CN103828722A - Method for blueberries seedling and cultivating blueberries in large area by applying DSE (Dark Septate Endophyte) fungus - Google Patents
Method for blueberries seedling and cultivating blueberries in large area by applying DSE (Dark Septate Endophyte) fungus Download PDFInfo
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Abstract
The invention discloses a method for blueberries seedling and cultivating blueberries in large area by applying a DSE (Dark Septate Endophyte) fungus. The method comprises the following steps: firstly, cultivating the DSE fungus and blueberry seedlings respectively; and then, carrying out symbiotic cultivation on the DSE fungus, blueberry cutting seedlings, blueberry seedlings and annual blueberry seedlings respectively. The method disclosed by the invention can promote rootage of the blueberry cutting seedlings and development of the blueberry seedling root system, so that the growing period of the seedlings is greatly shortened. According to the method, root development of the annual blueberry seedlings can be accelerated, the quantity of blueberry branches and the increment of the seedlings can be increased, and the drought resistance of the blueberry seedlings can be improved. The cultivating method disclosed by the invention is simple to operate, easy to popularize in a large area, less in investment and remarkable in yield-increasing effect.
Description
Technical field:
The invention belongs to biological technical field, be specifically related to a kind of application and carry out from separating the DSE fungi obtaining in Vaccinium plant blueberry root that blueberry is grown seedlings and the method for commerial growing.
Background technology:
Blueberry (Blueberry), formal name used at school blueberry, belong to Ericaceae, cowberry subfamily, Vaccinium (
vaccinium spp.) perennial fallen leaves or evergreen shrubs.Original producton location and the main producing region Dou U.S..In its fruit, contain abundant nutrient component, have and delay cerebral nerve aging, remove eye fatigue and increase eyesight, enhancing cardiac function and anticancer unique effects, FAO (Food and Agriculture Organization of the United Nation) is classified as one of the mankind's five large healthy food.
At present, the blueberry kind of cultivation mainly contains in the world: be suitable for the high-bush blueberry of temperate zone cultivation, be suitable for the short filling blueberry of severe cold areas cultivation and be suitable for subtropics or the Vaccinium ashei of torrid zone cultivation.The root system of blueberry is thin, is fibrous, there is no root hair, and absorbing capacity is little, and suitable to loosing soil, the cultivation of the region that moistening, the content of organic matter is high, its photosynthetic optimum temperature range is relatively little.Because blueberry is high to growing environment requirement, natural propagation rate is low, and poor growth, at the nutritive value that adds that blueberries itself is higher, constantly increases the demand of blueberry in recent years, causes the expensive of blueberry.
Although the plantlet bud propagation of blueberry has obtained success, incubation step complexity and the difficulty of test-tube plantlet are high, the especially seedling aspect of taking root, and general medium rooting rate is not high; After test-tube plantlet bottle outlet, the problem such as poor growth, growth cycle length becomes the major obstacle of restriction blueberry commerial growing.
Summary of the invention:
The object of the invention is to overcome the deficiency of above-mentioned prior art and provide a kind of method simple, be easy to spread, small investment, do not execute any fertilizer and agricultural chemicals, shorten the production cycle of growing seedlings, increase the numbers of branches of blueberry seedling and the amount of growth of seedling, improve the drought resistance of seedling, the application DSE fungi of realizing blueberry green high quality high yield carries out that blueberry is grown seedlings and the method for commerial growing.
Object of the present invention can reach by following measure: a kind of DSE of application fungi carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that it comprises the steps:
1), the cultivation of DSE fungi:
By DSE fungi after the slant tube actication of culture of low-temperature preservation, transfer in the plate of PDA medium, in 21-25 ℃ of constant temperature culture 11-19 days, punch into bacterium cake at colony edge respectively, and access liquid nutrient medium and cultivate or access solid culture medium and cultivate;
The composition of described DSE fungi liquid medium by weight percentage is counted: glucose 1.5-4.5%, and malt extract 1.5-4.5%, soy peptone 0.3-0.7%, excess water, Medium's PH Value is 50-6.0; Concussion is cultivated, culture vessel loading amount 10-40%; After liquid nutrient medium autoclaving, access DSE fungi; Seed bacterial classification inoculum concentration is the 10-20% of culture vessel loading amount; The condition of culture of liquid culture is: shaking table concussion rotating speed is 130-150r/min; Cultivate 26-30 days for 21-25 ℃; After cultivation finishes, can directly use or smash after thalline for subsequent use with refiner;
Described DSE fungus solids medium is made up of solid-based basal culture medium and liquid MEA, and the composition of solid-based basal culture medium is by weight: wheat bran: wheat husk: broad-leaved tree sawdust=1-2:1-3:1-3; The 20-40% that liquid MEA amount is above wheat bran, wheat husk, broad-leaved tree sawdust gross weight, medium pH is 5.0-6.0, leaves standstill and cultivates, culture vessel loading amount 30-70%; After solid culture medium sterilizing, access DSE fungi, bacterial classification inoculum concentration is the 10-20% of culture vessel loading amount; The condition of culture of solid culture is: cultivate 26-30 days for 21-25 ℃; In the time that mycelia is covered with whole culture matrix, cultivate and finish, can directly use or pulverize rear for subsequent use with cracker;
2), the cultivation of blueberry seedling:
(1) blueberry bud differentiation: tissue culture technology is cultivated routinely, medium used is take WPM medium as minimal medium, in every liter of WPM medium, all add sucrose or edible white granulated sugar 20-40g, agar 5-15g, the plant hormone adding is zeatin 1.0-3.0mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, medium used is take WPM medium as minimal medium, in every liter of WPM medium, all add sucrose or edible white granulated sugar 20-40g, agar 5-15g, the plant hormone adding is zeatin 0.5-2.5mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, medium used is take WPM medium as minimal medium, in every liter of WPM medium, all add sucrose or edible white granulated sugar 20-40g, agar 5-15g, the plant hormone adding is zeatin 0.5-2.5mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add sucrose or edible white granulated sugar 20-40g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is indolebutyric acid 0.5-2.5mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(5) blueberry cuttage seeding is cultivated or the cultivation of blueberry seedling or the cultivation of annual blueberry seedling:
Described blueberry cuttage seeding is cultivated: the blueberry tissue culture seedling of cultivating through subculture is after domestication by low temperature, in the stem section of clip robust growth 5-6cm is coiled take liver moss as cultivation matrix cuttage to nutrition cave, the 30-50 days that grows after cuttage, then the nutrition cave of the robust growth of clip 5-6cm, growing way homogeneous dish blueberry seedling without rhizome section as blueberry cuttage seeding;
Described blueberry seedling is cultivated: the blueberry tissue culture seedling after strengthening seedling and rooting is cultivated is transplanted to take liver moss and turfy soil in the nutritive cube of cultivation matrix after domestication by low temperature, and growth 20-40 days, is blueberry seedling;
Described annual blueberry seedling is cultivated: the above-mentioned blueberry seedling continued growth 11-13 month, be annual blueberry seedling;
3), the symbiotic cultivation of DSE fungi and blueberry:
Blueberry cuttage seeding or blueberry seedling or annual blueberry seedling and DSE fungi symbiotic cultivation by the following method:
(1) symbiotic cultivation of blueberry cuttage seeding and DSE fungi: there is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, the spacing in the rows of cultivation blueberry cuttage seeding is 3-10cm; Condition of culture is 18-25 ℃ of illumination cultivation, and intensity of illumination is 2000lx, light application time 16h/ days, and ambient humidity is 60%-80%; Place at the base portion of above-mentioned blueberry cuttage seeding after the cultivation matrix of 3/5 cave pore volume, adopt above-mentioned DSE fungal cultures and blueberry cuttage seeding symbiotic cultivation, finally with cultivation matrix, cuttage seeding is fixed, blueberry cuttage seeding starts to take root about 14 days, is grown to the blueberry seedling of well developed root system after 6 months;
(2) symbiotic cultivation of blueberry seedling and DSE fungi: there is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, the spacing in the rows of cultivation blueberry seedling is 10-30cm, condition of culture is that intermittent illumination is cultivated, intensity of illumination is 1000-3000lx, light application time 8-16h, and ambient humidity is 60-80%; Place after cultivation matrix, apply at above-mentioned blueberry seedling root DSE fungi liquid or the solid culture got ready, the addition of DSE fungi liquid culture is 30-100mL, or the addition of DSE fungus solids culture is 20-60g; Blueberry seedling and DSE mycosymbiosis all form mycorhiza structure after 60 days, continued growth is transferred to land for growing field crops and carries out commerial growing after 2 months;
(3) field production of annual blueberry seedling and DSE fungi blueberry: first dig out planting pit large Tanaka, spacing in the rows is 20-60cm, lay cultivation matrix the DSE fungi liquid culture 30-100mL that applies in planting pit again, or DSE fungus solids culture 20-60g, finally above-mentioned annual blueberry seedling is cultivated in planting pit; In blueberry process of growth, can mend every year and execute DSE fungi liquid or solid culture 1-7 time.
In order further to realize object of the present invention, described 3) cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry is liver moss.
In order further to realize object of the present invention, described 3) cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry is liver moss and turfy soil, and the ratio between these 2 kinds of material additions is by weight: liver moss: turfy soil=1-5:1-5.
In order further to realize object of the present invention, described 3) cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry is liver moss, turfy soil and vermiculite, and the ratio between these 3 kinds of material additions is by weight: liver moss: turfy soil: vermiculite=1-5:1-5:1-2.
In order further to realize object of the present invention, described 3) cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry is histosol and turfy soil, and the ratio between these 2 kinds of material additions is by weight: histosol: turfy soil=1-5:1-5.
In order further to realize object of the present invention, described 3) cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry is sandy soil and turfy soil, and the ratio between these 2 kinds of material additions is by weight: sandy soil: turfy soil=1-3:1-5.
In order further to realize object of the present invention, described 3) cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry is histosol;
In order further to realize object of the present invention, described 3) in, in DSE fungi and symbiotic cultivation step (1) the blueberry cuttage seeding of blueberry and the symbiotic cultivation of DSE fungi, place after the cultivation matrix of 3/5 cave pore volume at the base portion of blueberry cuttage seeding, be applied with the DSE fungi liquid culture 2-10mL or the DSE fungus solids culture 2-10g that get ready at blueberry cuttage seeding base portion, finally with cultivation matrix, cuttage seeding fixed.
In order further to realize object of the present invention, described 3) in, in DSE fungi and symbiotic cultivation step (1) the blueberry cuttage seeding of blueberry and the symbiotic cultivation of DSE fungi, place after 3/5 cultivation matrix of 3/5 cave pore volume at the base portion of blueberry cuttage seeding, first soak cuttage seeding with DSE fungi liquid culture, the interpolation standard of fungi liquid culture is complete submergence cuttage seeding, the time of soaking is 10-30min, then blueberry cuttage seeding is fixed with cultivation matrix.
In order further to realize object of the present invention, described DSE fungi is on October 8th, 2013 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and deposit number is: CGMCC No.8260.
The present invention can produce following good effect compared with the prior art: DSE fungi can be infected blueberry root system, and promote the growth of blueberry root system and the growth of seedling, improve the drought resistance of seedling, the present invention utilizes Mycorrhizal Technology by blueberry and DSE mycosymbiosis cultivation, solve the blueberry middle seedling rooting rate of growing seedlings low, the problems such as poor growth, can promote taking root and the growth of blueberry seedlings root of blueberry cuttage seeding, greatly shorten the growth cycle of growing seedlings; Increase the quantity of blueberry branch and the amount of growth of seedling, improve the drought resistance of blueberry seedling.DSE fungi is used for to growing seedlings and field production of blueberry, there is great theory significance and using value for the commerial growing of blueberry.Cultivation method of the present invention is simple to operate, be easy to spread, small investment, obvious effect of increasing production.
Embodiment:
Below in conjunction with accompanying drawing and instantiation, the present invention is further illustrated, protection scope of the present invention is not subject to the restriction of embodiment.
DSE fungi be a kind of Cladosporium (
cladosporium) the dendritic cladosporium of bud (
cladosporium cladosporioides), the DSE fungi below adopting is on October 8th, 2013 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and deposit number is: CGMCC No.8260.
Embodiment 1:
Apply DSE fungi and carry out the method that blueberry is grown seedlings, it is characterized in that it comprises the steps:
1, the cultivation of DSE fungi:
DSE fungi, after the slant tube actication of culture of low-temperature preservation, is transferred in the plate of PDA medium, in 23 ℃ of constant temperature culture 15 days, punch into bacterium cake at colony edge respectively with the card punch of φ 5mm, and access liquid nutrient medium and cultivate.The composition of liquid nutrient medium by weight percentage is counted: glucose 2%, and malt extract 3%, soy peptone 0.5%, excess water, Medium's PH Value is 5.5; By Erlenmeyer flask concussion cultivation, culture vessel loading amount 25%; ; After liquid nutrient medium autoclaving, access DSE fungi; Seed bacterial classification inoculum concentration is 15% of culture vessel loading amount; Shaking table concussion rotating speed is 140r/min; Cultivate 28 days for 23 ℃; Cultivation finishes to smash after thalline for subsequent use with refiner afterwards.
2, the cultivation of blueberry seedling:
(1) blueberry bud differentiation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 30g, agar 10g, zeatin 2.0mg, medium pH 5.5 in every liter of WPM medium; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 30g, agar 10g, zeatin 1.5mg, medium pH 5.5 in every liter of WPM medium; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 30g, agar 10g, zeatin 1.5mg, medium pH 5.5 in every liter of WPM medium; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add sucrose 30g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is IBA(indolebutyric acid) 1.5mg, medium pH 5.5; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(5) blueberry cuttage seeding is cultivated:
The blueberry tissue culture seedling of cultivating through subculture is after domestication by low temperature, and the stem section of clip robust growth 5-6cm is take liver moss as cultivation matrix cuttage in the nutrition cave dish in high 128 holes of 5cm, and the 30-50 days that grows after cuttage, is nutrition cave dish blueberry seedling.Then the nutrition cave of the robust growth of clip 5-6cm, growing way homogeneous dish blueberry seedling without rhizome section as blueberry cuttage seeding;
3, the symbiotic cultivation of DSE fungi and blueberry cuttage seeding:
(1) cultivation matrix: liver moss.
(2) blueberry cuttage seeding and DSE fungi symbiotic cultivation by the following method: there is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, cultivating tool is the nutrition cave dish in high 128 holes of 5 ㎝, vertical cultivation; The spacing in the rows of cultivation blueberry cuttage seeding is 3-10cm; In the liver moss of base portion tiling one deck 3/5 cave pore volume of blueberry cuttage seeding, by the above-mentioned DSE fungi liquid culture immersion cuttage seeding of getting ready, the interpolation standard of fungi liquid culture is complete submergence cuttage seeding, and the time of immersion is 10-30min, then blueberry cuttage seeding is fixed with liver moss; Cave dish is placed in glass greenhouse, and environmental temperature is 18 ~ 25 ℃, and intensity of illumination is 2000Lx, and light application time is 16h, and ambient humidity is 60%-80%.
(3) results of blueberry seedling:
Blueberry cuttage seeding starts to take root about 14 days, is grown to the blueberry seedling of well developed root system after 6 months, and can be market provides the blueberry seedling of commerial growing.
Embodiment 2:
Apply DSE fungi and carry out that blueberry is grown seedlings and a method for commerial growing, it is characterized in that it comprises the steps:
1, the cultivation of DSE fungi:
DSE fungi, after the slant tube actication of culture of low-temperature preservation, is transferred in the plate of PDA medium, in 21 ℃ of constant temperature culture 19 days, punch into bacterium cake at colony edge respectively with the card punch of φ 5mm, and access liquid nutrient medium and cultivate.The composition of liquid nutrient medium by weight percentage is counted: glucose 1.5%, and malt extract 4.5%, soy peptone 0.3%, excess water, Medium's PH Value is 5; By glass preserving jar concussion cultivation, culture vessel loading amount 10%; ; After liquid nutrient medium autoclaving, access DSE fungi; Seed bacterial classification inoculum concentration is 10% of culture vessel loading amount; Shaking table concussion rotating speed is 130r/min; Cultivate 30 days for 21 ℃; Cultivation finishes rear for subsequent use.
2, the cultivation of blueberry seedling:
(1) edible white granulated sugar 20g, agar 5g, zeatin 1.0mg, medium pH 5 are all added in blueberry bud differentiation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium in every liter of WPM medium; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 8h/ days, incubation time 120 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium is all added edible white granulated sugar 20g, agar 5g, zeatin 0.5mg, medium pH 5 in every liter of WPM medium; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 8h/ days, incubation time 120 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds edible white granulated sugar 20g, agar 5g, zeatin 0.5mg, medium pH 5 in every liter of WPM medium; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 8h/ days, incubation time 120 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add edible white granulated sugar 20g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is IBA(indolebutyric acid) 0.5mg, medium pH 5; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 8h/ days, incubation time 120 days;
(5) blueberry seedling is cultivated:
Blueberry tissue culture seedling after strengthening seedling and rooting is cultivated is transplanted to take liver moss and turfy soil in the nutritive cube of cultivation matrix after domestication by low temperature, and growth 20-40 days, is blueberry seedling;
3, the symbiotic cultivation of DSE fungi and blueberry seedling:
(1) cultivation matrix is liver moss and turfy soil, and the ratio between these 2 kinds of material additions is by weight: liver moss: turfy soil=1-5:1-5; First by liver moss with distilled water immersion 7-10 hour, make it fully absorb moisture, mix with turfy soil draining after excessive moisture.
(2) blueberry seedling and DSE fungi symbiotic cultivation by the following method:
There is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, the spacing in the rows of cultivation blueberry seedling is 10-30cm, and condition of culture is 25 ℃/18 ℃ illumination cultivation, and intensity of illumination is 1000-3000lx, light application time 8-16h, ambient humidity is 60-80%; With diameter × height at the bottom of 10cm × 20cm() nutritive cube, by after the liver moss and turfy soil mixed-matrix of the bottom placement 3/5 of nutritive cube, add the DSE fungi liquid culture 30-100mL getting ready at the root of blueberry seedling.
(3) results of blueberry seedling:
Blueberry seedling and DSE mycosymbiosis all form mycorhiza structure after 60 days, blueberry seedlings root is flourishing and grow vigorously, has branch to grow up on plant, and continued growth is transferred to land for growing field crops and carries out commerial growing after 2 months.
Embodiment 3:
Apply DSE fungi and carry out that blueberry is grown seedlings and a method for commerial growing, it is characterized in that it comprises the steps:
1, the cultivation of DSE fungi:
DSE fungi, after the slant tube actication of culture of low-temperature preservation, is transferred in the plate of PDA medium, in 25 ℃ of constant temperature culture 11 days, punch into bacterium cake at colony edge respectively with the card punch of φ 5mm, and access liquid nutrient medium and cultivate.The composition of liquid nutrient medium by weight percentage is counted: glucose 4.5%, and malt extract 1.5%, soy peptone 0.7%, excess water, Medium's PH Value is 6; By plastic bottle vessel concussion cultivation, culture vessel loading amount 40%; ; After liquid nutrient medium autoclaving, access DSE fungi; Seed bacterial classification inoculum concentration is 20% of culture vessel loading amount; Shaking table concussion rotating speed is 150r/min; Cultivate 26 days for 25 ℃; Cultivation finishes to smash after thalline for subsequent use with refiner afterwards.
2, the cultivation of blueberry seedling:
(1) blueberry bud differentiation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 40g, agar 15g, zeatin 3.0mg, medium pH 6 in every liter of WPM medium; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 40g, agar 15g, zeatin 2.5mg, medium pH 6 in every liter of WPM medium; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 40g, agar 15g, zeatin 2.5mg, medium pH 6 in every liter of WPM medium; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add sucrose 40g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is IBA(indolebutyric acid) 2.5mg, medium pH 6; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(5) blueberry seedling is cultivated:
Blueberry tissue culture seedling after strengthening seedling and rooting is cultivated is transplanted to take liver moss and turfy soil in the nutritive cube of cultivation matrix after domestication by low temperature, and growth 20-40 days, is blueberry seedling;
(6) annual blueberry seedling is cultivated: the above-mentioned blueberry seedling continued growth 11-13 month, be annual blueberry seedling.
3, the symbiotic cultivation that DSE fungi and annual blueberry seedling are cultivated:
(1) cultivation matrix is sandy soil and turfy soil, and the ratio between these 2 kinds of material additions is by weight: sandy soil: turfy soil=1-3:1-5;
(2) annual blueberry seedling and DSE fungi symbiotic cultivation by the following method: the abundant and suitable large Tanaka of soil pH cultivates at soil organic matter content, when cultivation, first dig out planting pit large Tanaka, spacing in the rows is 20-60cm, lay cultivation matrix apply DSE fungi liquid culture 30-100mL in planting pit, finally cultivates above-mentioned annual blueberry seedling in planting pit again.In blueberry process of growth, can mend every year and execute DSE fungi liquid culture 1-7 time.
(3) result of DSE fungi and annual blueberry seedling symbiotic cultivation: DSE fungi and the symbiosis of annual blueberry seedling can obviously see after 60 days that the vigorous and branch of blueberry seedling growth increases, and drought-resistant ability obviously strengthens.
Embodiment 4:
Apply DSE fungi and carry out the method that blueberry is grown seedlings, it is characterized in that it comprises the steps:
1, the cultivation of DSE fungi:
DSE fungi, after the slant tube actication of culture of low-temperature preservation, is transferred in the plate of PDA medium, in 23 ℃ of constant temperature culture 15 days, punch into bacterium cake at colony edge respectively with the card punch of φ 5mm, and access DSE fungus solids medium and cultivate.DSE fungus solids medium is made up of solid-based basal culture medium and liquid MEA, and the composition of solid-based basal culture medium is by weight: wheat bran: wheat husk: broad-leaved tree sawdust=1:1:1; Liquid MEA amount is 30% of above wheat bran, wheat husk, broad-leaved tree sawdust gross weight, and medium pH is 5.5, adopts Erlenmeyer flask 5 to leave standstill and cultivates, culture vessel loading amount 50%; After DSE fungus solids medium sterilization, access DSE fungi, bacterial classification inoculum concentration is 15% of culture vessel loading amount; The condition of culture of solid culture is: cultivate 28 days for 23 ℃; In the time that mycelia is covered with whole culture matrix, cultivate and finish, for subsequent use after pulverizing with cracker.
2, the cultivation of blueberry seedling:
(1) blueberry bud differentiation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 30g, agar 10g, zeatin 2.0mg, medium pH 5.5 in every liter of WPM medium; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 30g, agar 10g, zeatin 1.5mg, medium pH 5.5 in every liter of WPM medium; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 30g, agar 10g, zeatin 1.5mg, medium pH 5.5 in every liter of WPM medium; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add sucrose 30g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is IBA(indolebutyric acid) 1.5mg, medium pH 5.5; Leave standstill secretly and cultivate with Erlenmeyer flask, culture vessel loading amount 20%, the condition of illumination cultivation is 20 ℃ of temperature, intensity of illumination is 2000lx, light application time 12h/ days, incubation time 80 days;
(5) blueberry cuttage seeding is cultivated:
The blueberry tissue culture seedling of cultivating through subculture is after domestication by low temperature, and the stem section of clip robust growth 5-6cm is take liver moss as cultivation matrix cuttage in the nutrition cave dish in high 128 holes of 5cm, and the 30-50 days that grows after cuttage, is nutrition cave dish blueberry seedling.Then the nutrition cave of the robust growth of clip 5-6cm, growing way homogeneous dish blueberry seedling without rhizome section as blueberry cuttage seeding;
3, the symbiotic cultivation of DSE fungi and blueberry cuttage seeding:
(1) cultivation matrix: liver moss, turfy soil and vermiculite, the ratio between these 3 kinds of material additions is by weight: liver moss: turfy soil: vermiculite=1-5:1-5:1-2;
(2) blueberry cuttage seeding and DSE fungi symbiotic cultivation by the following method: there is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, cultivating tool is the nutrition cave dish in high 128 holes of 5 ㎝, vertical cultivation; The spacing in the rows of cultivation blueberry cuttage seeding is 3-10cm; At the mixed-matrix of liver moss, turfy soil and the vermiculite of base portion tiling one deck 3/5 cave pore volume of blueberry cuttage seeding, be applied with the DSE fungus solids culture 2-10g getting ready at blueberry cuttage seeding base portion, finally with the mixed-matrix of liver moss, turfy soil and vermiculite, cuttage seeding fixed; Cave dish is placed in glass greenhouse, and environmental temperature is 18 ~ 25 ℃, and intensity of illumination is 2000Lx, and light application time is 16h, and ambient humidity is 60%-80%.
(3) results of blueberry seedling:
Blueberry cuttage seeding starts to take root about 14 days, is grown to the blueberry seedling of well developed root system after 6 months, and can be market provides the blueberry seedling of commerial growing.
Embodiment 5:
Apply DSE fungi and carry out that blueberry is grown seedlings and a method for commerial growing, it is characterized in that it comprises the steps:
1, the cultivation of DSE fungi:
DSE fungi, after the slant tube actication of culture of low-temperature preservation, is transferred in the plate of PDA medium, in 21 ℃ of constant temperature culture 19 days, punch into bacterium cake at colony edge respectively with the card punch of φ 5mm, and access DSE fungus solids medium and cultivate.DSE fungus solids medium is made up of solid-based basal culture medium and liquid MEA, and the composition of solid-based basal culture medium is by weight: wheat bran: wheat husk: broad-leaved tree sawdust=1.5:2:2; Liquid MEA amount is 20% of above wheat bran, wheat husk, broad-leaved tree sawdust gross weight, and medium pH is 5.0, adopts glass preserving jar to leave standstill and cultivates, culture vessel loading amount 30%; After DSE fungus solids medium sterilization, access DSE fungi, bacterial classification inoculum concentration is 10% of culture vessel loading amount; The condition of culture of solid culture is: cultivate 30 days for 21 ℃; In the time that mycelia is covered with whole culture matrix, cultivate and finish, can directly use.
2, the cultivation of blueberry seedling:
(1) edible white granulated sugar 20g, agar 5g, zeatin 1.0mg, medium pH 5 are all added in blueberry bud differentiation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium in every liter of WPM medium; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 6h/ days, incubation time 120 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium is all added edible white granulated sugar 20g, agar 5g, zeatin 0.5mg, medium pH 5 in every liter of WPM medium; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 6h/ days, incubation time 120 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds edible white granulated sugar 20g, agar 5g, zeatin 0.5mg, medium pH 5 in every liter of WPM medium; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 6h/ days, incubation time 120 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add edible white granulated sugar 20g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is IBA(indolebutyric acid) 0.5mg, medium pH 5; Leave standstill secretly and cultivate with glass preserving jar, culture vessel loading amount 10%, the condition of illumination cultivation is 15 ℃ of temperature, intensity of illumination is 3000lx, light application time 8h/ days, incubation time 120 days;
(5) blueberry seedling is cultivated:
Blueberry tissue culture seedling after strengthening seedling and rooting is cultivated is transplanted to take liver moss and turfy soil in the nutritive cube of cultivation matrix after domestication by low temperature, and growth 20-40 days, is blueberry seedling;
3, the symbiotic cultivation of DSE fungi and blueberry seedling:
(1) cultivation matrix is histosol and turfy soil, and the ratio between these 2 kinds of material additions is by weight: histosol: turfy soil=1-5:1-5;
(2) blueberry seedling and DSE fungi symbiotic cultivation by the following method:
There is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, the spacing in the rows of cultivation blueberry seedling is 10-30cm, and condition of culture is 25 ℃/18 ℃ illumination cultivation, and intensity of illumination is 1000-3000lx, light application time 8-16h, ambient humidity is 60-80%; With diameter × height at the bottom of 10cm × 20cm() nutritive cube, by after the histosol and turfy soil mixed-matrix of the bottom placement 3/5 of nutritive cube, add DSE fungus solids culture 20-60g.
(3) results of blueberry seedling:
Blueberry seedling and DSE mycosymbiosis all form mycorhiza structure after 60 days, blueberry seedlings root is flourishing and grow vigorously, has branch to grow up on plant, and continued growth is transferred to land for growing field crops and carries out commerial growing after 2 months.
Embodiment 6:
Apply DSE fungi and carry out that blueberry is grown seedlings and a method for commerial growing, it is characterized in that it comprises the steps:
1, the cultivation of DSE fungi:
DSE fungi, after the slant tube actication of culture of low-temperature preservation, is transferred in the plate of PDA medium, in 25 ℃ of constant temperature culture 11 days, punch into bacterium cake at colony edge respectively with the card punch of φ 5mm, and access DSE fungus solids medium and cultivate.DSE fungus solids medium is made up of solid-based basal culture medium and liquid MEA, and the composition of solid-based basal culture medium is by weight: wheat bran: wheat husk: broad-leaved tree sawdust=2:3:3; Liquid MEA amount is 40% of above wheat bran, wheat husk, broad-leaved tree sawdust gross weight, and medium pH is 6.0, adopts plastic bag container to leave standstill and cultivates, culture vessel loading amount 70%; After DSE fungus solids medium sterilization, access DSE fungi, bacterial classification inoculum concentration is 120% of culture vessel loading amount; The condition of culture of solid culture is: cultivate 26 days for 25 ℃; In the time that mycelia is covered with whole culture matrix, cultivate and finish, for subsequent use after pulverizing with cracker.
2, the cultivation of blueberry seedling:
(1) blueberry bud differentiation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 40g, agar 15g, zeatin 3.0mg, medium pH 6 in every liter of WPM medium; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 40g, agar 15g, zeatin 2.5mg, medium pH 6 in every liter of WPM medium; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, and medium used is take WPM medium as minimal medium, all adds sucrose 40g, agar 15g, zeatin 2.5mg, medium pH 6 in every liter of WPM medium; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add sucrose 40g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is IBA(indolebutyric acid) 2.5mg, medium pH 6; Leave standstill secretly and cultivate with plastic bottle vessel, culture vessel loading amount 30%, the condition of illumination cultivation is 25 ℃ of temperature, intensity of illumination is 1000lx, light application time 16h/ days, incubation time 40 days;
(5) blueberry seedling is cultivated:
Blueberry tissue culture seedling after strengthening seedling and rooting is cultivated is transplanted to take liver moss and turfy soil in the nutritive cube of cultivation matrix after domestication by low temperature, and growth 20-40 days, is blueberry seedling;
(6) annual blueberry seedling is cultivated: the above-mentioned blueberry seedling continued growth 11-13 month, be annual blueberry seedling.
3, the symbiotic cultivation that DSE fungi and annual blueberry seedling are cultivated:
(1) cultivation matrix is histosol;
(2) annual blueberry seedling and DSE fungi symbiotic cultivation by the following method: the abundant and suitable large Tanaka of soil pH cultivates at soil organic matter content, when cultivation, first dig out planting pit large Tanaka, spacing in the rows is 20-60cm, lay cultivation matrix apply above-mentioned DSE fungus solids culture 20-60g in planting pit, finally cultivates above-mentioned annual blueberry seedling in planting pit again.In blueberry process of growth, can mend every year and execute DSE fungus solids culture 1-7 time.
(3) result of DSE fungi and annual blueberry seedling symbiotic cultivation: DSE fungi and the symbiosis of annual blueberry seedling can obviously see after 60 days that the vigorous and branch of blueberry seedling growth increases, and drought-resistant ability obviously strengthens, for the commerial growing of blueberry provides guarantee.
Comparative example one: the grow seedlings effect of DSE fungi to blueberry cuttage seeding
When cultivation with the growing state that adds the blueberry cuttage seeding of sterilized liquid medium in contrast.The blueberry cuttage seeding of clip is soaked 10-30min with sterilized liquid medium, then blueberry cuttage seeding is fixed with liver moss.The condition of culture of glass greenhouse is 18-25 ℃ of illumination cultivation, and intensity of illumination is 2000lx, light application time 16h/ days, and ambient humidity is 60%-80%.
Blueberry cuttage seeding is gathered in the crops part blueberry seedling (comprising the blueberry seedling of contrast and embodiment 1) after 1 month in greenhouse culture, take back fresh weight, height of seedling, root system overall length and the root surface area of laboratory statistical analysis acrial part together with cultivation matrix, the blueberry seedling of results, in 60 ℃ of oven for drying to constant weight, is measured to weight.
Statistic analysis result shows, compares with the nonvaccinated blueberry cuttage seeding that contrasts, and has increased 29%-43% with the acrial part height of the blueberry cuttage seeding of DSE mycosymbiosis cultivation, and fresh weight has increased 50%-80%, and dry weight has increased 14%-221%.
Comparative example two: the growth effects of DSE fungi to blueberry seedling
When cultivation with the growing state that adds the blueberry seedling of sterilized liquid medium in contrast.Symbiotic cultivation matrix treatments method, first uses liver moss distilled water immersion 7-10 hour, makes it fully absorb moisture, draining excessive moisture, and turfy soil mixes in the ratio of 1:1.By the matrix of nutritive cube bottom placement 3/5, then place at the root of blueberry seedling the medium of the sterilized liquid 30mL preparing, the root of blueberry seedling is fully contacted with it, finally with liver moss and turfy soil mixed-matrix, blueberry seedling is fixed.All blueberry seedling are all cultivated in glass greenhouse, and condition of culture is 18-25 ℃ of illumination cultivation, and intensity of illumination is 2000lx, light application time 16h/ days, and ambient humidity is 60%-80%.
Part blueberry seedling (comprising the blueberry seedling of contrast and embodiment 2) is gathered in the crops in the cultivation of blueberry seedling after 2 months, take back fresh weight, height of seedling, root system overall length and the root surface area of laboratory statistical analysis acrial part together with matrix, by the blueberry seedling of results in 60 ℃ of oven for drying to constant weight and measure weight.Statistic analysis result shows, contrasts blueberry seedling and compares with nonvaccinated, increased 40%-65% with the acrial part height of the blueberry seedling of DSE mycosymbiosis cultivation, and fresh weight has increased 60%-70%, and dry weight has increased 50%-60%.
Can find out from above-mentioned comparative example, DSE fungi significantly promotes the growth of taking root of blueberry cuttage seeding and blueberry seedling, the blueberry seedling that connects bacteria cultivation is compared with contrasting blueberry seedling, and the content that connects rooting rate, fresh weight and the dry weight of the blueberry seedling of bacterium does not more connect all improving a lot of bacterium.On the other hand, the present invention can significantly shorten the growing-seedling period of blueberry, the various resistances of the blueberry seedling after Mycorrhizal contrast blueberry seedling simultaneously very large enhancing, especially drought-resistant ability, reduce the requirement to planting environment, the green high quality high yield that guarantees blueberry, this invention is applicable to the whole process of blueberry from the field production of growing seedlings.
Above embodiment is described the preferred embodiment of the present invention; not scope of the present invention is limited; design under the prerequisite of spirit not departing from the present invention; various distortion and improvement that the common engineers and technicians in this area make technical scheme of the present invention, all should fall in the definite protection domain of claims of the present invention.
Claims (10)
1. apply DSE fungi and carry out that blueberry is grown seedlings and a method for commerial growing, it is characterized in that it comprises the steps:
1), the cultivation of DSE fungi:
By DSE fungi after the slant tube actication of culture of low-temperature preservation, transfer in the plate of PDA medium, in 21-25 ℃ of constant temperature culture 11-19 days, punch into bacterium cake at colony edge respectively, and access liquid nutrient medium and cultivate or access solid culture medium and cultivate;
The composition of described DSE fungi liquid medium by weight percentage is counted: glucose 1.5-4.5%, and malt extract 1.5-4.5%, soy peptone 0.3-0.7%, excess water, Medium's PH Value is 50-6.0; Concussion is cultivated, culture vessel loading amount 10-40%; After liquid nutrient medium autoclaving, access DSE fungi; Seed bacterial classification inoculum concentration is the 10-20% of culture vessel loading amount; The condition of culture of liquid culture is: shaking table concussion rotating speed is 130-150r/min; Cultivate 26-30 days for 21-25 ℃; After cultivation finishes, can directly use or smash after thalline for subsequent use with refiner;
Described DSE fungus solids medium is made up of solid-based basal culture medium and liquid MEA, and the composition of solid-based basal culture medium is by weight: wheat bran: wheat husk: broad-leaved tree sawdust=1-2:1-3:1-3; The 20-40% that liquid MEA amount is above wheat bran, wheat husk, broad-leaved tree sawdust gross weight, medium pH is 5.0-6.0, leaves standstill and cultivates, culture vessel loading amount 30-70%; After solid culture medium sterilizing, access DSE fungi, bacterial classification inoculum concentration is the 10-20% of culture vessel loading amount; The condition of culture of solid culture is: cultivate 26-30 days for 21-25 ℃; In the time that mycelia is covered with whole culture matrix, cultivate and finish, can directly use or pulverize rear for subsequent use with cracker;
2), the cultivation of blueberry seedling:
(1) blueberry bud differentiation: tissue culture technology is cultivated routinely, medium used is take WPM medium as minimal medium, in every liter of WPM medium, all add sucrose or edible white granulated sugar 20-40g, agar 5-15g, the plant hormone adding is zeatin 1.0-3.0mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(2) blueberry clump bud propagation: tissue culture technology is cultivated routinely, medium used is take WPM medium as minimal medium, in every liter of WPM medium, all add sucrose or edible white granulated sugar 20-40g, agar 5-15g, the plant hormone adding is zeatin 0.5-2.5mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(3)) blueberry seedling subculture is cultivated: tissue culture technology is cultivated routinely, medium used is take WPM medium as minimal medium, in every liter of WPM medium, all add sucrose or edible white granulated sugar 20-40g, agar 5-15g, the plant hormone adding is zeatin 0.5-2.5mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(4) blueberry strengthening seedling and rooting: tissue culture technology is cultivated routinely, medium used is take 1/2WPM medium as minimal medium, in every liter of 1/2WPM medium, all add sucrose or edible white granulated sugar 20-40g, be paved with the bottom of culture vessel used with filter paper ball or little cotton mass, the plant hormone adding is indolebutyric acid 0.5-2.5mg, medium pH 5.0-6.0; Leave standstill dark cultivation, culture vessel loading amount 10-30%, the condition of illumination cultivation is temperature 15-25 ℃, intensity of illumination is 1000-3000lx, light application time 8-16h/ days, incubation time 40-120 days;
(5) blueberry cuttage seeding is cultivated or the cultivation of blueberry seedling or the cultivation of annual blueberry seedling:
Described blueberry cuttage seeding is cultivated: the blueberry tissue culture seedling of cultivating through subculture is after domestication by low temperature, in the stem section of clip robust growth 5-6cm is coiled take liver moss as cultivation matrix cuttage to nutrition cave, the 30-50 days that grows after cuttage, then the nutrition cave of the robust growth of clip 5-6cm, growing way homogeneous dish blueberry seedling without rhizome section as blueberry cuttage seeding;
Described blueberry seedling is cultivated: the blueberry tissue culture seedling after strengthening seedling and rooting is cultivated is transplanted to take liver moss and turfy soil in the nutritive cube of cultivation matrix after domestication by low temperature, and growth 20-40 days, is blueberry seedling;
Described annual blueberry seedling is cultivated: the above-mentioned blueberry seedling continued growth 11-13 month, be annual blueberry seedling;
3), the symbiotic cultivation of DSE fungi and blueberry:
Blueberry cuttage seeding or blueberry seedling or annual blueberry seedling and DSE fungi symbiotic cultivation by the following method:
(1) symbiotic cultivation of blueberry cuttage seeding and DSE fungi: there is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, the spacing in the rows of cultivation blueberry cuttage seeding is 3-10cm; Condition of culture is 18-25 ℃ of illumination cultivation, and intensity of illumination is 2000lx, light application time 16h/ days, and ambient humidity is 60%-80%; Place at the base portion of above-mentioned blueberry cuttage seeding after the cultivation matrix of 3/5 cave pore volume, adopt above-mentioned DSE fungal cultures and blueberry cuttage seeding symbiotic cultivation, finally with cultivation matrix, cuttage seeding is fixed, blueberry cuttage seeding starts to take root about 14 days, is grown to the blueberry seedling of well developed root system after 6 months;
(2) symbiotic cultivation of blueberry seedling and DSE fungi: there is the outdoor booth of sunshade net, or Plastic film greenhouse, or in glass greenhouse, grow seedlings to carry and train, the spacing in the rows of cultivation blueberry seedling is 10-30cm, condition of culture is that intermittent illumination is cultivated, intensity of illumination is 1000-3000lx, light application time 8-16h, and ambient humidity is 60-80%; Place after cultivation matrix, apply at above-mentioned blueberry seedling root DSE fungi liquid or the solid culture got ready, the addition of DSE fungi liquid culture is 30-100mL, or the addition of DSE fungus solids culture is 20-60g; Blueberry seedling and DSE mycosymbiosis all form mycorhiza structure after 60 days, continued growth is transferred to land for growing field crops and carries out commerial growing after 2 months;
(3) field production of annual blueberry seedling and DSE fungi blueberry: first dig out planting pit large Tanaka, spacing in the rows is 20-60cm, lay cultivation matrix the DSE fungi liquid culture 30-100mL that applies in planting pit again, or DSE fungus solids culture 20-60g, finally above-mentioned annual blueberry seedling is cultivated in planting pit;
In blueberry process of growth, can mend every year and execute DSE fungi liquid or solid culture 1-7 time.
2. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry be liver moss.
3. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry be liver moss and turfy soil, the ratio between these 2 kinds of material additions is by weight: liver moss: turfy soil=1-5:1-5.
4. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry be liver moss, turfy soil and vermiculite, the ratio between these 3 kinds of material additions is by weight: liver moss: turfy soil: vermiculite=1-5:1-5:1-2.
5. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry be histosol and turfy soil, the ratio between these 2 kinds of material additions is by weight: histosol: turfy soil=1-5:1-5.
6. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry be sandy soil and turfy soil, the ratio between these 2 kinds of material additions is by weight: sandy soil: turfy soil=1-3:1-5.
7. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in cultivation matrix in the symbiotic cultivation of DSE fungi and blueberry be histosol.
8. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in place after the cultivation matrix of 3/5 cave pore volume at the base portion of blueberry cuttage seeding in DSE fungi and symbiotic cultivation step (1) the blueberry cuttage seeding of blueberry and the symbiotic cultivation of DSE fungi, be applied with the DSE fungi liquid culture 2-10mL or the DSE fungus solids culture 2-10g that get ready at blueberry cuttage seeding base portion, finally with cultivation matrix, cuttage seeding fixed.
9. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, it is characterized in that described 3) in place after 3/5 cultivation matrix of 3/5 cave pore volume at the base portion of blueberry cuttage seeding in DSE fungi and symbiotic cultivation step (1) the blueberry cuttage seeding of blueberry and the symbiotic cultivation of DSE fungi, first soak cuttage seeding with DSE fungi liquid culture, the interpolation standard of fungi liquid culture is complete submergence cuttage seeding, the time of soaking is 10-30min, then blueberry cuttage seeding is fixed with cultivation matrix.
10. a kind of DSE of application fungi according to claim 1 carries out that blueberry is grown seedlings and the method for commerial growing, DSE fungi described in it is characterized in that is on October 8th, 2013 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and deposit number is: CGMCC No.8260.
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| CN104823739A (en) * | 2015-04-20 | 2015-08-12 | 安徽师范大学 | Drought-resistant blueberry and cultivation method thereof |
| CN105052498A (en) * | 2015-08-20 | 2015-11-18 | 云南大学 | Liquid culturing method for establishing DSE and corn symbiotic system |
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| CN105613232A (en) * | 2015-12-17 | 2016-06-01 | 青岛沃林蓝莓果业有限公司 | Blueberry tissue culture seedling transplantation substrate and preparation method thereof |
| CN105918127B (en) * | 2016-05-20 | 2018-05-01 | 武汉理工大学 | A kind of method that DSE- plant symbiosis systems are established based on DSE fast-propagations |
| CN105918127A (en) * | 2016-05-20 | 2016-09-07 | 武汉理工大学 | Method for building DSE-plant symbiosis system based on DSE rapid expanding propagation |
| CN106550750A (en) * | 2016-11-09 | 2017-04-05 | 广东融和生态农业集团有限公司 | It is a kind of to improve the method that blue berry seedling cultivates quality |
| CN107083335A (en) * | 2017-04-25 | 2017-08-22 | 鲁东大学 | One plant of DSE fungi and the method for the quick Mycorrhizal of blueberry tissue culture seedling |
| CN107129935A (en) * | 2017-04-25 | 2017-09-05 | 鲁东大学 | A kind of DSE bacterium and its application in blueberry growth and drought resisting is improved |
| CN107083335B (en) * | 2017-04-25 | 2020-05-26 | 鲁东大学 | Method for rapidly mycorrhizating DSE fungi and blueberry tissue culture seedlings |
| CN107129935B (en) * | 2017-04-25 | 2020-06-19 | 鲁东大学 | DSE (Deuterorhizobium-beta) bacterium and application thereof in improving growth and drought resistance of blueberries |
| CN110200018A (en) * | 2019-06-12 | 2019-09-06 | 中国矿业大学(北京) | It is a kind of promote plant establishment best DSE connect bacterium amount |
| CN110200018B (en) * | 2019-06-12 | 2020-10-16 | 中国矿业大学(北京) | Optimal DSE inoculation amount for promoting plant rooting |
| CN110591923A (en) * | 2019-08-09 | 2019-12-20 | 云南大学 | Method for quickly obtaining mass DSE mycelium inoculant |
| CN112790107A (en) * | 2021-01-18 | 2021-05-14 | 安徽师范大学 | Method for promoting growth of blueberry seedlings |
| CN116463220A (en) * | 2023-03-06 | 2023-07-21 | 贵州大学 | Dark-color DSE fungus for promoting blueberry growth and application thereof |
| CN116463220B (en) * | 2023-03-06 | 2024-03-19 | 贵州大学 | Dark-color DSE fungus for promoting blueberry growth and application thereof |
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