.6423.
Second purpose of the present invention has been to provide the application of above-mentioned Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 in suppressing pathogenic bacteria, and described pathogenic bacteria is capsicum rhizoctonia solani or Sclerotinia sclerotiorum; The bacteriostasis rate of described general city Serratia strain GH010 is respectively capsicum rhizoctonia solani 67.5%, Sclerotinia sclerotiorum 69.5%.
Further, the application of above-mentioned Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 in suppressing pathogenic bacteria, described pathogenic bacteria are cotton rhizoctonia solani, eggplant sclerotium germ, botrytis cinerea, cucumber fusarium axysporum, Exserohilum turcicum, southern corn leaf blight or the wheat length spore bacterium that wriggles; The bacteriostasis rate of described general city Serratia strain GH010 is more than or equal to 57.5%.
Further, the application of above-mentioned Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 in suppressing pathogenic bacteria, the amount of described Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 secretion indolylacetic acid in containing tryptophane and not containing the Jin Shi substratum of tryptophane is respectively 63.82mg/L and 5.38mg/L, in inorganic phosphorus and organophosphorus nutrient solution, the available phosphorus increment is respectively 88.63 mg/L and 86.05 mg/L.
The 3rd purpose of the present invention has been to provide the microbiobacterial agent of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation, the composition of described microbiobacterial agent is counted general city Serratia strain GH010 5-10% according to weight percent, general city Serratia strain GH010 substratum 90%-95%.
Further, the microbiobacterial agent of above-mentioned Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation, described general city Serratia strain GH010 substratum comprises following component by weight percentage: Semen Maydis powder 1.5-2.5%, analysis for soybean powder 1-1.5%, peptone 0.2-0.5%, distilled water or deionized water 1000ml; The pH value of described general city Serratia strain GH010 substratum is 7.5-8.5.
Further, the microbiobacterial agent of above-mentioned Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation, described general city Serratia strain GH010 substratum comprises following component by weight percentage: Semen Maydis powder 2%, analysis for soybean powder 1.25%, peptone 0.35%, distilled water or deionized water 1000ml; The pH value of described general city Serratia strain GH010 substratum is 7.5.
The 4th purpose of the present invention has been to provide the preparation method of the microbiobacterial agent of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation, and Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 was obtained on the general city Serratia strain GH010 substratum in liquid fermenting 16-22 hour; Described liquid fermentation condition is temperature 25-29 ℃, rotating speed 200-300r/min, air flow 800-1000L/h; The viable count of described general city Serratia strain GH010 is 9.86 * 10
11Cfu/mL-1.06 * 10
13Cfu/mL.
Further, the preparation method of the microbiobacterial agent of above-mentioned Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation, described liquid fermentation condition is 27 ℃ of temperature, rotating speed 250r/min, air flow 900L/h, fermentation time 19 hours.
The 5th purpose of the present invention has been to provide the application of microbiobacterial agent in preparation microbial pesticide or microbial fertilizer of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation.
Specific operation process of the present invention is as follows:
One, to Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010
Serratia plymuthicaThe identification of morphology of (CGMCC No.6423).
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) be inoculated at beef extract-peptone plate culture medium (extractum carnis 3g, peptone 5g, agar 10-12g, water 1000ml, pH7.0-7.2) on, 28 ℃ of cultivation proterties of cultivating 5d are: Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) bacterium colony is rounded, neat in edge, and the umbilical projection, the surface is glossy, cream color.At NA liquid nutrient medium (extractum carnis 3g, peptone 5g, water 1000ml, pH7.0-7.2), after 28 ℃ of shaking tables are cultivated 3d, growth is abundant, and is highly muddy, homogeneous, flocks, and vibration disperses fully, surface nothing growth, scent of, gramstaining is negative, rod-short.
Two, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 16S rDNA gene order is identified.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 16S rDNA gene order in Genebank with
Serratia plymuthica(EU266583) 16S rDNA gene order is carried out homology relatively.Comparative result shows, the Serratia strain of general city
Serratia plymuthicaGH010(CGMCC No.6423) with
Serratia plymuthica(EU266583) homology can reach more than 99%, can prove Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) really belong to Serratia
Serratia
Three, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) indoor bacteriostatic activity is measured.
Filter out first the general city Serratia strain of Alpine Grasslands herbage endophyte
Serratia plymuthicaGH010(CGMCC No.6423) antagonistic strain is inoculated in GH010(CGMCC No.6423 with this antagonistic strain) cultivate in substratum; Simultaneously, indicator strain is inoculated in potato dextrose agar (being PDA) substratum (potato 200g, glucose 20g, agar 15g, distilled water 1000m1, pH6.7-7.0) and cultivates.When antagonistic strain and indicator strain are cultivated activation in substratum separately after, break into punch tool the bacterium cake that diameter is 7mm respectively.Choose one of the bacterium cake of antagonistic strain, be placed in the PDA substratum; Concrete, can be with the middle position (plate diameter 9cm) of this pure culture biscuits involvng inoculation in the plate of splendid attire PDA substratum; Choose four of the bacterium cakes that obtained by indicator strain, according to the face-off method, equidistantly being inoculated in this four ferfas cake apart from this plate middle position is on the circumference of 3.5cm again.This culture dish after cultivating 5 days under the envrionment temperature of 26 ℃, is used the antibacterial circle diameter of above-mentioned bacterial strains in cross fado time this PDA substratum of replication, in this test, replication 3 times.
The Serratia strain of Alpine Grasslands herbage Nei Shengpu city
Serratia plymuthicaGH010(CGMCC No.6423) sterile liquid can be referring to table 1 to the measurement result of part pathogenic bacteria bacteriostasis.
As shown in Table 1, Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) the capsicum miliary damping-off germ bacteriostasis rate in his-and-hers watches 1 reaches 67.5%, and the Sclerotinia sclerotiorum bacteriostasis rate reaches 69.5%, can reach 57.5% at least to the inhibiting rates of all the other 7 kinds of frequently seen plants germs, and DEVELOPMENT PROSPECT is better.
Four, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) generation indolylacetic acid and molten phosphorus ability detect and the short ability of giving birth to is measured.
1, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) generation indolylacetic acid (IAA) ability is measured.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) be inoculated in Jin Shi (King) nutrient solution (peptone 20g, the K that contains 100mg/L tryptophane (perhaps not containing tryptophane)
2HPO
41.15g, MgSO
4.7H
2O 1.5g, glycerol 15ml, L-Trp 100mg (or not adding tryptophane)) cultivate after 12 days bacterium suspension and centrifugal 10 min of blank, rotating speed is 10000 r/min, get supernatant liquor 4 mL and add respectively the equivalent color solution, use immediately spectrophotometric determination OD530 value, the Serratia strain of general city in dark after standing 30min
Serratia plymuthicaGH010(CGMCC No.6423) amount of producing IAA is respectively 63.82mg/L and 5.38mg/L in containing tryptophane and not containing the Jin Shi substratum of tryptophane.
2, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) molten phosphorus ability is measured.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) be inoculated in PKO(or Meng Jinna) nutrient solution (Ca
3(P0
4)
23.0-5.0g, sucrose 10.0g, (NH
4)
2SO
40.5g, NaCl 0.2g, MgSO
4.7H
2O 0.1g, KC1 0.2g, yeast powder 0.5g, MnSO
41mL (0.004 g/L), FeSO
4(Fe.EDTA) 0.1mL (0.002g/L), agar 15g, pH value 7.0) in, liquid amount is 50/150ml, 121 ℃ of sterilization 20min are take the basic culture solution that do not connect bacterium as contrast.Then be placed in 28 ℃, 160 r/min constant temperature oscillators and cultivate centrifugal 15 min after 10d, rotating speed is 10000 r/min, and supernatant liquor adopts molybdenum antimony resistance colorimetric method to measure available phosphorus increment, the Serratia strain of general city
Serratia plymuthicaGH010(CGMCC No.6423) in inorganic phosphorus and organophosphorus nutrient solution, the available phosphorus increment is respectively 88.63 mg/L and 86.05 mg/L.
3, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) the short ability of giving birth to is measured.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) be inoculated in beef extract-peptone nutrient solution (extractum carnis 3g, peptone 5g, agar 10-12g, water 1000ml, pH7.0-7.2), 48h is cultivated in concussion under 28 ℃, is diluted to the concentration of 1:30,1:40,1:50 with sterilized water.Seed soaking 24h is treated to contrast with clear water, is placed on the filter paper of culture dish, and 100, every ware, every processing repeats 3 times, the germination that observe, record supplies the examination crop seed.After showing money or valuables one carries unintentionally for the examination crop seed, the crop seed that has showed money or valuables one carries unintentionally to be planted to small flower, every day, the bacterium liquid with different concns watered seedling, recorded the upgrowth situation of its plant height.Result shows, 1:50 bacterium liquid has significant promoter action to budding rate and plant height.
The Serratia strain of Alpine Grasslands herbage Nei Shengpu city
Serratia plymuthicaGH010(CGMCC No.6423) to the measurement result of part rate of emergence referring to table 2.
The Serratia strain of Alpine Grasslands herbage Nei Shengpu city
Serratia plymuthicaGH010(CGMCC No.6423) to the measurement result of part crop plant height referring to table 3.
Five, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) optimum medium and the performance of microbiobacterial agent thereof detect.
1, determine Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) optimum medium.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) be inoculated in respectively in the substratum that label is followed successively by A, B, C, D, E, F and G, observe the Serratia strain of general city
Serratia plymuthicaGH010(CGMCC No.6423) energy for growth in these 7 kinds of substratum.
Through observation, find Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) all can grow in these 7 kinds of substratum, but viable count is variant.Wherein, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) measurement result of viable count can be referring to table 4 in these 7 kinds of substratum.
In table 4, " A " expression nutrient agar medium (Nutrient Agar is called for short NA) substratum in the NA substratum, comprises beef peptone 5.0g, peptone 10g, sucrose 20g and distilled water/deionized water 1000mL; " B " expression PDA substratum in the PDA substratum, comprises potato 200g, peptone 10g, sucrose 20g and distilled water/deionized water 1000mL; " C " expression nutrient broth (Nutrient Broth is called for short NB) substratum in the NB substratum, comprises beef peptone 3g, peptone 5.0g, glucose 2.5g and distilled water/deionized water 1000mL; " D " expression yeast glucose solids (being NYDA) substratum in the NYDA substratum, comprises beef peptone 8g, yeast extract 5.0g, glucose 10g and distilled water/deionized water 1000mL; " E " represents the first W-Gum substratum, in the first W-Gum substratum, comprises W-Gum 2.5g, (NH4) 2SO4 10g, sucrose 10g and distilled water/deionized water 1000mL; " F " represents the second W-Gum substratum, in the second W-Gum substratum, comprises W-Gum 3.0g, (NH4) 2SO4 10g, KH2PO4 15g, sucrose 10g and distilled water/deionized water 1000mL; " G " represents protein culture medium, in protein culture medium, comprises peptone 10g, yeast extract 5.0g, NaCl 10g and distilled water/deionized water 1000mL.
As shown in Table 4, under identical condition, select 7 kinds of different nutrient solutions to cultivate GH010(CGMCC No.6423), its viable bacteria amount has notable difference.In nutrient solution E, viable count is 1.92 * 10
11Cfu/mL, and the viable count of nutrient solution A reaches 1.06 * 10
12Cfu/mL is 5.52 times of nutrient solution E, has significant difference between the two.The viable count of other 5 kinds of nutrient solutions is 9.26 * 10
11Cfu/mL～6.36 * 10
11All there is utmost point significant difference in cfu/mL with nutrient solution A.As seen, nutrient solution A is comparatively desirable, and therefore, extractum carnis 5.0g/L, peptone 10g/L, sucrose 20g/L are GH010(CGMCC No.6423) basic medium.
Be the most suitable general city Serratia strain at definite NA substratum
Serratia plymuthicaGH010(CGMCC No.6423) on the basis of the substratum of growth, further utilize Orthogonal Method that carbon nitrogen source content in the NA substratum and pH value are optimized.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) with 5% inoculum size, with the rotating speed of 140r/min, after the temperature condition bottom fermentation 24h of 28 ℃, take Semen Maydis powder, yam starch, W-Gum, white sugar with do not add carbon source as processing, measure the viable count in fermented liquid.
Through synthesis measuring, to cultivate with 4 kinds of different carbon sources, its viable bacteria amount has notable difference, and the substratum take Semen Maydis powder as carbon source is more suitable for the Serratia strain of general city
Serratia plymuthicaGH010(CGMCC No.6423) growth, the viable bacteria amount reaches 6.89 * 10
11Cfu/mL, namely Semen Maydis powder is the more excellent carbon source that filters out.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) with 5% inoculum size, with the rotating speed of 140r/min, after the temperature condition bottom fermentation 24h of 28 ℃, with analysis for soybean powder, NH
4Cl, (NH
4)
2SO
4, KNO
3Do not add nitrogenous source for processing, measure the viable count in fermented liquid.
Through synthesis measuring, to cultivate with 4 kinds of different nitrogenous sources, its viable bacteria amount has notable difference, and is more suitable for Alpine Grasslands herbage Nei Shengpu city Serratia strain during take analysis for soybean powder as nitrogenous source
Serratia plymuthicaGH010(CGMCC No.6423) growth, the viable bacteria amount reaches 7.83 * 10
11Cfu/mL, namely analysis for soybean powder is the more excellent nitrogenous source that filters out.
Carry out orthogonal test with Semen Maydis powder, analysis for soybean powder, peptone and pH, through synthesis measuring, the substratum after finding to optimize is more suitable for the Serratia strain of general city than above-mentioned NA substratum
Serratia plymuthicaGH010(CGMCC No.6423) growth and antibacterial.Wherein, the component of the substratum after above-mentioned optimization, content and pH value are respectively: Semen Maydis powder 2%, analysis for soybean powder 1.25%, peptone 0.35%, pH value 7.5, distilled water/deionized water 1000mL.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) ferment in the substratum after above-mentioned optimization, in fermented liquid, viable count can reach 5.43 * 10
11Cfu/mL-7.33 * 10
12Cfu/mL.In addition, with the Serratia strain of general city
Serratia plymuthicaGH010(CGMCC No.6423) comprising Semen Maydis powder 2%, analysis for soybean powder 1.25%, peptone 0.35%, pH value 7.5, ferment in the substratum of distilled water/deionized water 1000ml, in fermented liquid, viable count can reach 7.33 * 10
12Cfu/mL.
2, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) activity of microbiobacterial agent detects.
Determined Alpine Grasslands herbage Nei Shengpu city Serratia strain in above-mentioned test one
Serratia plymuthicaOn the basis of optimum medium GH010(CGMCC No.6423), with the Alpine Grasslands herbage Nei Shengpu city Serratia strain that is kept in slant medium
Serratia plymuthicaGH010(CGMCC No.6423), after cultivating activation 24h on the plate of nutrient agar medium (Nutrient Agar is called for short NA) substratum, be inoculated in nutrient broth (Nutrient Broth is called for short NB) substratum; After cultivating 24h, after cultivating 20h in 5% inoculum size access seed fermentation tank, turn in the fermentor tank that is inoculated in 10L and ferment, viable count is as index in the fermented liquid, respectively air flow, rotating speed, inoculum size and liquid amount are optimized, finally determine Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) the most adaptable method in the 10L fermentor tank is: temperature 25-29 ℃, and inoculum size 5%-10%, pH7.5-8.5, air flow 800-1000L/h, rotating speed 200-300 r/min, incubation time are 16-22h, viable count is 9.859 * 10
11/ mL-1.06 * 10
13Cfu/mL.
With Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) in the fermentation cylinder for fermentation of above-mentioned fermentation condition, can make the microbiobacterial agent with good short living ability and bacteriostasis.Wherein, be 28 ℃ in temperature, inoculum size is that 10%, pH value is 7.5, and air flow is 1000L/h, and rotating speed is that 200r/min and incubation time are under the fermentation condition of 19h, and in fermented liquid, viable count can reach 1.06 * 10
13Cfu/mL.
3, to Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) quality determining method of microbiobacterial agent is verified.
The method of plate culture count is used for the density of assay plate bacterium colony viable bacteria body, and turbidimetry is used for the number of assay plate bacterium colony viable bacteria body.Here, with density and the number of viable bacteria body in the method for plate culture count and turbidimetry for Determination flat-plate bacterial colony, measurement result is basically identical respectively, and namely on flat-plate bacterial colony, the density of viable bacteria body is larger, number is more, and ultraviolet spectrophotometer is measured OD
600The absorption value at nm place is higher.
As seen, Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) quality of microbiobacterial agent can adopt the method for plate culture count to detect, and also can adopt turbidimetry to detect.
beneficial effect of the present invention is: general city Serratia strain GH010 provided by the invention reaches the microbiobacterial agent with its preparation, multiple pathogenic bacteria all there is good inhibition, it suppresses result is capsicum rhizoctonia solani 67.5%, Sclerotinia sclerotiorum 69.5%, cotton rhizoctonia solani, eggplant sclerotium germ, botrytis cinerea, cucumber fusarium axysporum, Exserohilum turcicum, the wriggle bacteriostasis rate of spore of southern corn leaf blight or wheat length is more than or equal to 57.5%, and the crops such as alfalfa there is growth-promoting functions, microbial pesticide and the microbial fertilizer feature of environmental protection with its preparation are good, be difficult for developing immunity to drugs, security is good.
Description of drawings
Fig. 1 is colonial morphology (left side) and the thalli morphology (right side) of general city Serratia strain GH010 provided by the invention.
Embodiment
Embodiment 1:
One strain general city Serratia strain, the Latin name of this general city Serratia strain is called
Serratia plymuthicaGH010(CGMCC No.6423), by the center preservation of Chinese microorganism strains preservation management committee's common micro-organisms, deposit number is CGMCC No.6423.
As shown in Figure 1, provide Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) optical microscope photograph figure.
Embodiment 2:
General city Serratia strain GH010 can be to the inhibition of the pathogenic bacterias such as capsicum rhizoctonia solani and Sclerotinia sclerotiorum, Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) be 69.5% to the bacteriostasis rate of Sclerotinia sclerotiorum; Bacteriostasis rate to the capsicum rhizoctonia solani is 67.5%.
Embodiment 3:
The Serratia strain of Alpine Grasslands herbage Nei Shengpu city
Serratia plymuthicaGH010(CGMCC No.6423) cotton rhizoctonia solani, eggplant sclerotium germ, botrytis cinerea, cucumber fusarium axysporum, Exserohilum turcicum, southern corn leaf blight and wheat length are wriggled pathogenic bacterias such as spore are inhibited, and bacteriostasis rate is at least 57.5%.
Embodiment 4:
The Serratia strain of Alpine Grasslands herbage Nei Shengpu city
Serratia plymuthicaGH010(CGMCC No.6423) fermented liquid can make the alfalfa seed percentage of germination improve 18.9%, makes plant height increase 1.01cm.
Embodiment 5:
A kind of general city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) microbiobacterial agent, biological activity component and the weight percent content of this microbiobacterial agent comprise: Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 5% and GH010(CGMCC No.6423) the substratum surplus.
GH010(CGMCC No.6423) component of substratum and content comprise: Semen Maydis powder 1.5%, analysis for soybean powder 1.0%, peptone 0.2%, pH value 7.5, distilled water/deionized water 1000ml.
Embodiment 6:
Different from above-described embodiment is, in the present embodiment, and Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) component of microbiobacterial agent and weight percent content comprise: Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 8% and GH010(CGMCC No.6423) the substratum surplus.
In the present embodiment, GH010(CGMCC No.6423) component and the content of substratum comprise: Semen Maydis powder 2%, analysis for soybean powder 1.25%, peptone 0.35%, pH value 8.0, distilled water/deionized water 1000ml.
Embodiment 7:
Different from above-described embodiment is, in the present embodiment, and Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) component of microbiobacterial agent and weight percent content comprise: Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 10% and GH010(CGMCC No.6423) the substratum surplus.
In the present embodiment, GH010(CGMCC No.6423) component and the content of substratum comprise: Semen Maydis powder 2.5%, analysis for soybean powder 1.5%, peptone 0.5%, pH value 8.5, distilled water/deionized water 1000ml.
Embodiment 8:
A kind of Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) preparation method of microbiobacterial agent, this preparation method comprises: with Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 5% be inoculated in GH010(CGMCC No.6423) in substratum, be that 25 ℃, rotating speed are that 200r/min and air flow are under the condition of 800L/h in temperature, carry out liquid fermenting 16h, the fermented liquid that obtains can be directly as Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) microbiobacterial agent uses.
In the present embodiment, GH010(CGMCC No.6423) the pH value of substratum is 7.5, and component and content can comprise: Semen Maydis powder 1.0%, analysis for soybean powder 1.0%, peptone 0.2%, pH value 7.5, distilled water/deionized water 1000ml.
After measured, Alpine Grasslands herbage Nei Shengpu city Serratia strain in above-mentioned fermented liquid
Serratia plymuthicaGH010(CGMCC No.6423) viable count can reach 5.43 * 10
12Cfu/mL.
Embodiment 9:
Different from above-described embodiment is, in the present embodiment, and Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) preparation method of microbiobacterial agent comprises: with Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 8% be inoculated in GH010(CGMCC No.6423) in substratum, be that 27 ℃, rotating speed are that 250r/min and air flow are under the condition of 900L/h in temperature, carry out liquid fermenting 19h, the fermented liquid that obtains can be directly as Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) microbiobacterial agent uses.
In the present embodiment, GH010(CGMCC No.6423) the pH value of substratum is 8.0, and component and content can comprise: Semen Maydis powder 2%, analysis for soybean powder 1.25%, peptone 0.35%, pH value 8.0, distilled water/deionized water 1000ml.
After measured, Alpine Grasslands herbage Nei Shengpu city Serratia strain in above-mentioned fermented liquid
Serratia plymuthicaGH010(CGMCC No.6423) viable count can reach 1.06 * 10
13Cfu/mL.
Embodiment 10:
Different from above-described embodiment is, in the present embodiment, and Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) preparation method of microbiobacterial agent comprises: with Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) 10% be inoculated in GH010(CGMCC No.6423) in substratum, be that 29 ℃, rotating speed are that 300r/min and air flow are under the condition of 1000L/h in temperature, carry out liquid fermenting 22h, the fermented liquid that obtains can be directly as the Serratia strain of general city
Serratia plymuthicaGH010(CGMCC No.6423) microbiobacterial agent uses.
In the present embodiment, GH010(CGMCC No.6423) the pH value of substratum is 8.5, and component and content can comprise: Semen Maydis powder 2.5%, analysis for soybean powder 1.5%, peptone 0.5%, pH value 8.5, distilled water/deionized water 1000ml.
After measured, Alpine Grasslands herbage Nei Shengpu city Serratia strain in above-mentioned fermented liquid
Serratia plymuthicaGH010(CGMCC No.6423) viable count can reach 9.859 * 10
12Cfu/mL.
Through verification experimental verification, above-mentioned Alpine Grasslands herbage Nei Shengpu city Serratia strain
Serratia plymuthicaGH010(CGMCC No.6423) the Alpine Grasslands herbage Nei Shengpu city Serratia strain of microbiobacterial agent and preparation method thereof embodiment
Serratia plymuthicaGH010(CGMCC No.6423) microbiobacterial agent, suitable to arid, the semiarid and area use that cools.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment, the present invention is had been described in detail, for a person skilled in the art, it still can be modified to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010, bacterial strain GH010 be serratia (
Serratia plymuthica), being preserved in Chinese common micro-organisms culture presevation administrative center on 08 13rd, 2012, culture presevation is numbered CGMCC NO.6423.
2. the application of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 according to claim 1 in suppressing pathogenic bacteria is characterized in that described pathogenic bacteria is capsicum rhizoctonia solani or Sclerotinia sclerotiorum; The bacteriostasis rate of described general city Serratia strain GH010 is respectively capsicum rhizoctonia solani 67.5%, Sclerotinia sclerotiorum 69.5%.
3. the application of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 according to claim 1 in suppressing pathogenic bacteria, it is characterized in that, described pathogenic bacteria is cotton rhizoctonia solani, eggplant sclerotium germ, botrytis cinerea, cucumber fusarium axysporum, Exserohilum turcicum, southern corn leaf blight or the wheat length spore bacterium that wriggles; The bacteriostasis rate of described general city Serratia strain GH010 is more than or equal to 57.5%.
4. the application of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 according to claim 1 in suppressing pathogenic bacteria, it is characterized in that, the amount of described Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 secretion indolylacetic acid in containing tryptophane and not containing the Jin Shi substratum of tryptophane is respectively 63.82mg/L and 5.38mg/L, in inorganic phosphorus and organophosphorus nutrient solution, the available phosphorus increment is respectively 88.63 mg/L and 86.05 mg/L.
5. the microbiobacterial agent of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation, it is characterized in that, the composition of described microbiobacterial agent is counted general city Serratia strain GH010 5-10% according to weight percent, general city Serratia strain GH010 substratum 90%-95%.
6. microbiobacterial agent according to claim 5, it is characterized in that, described general city Serratia strain GH010 substratum comprises following component by weight percentage: Semen Maydis powder 1.5-2.5%, analysis for soybean powder 1-1.5%, peptone 0.2-0.5%, distilled water or deionized water 1000ml; The pH value of described general city Serratia strain GH010 substratum is 7.5-8.5.
7. microbiobacterial agent according to claim 6, is characterized in that, described general city Serratia strain GH010 substratum comprises following component by weight percentage: Semen Maydis powder 2%, analysis for soybean powder 1.25%, peptone 0.35%, distilled water or deionized water 1000ml; The pH value of described general city Serratia strain GH010 substratum is 7.5.
8. the preparation method of the microbiobacterial agent of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation, it is characterized in that, Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 was obtained on general city Serratia strain GH010 substratum in liquid fermenting 16-22 hour; Described liquid fermentation condition is temperature 25-29 ℃, rotating speed 200-300r/min, air flow 800-1000L/h; The viable count of described general city Serratia strain GH010 is 9.86 * 10
11Cfu/mL-1.06 * 10
13Cfu/mL.
9. the preparation method of the microbiobacterial agent of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 according to claim 8 preparation, it is characterized in that, described liquid fermentation condition is 27 ℃ of temperature, rotating speed 250r/min, air flow 900L/h, fermentation time 19 hours.
10. the application of microbiobacterial agent in preparation microbial pesticide or microbial fertilizer of Alpine Grasslands herbage Nei Shengpu city Serratia strain GH010 preparation.
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| CN106119168A (en) * | 2016-07-22 | 2016-11-16 | 湖北大学 | One strain has rich phosphorus, degrading organic phosphor and the Serratieae of suppression plant pathogenic fungi |
| CN110699301A (en) * | 2019-11-15 | 2020-01-17 | 云南大学 | Bacterial strain and application thereof in preventing and treating root rot of panax notoginseng |
| CN113667617A (en) * | 2021-08-06 | 2021-11-19 | 甘肃农业大学 | Special microbial agent for natural high-cold grassland and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119168A (en) * | 2016-07-22 | 2016-11-16 | 湖北大学 | One strain has rich phosphorus, degrading organic phosphor and the Serratieae of suppression plant pathogenic fungi |
| CN106119168B (en) * | 2016-07-22 | 2019-06-18 | 湖北大学 | One plant has rich phosphorus, degrading organic phosphor and the Serratieae for inhibiting plant pathogenic fungi |
| CN110699301A (en) * | 2019-11-15 | 2020-01-17 | 云南大学 | Bacterial strain and application thereof in preventing and treating root rot of panax notoginseng |
| CN110699301B (en) * | 2019-11-15 | 2022-11-08 | 云南大学 | Bacterial strain and application thereof in preventing and treating root rot of panax notoginseng |
| CN113667617A (en) * | 2021-08-06 | 2021-11-19 | 甘肃农业大学 | Special microbial agent for natural high-cold grassland and preparation method and application thereof |
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| Publication number | Publication date |
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| CN103122330B (en) | 2015-04-08 |
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