CN105296377A - Bacillus licheniformis and microbial agent and application of bacillus licheniformis and microbial agent to prevention and treatment of plant continuous cropping - Google Patents
Bacillus licheniformis and microbial agent and application of bacillus licheniformis and microbial agent to prevention and treatment of plant continuous cropping Download PDFInfo
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Abstract
The invention provides bacillus licheniformis with the preservation number being CCTCC No. M2013495. The invention further provides a microbial agent containing the bacillus licheniformis, other strains and auxiliaries. The invention also provides application of the bacillus licheniformis or the microbial agent to the prevention and treatment of plant continuous cropping. By the technical scheme, the prevention and treatment level of the plant continuous cropping can be increased greatly.
Description
Technical field
The present invention relates to agricultural biological technical field, particularly, relate to a kind of Bacillus licheniformis, a kind of microbiobacterial agent and their application in control plant continuous cropping disease.
Background technology
In recent years, along with global Desertification, desertification situation increasingly serious, can plough soil fewer and feweri, and the contradiction that China has a large population and a few land is more outstanding, raise the multiple-cropping index (namely use continuous cropping farming) is unique way of dealing with problems.But the continuous cropping diseases such as consequent soil-borne disease cause crop to drop in production over a large area, have no harvest.Continuous cropping continuous cropping makes many crops as leguminous plants, melon, vegetables, strawberry and some herbal medicine etc., under the envrionment conditions of continuous cropping, the pathogenic micro-organisms such as fungus and bacterium are through amount reproduction, the root system of continuous infringement plant, causes plant to fall ill, poor growth, and then cause crop blight, leaf blight, the harm such as virus disease, have a strong impact on plant growth.
At present, to continuous cropping disease process mode comprise: crop rotation plantation, vexed canopy sterilizing, applying organic manure, plough deeply vegetable soil, chemical agent sterilize and select high disease resistant plant etc.Also occurred that use microorganism prevents and treats continuous cropping disease recently, such as CN102382810A provides one, this microbial inoculum is by subtilis (Bacillussubtilis), Bacillus licheniformis (Bacilluslicheniformis), shellfish thunder trichosporon (Trichosporonbehrendii), Paecilomyces lilacinus (Paecilomyceslilacinus), streptomyces microflavus (Streptomycesmicroflavus), bacillus polymyxa (Paenibacilluspolymyxa) and Metarhizium anisopliae (Metarhiziumanisopliae) mixing.But experiment proves, the sick prevention and control capability of continuous cropping of the bacterial classification in this microbial inoculum also needs further raising.
Summary of the invention
In order to the sick ability of the anti-continuous cropping improving microbial strains further, the present inventor, through screening repeatedly for many years, obtains a strain has very excellent prevention and control capability Bacillus licheniformis to continuous cropping disease, resulting in the present invention.The invention provides a kind of Bacillus licheniformis, this Bacillus licheniformis has very excellent prevention and control capability to continuous cropping disease.
The invention provides a kind of Bacillus licheniformis (Bacilluslicheniformis), the deposit number of this Bacillus licheniformis is CCTCCNO.M2013495.
Present invention also offers a kind of microbiobacterial agent, this microbiobacterial agent contains Bacillus licheniformis as above and auxiliary material.
Present invention also offers Bacillus licheniformis as above or the application of microbiobacterial agent as above in control plant continuous cropping disease.
By technique scheme, the present invention can promote significantly and prevent and treat level to plant continuous cropping disease.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Biomaterial preservation
Bacillus licheniformis of the present invention (Bacilluslicheniformis) is the pure growth that the present inventor is separated from the practice ground soil of Nong Feng Technology Park, Tianjin Jinghai County, its deposit number is CCTCCNO:M2013495, preservation date is on October 24th, 2013, depositary institution is China typical culture collection center, address is positioned at Wuhan, China Wuhan University, and Classification And Nomenclature is Bacilluslicheniformis.
Subtilis of the present invention (Bacillussubtilis) is the pure growth that the present inventor is separated from the practice ground soil of Nong Feng Technology Park, Tianjin Jinghai County, its deposit number is CCTCCNO:M2013496, preservation date is on October 24th, 2013, depositary institution is China typical culture collection center, address is positioned at Wuhan, China Wuhan University, and Classification And Nomenclature is Bacillussubtilis.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a kind of Bacillus licheniformis (Bacilluslicheniformis), the deposit number of this Bacillus licheniformis is CCTCCNO.M2013495.
Bacillus licheniformis as above provided by the invention can survive and growth and breeding in the bacteria culture medium of routine.Such as, described bacteria culture medium can comprise at least one in Zulkovsky starch substratum, LB substratum and maize powder medium.Under preferable case, described bacteria culture medium contains the ferrous sulfate of the bean powder of 30-250g/L, the starch of 20-120g/L, the sodium-chlor of 0.2-4g/L, the calcium carbonate of 0.3-1g/L, the potassium primary phosphate of 0.1-0.8g/L and 3-15g/L.
The present invention Bacillus licheniformis as above is the very excellent plant root probiotic bacterium of a strain, effectively can suppress the generation of continuous cropping disease.
In order to preserve described Bacillus licheniformis for a long time, Bacillus licheniformis can be kept in glycerine conserving liquid, storage temperature is-85 DEG C to-20 DEG C.
Present invention also offers a kind of microbiobacterial agent, this microbiobacterial agent contains Bacillus licheniformis as above and auxiliary material.
Wherein, the not special requirement of content of the Bacillus licheniformis in this microbiobacterial agent, can be the content of microbial fertilizer field and microbial pesticide field routine, such as, relative to the microbiobacterial agent of 1g, the content of described Bacillus licheniformis be 10
6-10
11cFU; More preferably, relative to the microbiobacterial agent of 1g, the content of described Bacillus licheniformis is 10
8-10
9cFU.
Wherein, in the particularly preferred a kind of embodiment of the present invention, this microbiobacterial agent is also containing subtilis, and the deposit number of described subtilis is CCTCCNO.M2013496; And described Bacillus licheniformis is 1:(0.1-0.3 with the CFU ratio of described subtilis).In this preferred implementation, described Bacillus licheniformis and described subtilis can play synergy, and microbiobacterial agent of the present invention can obtain the sick prevention effect of good continuous cropping with lower amount of application.
Wherein, the not special requirement of content of the auxiliary material in this microbiobacterial agent, such as, relative to 10
9the described Bacillus licheniformis of CFU, the content of auxiliary material can be 0.01-100g, is preferably 0.1-10g.
Wherein, described auxiliary material can be the selection of microbial fertilizer field and microbial pesticide field routine, and such as, described auxiliary material can contain at least one in dispersion agent, nutrition agent and inert support.
Wherein, the not special requirement of content of the dispersion agent in this microbiobacterial agent, nutrition agent and inert support, such as, relative to 10
9the described Bacillus licheniformis of CFU, the content of auxiliary material can be 0.01-100g, is preferably 0.1-10g.
Described dispersion agent for material particles can be impelled to be dispersed in medium the material forming stable suspersion system, such as, can comprise at least one in polycarboxylate, sodium lignosulfonate, calcium lignin sulphonate, dialkyl sodium sulfosuccinate, sodium alkylarysulfonate, polyoxyethylene alkyl phenyl ether, tripoly phosphate sodium STPP, polyxyethylated aryl phosphide, polyoxyethylene alkylaryl ether, polyxyethylated aryl polymer, polyalkylene glycol alkyl phenyl ether polyoxyethylene nonylphenol ether, sulfonation formaldehyde sodium naphthalene, Triton X-100 and tween 80.
Described nutrition agent for providing the material of carbon source for the growth of Bacillus licheniformis, such as, can comprise at least one in the peat composed of rotten mosses, soyflour, rice, wheat, dextrin, collagen, grape sugar and starch.
Described inert support for providing supporting matrix but having the material of inertia, such as, can comprise at least one in calcium carbonate, loess, diatomite, wilkinite, talcum powder, mica powder, kaolin and calcite in powder.
Wherein, can also containing at least one be selected from preservatives, wetting agent, attractive substance, capsule agent, bonding agent, emulsifying agent, dyestuff, ultraviolet protective agent, buffer reagent and flow agent in microbiobacterial agent of the present invention.
Wherein, the not special requirement of formulation of described microbiobacterial agent, can be the formulation of the conventional various microbiobacterial agents used, such as, can be pulvis and/or granule.
Wherein, under preferable case, so-called microbiobacterial agent is granule, and described auxiliary material contains the peat composed of rotten mosses, collagen, tripoly phosphate sodium STPP, calcium carbonate and starch; Relative to 10
9the described Bacillus licheniformis of CFU, the content of the peat composed of rotten mosses is 0.3-3g, and the content of collagen is 0.01-0.1g, and the content of tripoly phosphate sodium STPP is the content of calcium carbonate is 0.1-0.5g, and the content of starch is 0.1-1g.
The Bacillus licheniformis (Bacilluslicheniformis) that the present invention's microbiobacterial agent as above can use deposit number to be CCTCCNO.M2013495, prepared by the microbiobacterial agent preparation method of routine, such as, can be that the Bacillus licheniformis of CCTCCNO.M2013495 is cultivated in liquid bacteria culture medium by deposit number, then nutrient solution be mixed with auxiliary material.
Wherein, bacteria culture medium can be the conventional bacteria culture medium used, such as, can comprise at least one in Zulkovsky starch substratum, LB substratum and maize powder medium.Zulkovsky starch substratum can contain the Zulkovsky starch of 10-30g/L, the KNO of 0.5-2g/L
3, 0.1-1g/L K
2hPO
4, 0.1-1g/L MgSO
4with the NaCl of 0.1-1g/L; LB substratum can contain the NaCl of the peptone of 5-20g/L, the yeast extract of 3-7g/L and 3-7g/L; Maize powder medium can contain the Semen Maydis powder of 10-30g/L.Under preferable case, described bacteria culture medium contains the ferrous sulfate of the bean powder of 30-250g/L, the starch of 20-120g/L, the sodium-chlor of 0.2-4g/L, the calcium carbonate of 0.3-1g/L, the potassium primary phosphate of 0.1-0.8g/L and 3-15g/L.
Wherein, the process of cultivation can comprise plate streaking activation, first order seed is cultivated, secondary seed is cultivated and three grades of enlarged culturing.Such as, the process of cultivation can comprise: the slant strains plate streak of preservation activates by (1); (2) from picking list bacterium colony flat board, and enlarged culturing makes the first order seed of 10-50mL; (3) first order seed is carried out secondary enlarged culturing according to 5-30 enlarged culturing multiple doubly and make secondary seed; (4) secondary seed is carried out three grades of enlarged culturing according to 5-30 enlarged culturing multiple doubly.
Wherein, the condition of activation and cultivation at different levels can comprise: temperature is 30-37 DEG C, and pH value is 7.2-7.6, and incubation time is 8-20h, and ventilation is 8-10m
3/ h, needs rotating speed when stirring or shake to be 200-300r/min.
Wherein, the condition mixed with auxiliary material by nutrient solution can comprise: the temperature of mixing can be 10-35 DEG C, and the time of mixing can be 10-30 minute.Wherein, 1-10 hour can be left standstill after having mixed.Wherein, after being mixed with auxiliary material by nutrient solution, mixed material can be carried out drying, pulverizing or granulation, to facilitate storage, transport and to use.
Wherein, in the particularly preferred a kind of embodiment of the present invention, this microbiobacterial agent is also containing subtilis, and the deposit number of described subtilis is CCTCCNO.M2013496; And described Bacillus licheniformis is 1:(0.1-0.3 with the CFU ratio of described subtilis).The subtilis of can be the Bacillus licheniformis of CCTCCNO.M2013495 and deposit number by deposit number the be CCTCCNO.M2013496 of the microbiobacterial agent in this preferred implementation is cultivated in liquid bacteria culture medium, is then mixed with auxiliary material by nutrient solution.Such as, the secondary seed solution of the secondary seed solution of described Bacillus licheniformis and described subtilis can be mixed, then carry out three grades of enlarged culturing according to 5-30 enlarged culturing multiple doubly; Then nutrient solution is mixed with auxiliary material.
Present invention also offers Bacillus licheniformis as above or the application of microbiobacterial agent as above in control plant continuous cropping disease.
Wherein, described plant is at least one in plant of Solanaceae, cucurbitaceous plant, Malvaceae plant, rosaceous plant and vitaceae.
Wherein, described plant of Solanaceae comprises at least one in tomato, capsicum and eggplant; Described cucurbitaceous plant comprises at least one in cucumber, watermelon and muskmelon; Described Malvaceae plant comprises at least one in cotton, piemarker and hibiscus cannabinus; Described rosaceous plant comprises at least one in apple, Chinese pear-leaved crabapple, Malus spectabilis, pears, peach, Lee, apricot, plum, cherry, loquat, hawthorn, strawberry and raspberry; Described vitaceae comprises grape.
Wherein, in use, can the nutrient solution of Bacillus licheniformis as above or microbiobacterial agent as above be sprayed onto in the root soil of plant, or use the nutrient solution of Bacillus licheniformis as above microbiobacterial agent as above to dip in root before transplantation, or microbiobacterial agent as above is embedded in plant soil.
Below, by embodiment the present invention for durther example details, but scope of the present invention is not restricted in following examples.
Embodiment 1
The cultivation of the present embodiment for illustration of Bacillus licheniformis of the present invention and the preparation of microbiobacterial agent.
Be that the slant strains of the Bacillus licheniformis (Bacilluslicheniformis) of CCTCCNO:M2013495 is at the LB flat board (peptone containing 10g/L by deposit number, the yeast extract of 5g/L, the agarose of NaCl and 3g/L of 5g/L, pH value 7.4, identical below) upper line, and cultivate 15 hours at 37 DEG C, picking list bacterium colony from flat board, be inoculated in the LB liquid nutrient medium (peptone containing 10g/L of 25mL, the yeast extract of 5g/L and the NaCl of 5g/L, pH value 7.4, identical below) in, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain first order seed, 20mL first order seed is added in the LB liquid nutrient medium of 500mL, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain secondary seed, the secondary seed of 2L is added in the LB liquid nutrient medium of 50L, at 37 DEG C 250r/min stirring under and 10m
3cultivate 15 hours under the ventilation of/h, obtain nutrient solution.The blood cell plate counting process cell concentration recorded in nutrient solution is adopted to be 10
9cFU/mL.
The starch of the tripoly phosphate sodium STPP of the collagen of the peat composed of rotten mosses of 1 weight part, 0.05 weight part, 0.005 weight part, the calcium carbonate of 0.3 weight part and 0.5 weight part is mixed to get auxiliary material.Above-mentioned for 50L nutrient solution is mixed with 50kg auxiliary material, and is 75kg by mixed dry materials ad pond om, then granulate as diameter is about the particle of about 8mm, namely obtain microbiobacterial agent of the present invention.
Comparative example 1
Glad composite microbiological fertilizer DX-01 is buied from Shijiazhuang Gold Sun Biological Organic Fertilizer Co., Ltd., get this fertilizer 1g, add 10mL physiological saline, soak homogenate after 10 minutes, by homogenate filtered through gauze, filtrate is inoculated on LB flat board, after cultivating 12h at 37 DEG C, single bacterium colony of picking genus bacillus, and the classification differentiating genus bacillus by 16SrDNA method, obtain a bacillus licheniformis.
By the slant strains of above-mentioned Bacillus licheniformis at the LB flat board (peptone containing 10g/L, the yeast extract of 5g/L, the agarose of NaCl and 3g/L of 5g/L, pH value 7.4, identical below) upper line, and cultivate 15 hours at 37 DEG C, picking list bacterium colony from flat board, be inoculated in the LB liquid nutrient medium (peptone containing 10g/L of 25mL, the yeast extract of 5g/L and the NaCl of 5g/L, pH value 7.4, identical below) in, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain first order seed, 20mL first order seed is added in the LB liquid nutrient medium of 500mL, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain secondary seed, the secondary seed of 2L is added in the LB liquid nutrient medium of 50L, at 37 DEG C 250r/min stirring under and 10m
3cultivate 18 hours under the ventilation of/h, obtain nutrient solution.The blood cell plate counting process cell concentration recorded in nutrient solution is adopted to be 10
9cFU/mL.
The starch of the tripoly phosphate sodium STPP of the collagen of the peat composed of rotten mosses of 1 weight part, 0.05 weight part, 0.005 weight part, the calcium carbonate of 0.3 weight part and 0.5 weight part is mixed to get auxiliary material.Above-mentioned for 50L nutrient solution is mixed with 50kg auxiliary material, and is 75kg by mixed dry materials ad pond om, then granulate as diameter is about the particle of about 8mm, namely obtain the microbiobacterial agent of this comparative example.
Comparative example 2
Buy a bacillus licheniformis from U.S. ATCC, the article No. of this Bacillus licheniformis is 10716.
By the slant strains of above-mentioned Bacillus licheniformis at the LB flat board (peptone containing 10g/L, the yeast extract of 5g/L, the agarose of NaCl and 3g/L of 5g/L, pH value 7.4, identical below) upper line, and cultivate 15 hours at 37 DEG C, picking list bacterium colony from flat board, be inoculated in the LB liquid nutrient medium (peptone containing 10g/L of 25mL, the yeast extract of 5g/L and the NaCl of 5g/L, pH value 7.4, identical below) in, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain first order seed, 20mL first order seed is added in the LB liquid nutrient medium of 500mL, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain secondary seed, the secondary seed of 2L is added in the LB liquid nutrient medium of 50L, at 37 DEG C 250r/min stirring under and 10m
3cultivate 20 hours under the ventilation of/h, obtain nutrient solution.The blood cell plate counting process cell concentration recorded in nutrient solution is adopted to be 10
9cFU/mL.
The starch of the tripoly phosphate sodium STPP of the collagen of the peat composed of rotten mosses of 1 weight part, 0.05 weight part, 0.005 weight part, the calcium carbonate of 0.3 weight part and 0.5 weight part is mixed to get auxiliary material.Above-mentioned for 50L nutrient solution is mixed with 50kg auxiliary material, and is 75kg by mixed dry materials ad pond om, then granulate as diameter is about the particle of about 8mm, namely obtain the microbiobacterial agent of this comparative example.
Testing example 1
This testing example chooses peasant household of municipal each village village of river, Hebei province Gaobeidian City through the continuous plantation tomato tomato booth of 5 years, the microbiobacterial agent of embodiment 1 and comparative example 1 and 2 is carried out to the test of anti-tomato continuous cropping disease.
(1) the using of tomato planting and microbiobacterial agent: tomato planting mode is culturing and transplanting seedlings, line-spacing 85cm, spacing in the rows 26cm.Time before tomato is transplanted, perforated method is adopted to use the microbiobacterial agent of embodiment 1 and comparative example 1 and 2.Test facilities consumption is respectively control group amount of application 0g/ cave, the first experimental group amount of application 0.6g/ cave, the second experimental group amount of application 1.2g/ cave and the 3rd experimental group amount of application 1.6g/ cave.The area often organizing trial plot used is 40m
2(long 8m, wide 5m), often organizes the planting density that trial plot used adopts 5 row/district, and random processing arranges, and often organizes 4 times and repeats.Often organize both sides, trial plot used and establish protection row, and other field management measures are with conventional.
(2) state of an illness of continuous cropping disease characterizes: protection effect is investigated.When control group death rate reaches 60%, investigate the dead seedling number of each experimental group respectively, calculate sickness rate and prevention effect by formula (1) and formula (2).
Formula (1): sickness rate (%)=dead seedling number/planting stock number × 100%
Formula (2): prevention effect (%)=(contrast sickness rate-experiment sickness rate)/contrast sickness rate × 100%
Survey the output of each process community during results, calculate stimulation ratio by formula (3).
Formula (3): stimulation ratio (%)=(experimental yield-contrast output)/check plot output × 100%.
The result more than obtained is as shown in table 1:
Table 1
Visible according to the data of table 1, lichem bacillus strain of the present invention significantly increases the prevention effect of continuous cropping disease, reduces the death rate of tomato continuous cropping disease, and improves stimulation ratio, prevents and treats level therefore, it is possible to promote significantly to plant continuous cropping disease.
Testing example 2
This testing example chooses Luohe City peasant household of Henan Province through the continuous plantation capsicum capsicum booth of 6 years, the microbiobacterial agent of embodiment 1 and comparative example 1 and 2 is carried out to the test of anti-capsicum continuous cropping disease.
(1) the using of pepper planting and microbiobacterial agent: pepper planting mode is culturing and transplanting seedlings, line-spacing 55cm, spacing in the rows 15cm.Time before capsicum is transplanted, perforated method is adopted to use the microbiobacterial agent of embodiment 1 and comparative example 1 and 2.Test facilities consumption is respectively control group amount of application 0g/ cave, the first experimental group amount of application 0.3g/ cave, the second experimental group amount of application 0.6g/ cave and the 3rd experimental group amount of application 1g/ cave.The area often organizing trial plot used is 30m
2(long 6m, wide 5m), often organizes the planting density that trial plot used adopts 6 row/district, and random processing arranges, and often organizes 3 times and repeats.Often organize both sides, trial plot used and establish protection row, and other field management measures are with conventional.
(2) state of an illness of continuous cropping disease characterizes: calculate microbiobacterial agent to the prevention effect of capsicum continuous cropping disease and stimulation ratio according to the method for testing example 1, the result obtained is as shown in table 2:
Table 2
Visible according to the data of table 2, lichem bacillus strain of the present invention significantly increases the prevention effect to capsicum continuous cropping disease, reduce the death rate of capsicum continuous cropping disease, and improve stimulation ratio, therefore, it is possible to promote significantly level is prevented and treated to plant continuous cropping disease.
Testing example 3
This testing example chooses peasant household of Daxing district, Beijing, China through the continuous plantation watermelon watermelon land for growing field crops of 3 years, the microbiobacterial agent of embodiment 1 and comparative example 1 and 2 is carried out to the test of anti-watermelon continuous cropping disease.
(1) the using of growth of watermelon and microbiobacterial agent: growth of watermelon mode is culturing and transplanting seedlings, line-spacing 130cm, spacing in the rows 50cm.Time before watermelon is transplanted, perforated method is adopted to use the microbiobacterial agent of embodiment 1 and comparative example 1 and 2.Test facilities consumption is respectively control group amount of application 0g/ cave, the first experimental group amount of application 1g/ cave, the second experimental group amount of application 2g/ cave and the 3rd experimental group amount of application 4g/ cave.The area often organizing trial plot used is 100m
2(long 20m, wide 5m), often organizes the planting density that trial plot used adopts 5 row/district, and random processing arranges, and often organizes 3 times and repeats.Often organize both sides, trial plot used and establish protection row, and other field management measures are with conventional.
(2) state of an illness of continuous cropping disease characterizes: calculate microbiobacterial agent to the prevention effect of watermelon continuous cropping disease and stimulation ratio according to the method for testing example 1, the result obtained is as shown in table 3:
Table 3
Visible according to the data of table 3, lichem bacillus strain of the present invention significantly increases the prevention effect to watermelon continuous cropping disease, reduce the death rate of watermelon continuous cropping disease, and improve stimulation ratio, therefore, it is possible to promote significantly level is prevented and treated to plant continuous cropping disease.
Testing example 4
This testing example chooses Jining City in Shandong Province peasant household through the continuous plant cotton cotton land for growing field crops of 4 years, the microbiobacterial agent of embodiment 1 and comparative example 1 and 2 is carried out to the test of anti-cotton continuous cropping disease.
(1) the using of cotton planting and microbiobacterial agent: the line-spacing 110cm of cotton planting, spacing in the rows 50cm.Before sowing cotton seed, when ploughing, use the microbiobacterial agent of embodiment 1 and comparative example 1 and 2.Test facilities consumption is respectively control group amount of application 0kg/ mu, the first experimental group amount of application 30kg/ mu, the second experimental group amount of application 60kg/ mu and the 3rd experimental group amount of application 90kg/ mu.The area often organizing trial plot used is 100m
2(long 20m, wide 5m), often organizes the planting density that trial plot used adopts 5 row/district, and random processing arranges, and often organizes 2 times and repeats.Often organize both sides, trial plot used and establish protection row, and other field management measures are with conventional.
(2) state of an illness of continuous cropping disease characterizes: calculate microbiobacterial agent to the prevention effect of cotton continuous cropping disease and stimulation ratio according to the method for testing example 1, the result obtained is as shown in table 4:
Table 4
Visible according to the data of table 4, lichem bacillus strain of the present invention significantly increases the prevention effect to cotton continuous cropping disease, reduce the death rate of cotton continuous cropping disease, and improve stimulation ratio, therefore, it is possible to promote significantly level is prevented and treated to plant continuous cropping disease.
Embodiment 2
The present embodiment is for illustration of Bacillus licheniformis of the present invention and the cultivation of subtilis and the preparation of microbiobacterial agent.
Be that the slant strains of the Bacillus licheniformis (Bacilluslicheniformis) of CCTCCNO:M2013495 is at the LB flat board (peptone containing 10g/L by deposit number, the yeast extract of 5g/L, the agarose of NaCl and 3g/L of 5g/L, pH value 7.4, identical below) upper line, and cultivate 15 hours at 37 DEG C, picking list bacterium colony from flat board, be inoculated in the LB liquid nutrient medium (peptone containing 10g/L of 25mL, the yeast extract of 5g/L and the NaCl of 5g/L, pH value 7.4, identical below) in, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain first order seed, 20mL first order seed is added in the LB liquid nutrient medium of 500mL, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain the secondary seed of Bacillus licheniformis.
Be that the slant strains of the subtilis (Bacillussubtilis) of CCTCCNO:M2013496 is at the LB flat board (peptone containing 10g/L by deposit number, the yeast extract of 5g/L, the agarose of NaCl and 3g/L of 5g/L, pH value 7.4, identical below) upper line, and cultivate 15 hours at 37 DEG C, picking list bacterium colony from flat board, be inoculated in the LB liquid nutrient medium (peptone containing 10g/L of 25mL, the yeast extract of 5g/L and the NaCl of 5g/L, pH value 7.4, identical below) in, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain first order seed, 20mL first order seed is added in the LB liquid nutrient medium of 500mL, at 37 DEG C 250r/min shaking table on cultivate 15 hours, obtain the secondary seed of subtilis.
By the secondary seed of the Bacillus licheniformis of 2L and 0.4L subtilis secondary seed mixing, then add in the LB liquid nutrient medium of 60L, at 37 DEG C 250r/min stirring under and 10m
3cultivate 15 hours under the ventilation of/h, obtain nutrient solution.The blood cell plate counting process total cell concentration recorded in nutrient solution is adopted to be 10
9cFU/mL.Use the nucleic acid quantification method based on 16SrDNA to record in nutrient solution, described Bacillus licheniformis is 1:0.2 with the CFU ratio of described subtilis.
The starch of the tripoly phosphate sodium STPP of the collagen of the peat composed of rotten mosses of 1 weight part, 0.05 weight part, 0.005 weight part, the calcium carbonate of 0.3 weight part and 0.5 weight part is mixed to get auxiliary material.Above-mentioned for 50L nutrient solution is mixed with 50kg auxiliary material, and is 75kg by mixed dry materials ad pond om, then granulate as diameter is about the particle of about 8mm, namely obtain microbiobacterial agent of the present invention.
Embodiment 3
Microbiobacterial agent is prepared according to the method for embodiment 2, unlike, by the secondary seed of the Bacillus licheniformis of 1.2L and the mixing of 1.2L subtilis secondary seed, then carry out three grades of enlarged culturing.
In the present embodiment, use the nucleic acid quantification method based on 16SrDNA to record in nutrient solution, described Bacillus licheniformis is 1:0.9 with the CFU ratio of described subtilis.
Testing example 5
This testing example chooses peasant household of municipal each village village of river, Hebei province Gaobeidian City through planting the tomato tomato booth of 5 years continuously, the microbiobacterial agent of embodiment 1-3 is carried out to the test of anti-tomato continuous cropping disease.
(1) the using of tomato planting and microbiobacterial agent: tomato planting mode is culturing and transplanting seedlings, line-spacing 85cm, spacing in the rows 26cm.Time before tomato is transplanted, perforated method is adopted to use the microbiobacterial agent of embodiment 1-3.Test facilities consumption is respectively control group amount of application 0g/ cave, the first experimental group amount of application 0.6g/ cave, the second experimental group amount of application 1.2g/ cave and the 3rd experimental group amount of application 1.6g/ cave.The area often organizing trial plot used is 40m
2(long 8m, wide 5m), often organizes the planting density that trial plot used adopts 5 row/district, and random processing arranges, and often organizes 4 times and repeats.Often organize both sides, trial plot used and establish protection row, and other field management measures are with conventional.
(2) state of an illness of continuous cropping disease characterizes: protection effect is investigated.When control group death rate reaches 60%, investigate the dead seedling number of each experimental group respectively, calculate sickness rate and prevention effect by formula (1) and formula (2).
Formula (1): sickness rate (%)=dead seedling number/planting stock number × 100%
Formula (2): prevention effect (%)=(contrast sickness rate-experiment sickness rate)/contrast sickness rate × 100%
Survey the output of each process community during results, calculate stimulation ratio by formula (3).
Formula (3): stimulation ratio (%)=(experimental yield-contrast output)/check plot output × 100%.
The result more than obtained is as shown in table 5:
Table 5
Visible according to the data of table 5, at this microbiobacterial agent preferred also containing subtilis, the deposit number of described subtilis is CCTCCNO.M2013496; And described Bacillus licheniformis is 1:(0.1-0.3 with the CFU ratio of described subtilis) when, Bacillus licheniformis of the present invention and described subtilis can play synergy, can obtain the sick prevention effect of better continuous cropping with lower amount of application.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. a Bacillus licheniformis (Bacilluslicheniformis), is characterized in that: the deposit number of this Bacillus licheniformis is CCTCCNO.M2013495.
2. a microbiobacterial agent, is characterized in that: this microbiobacterial agent is containing, for example Bacillus licheniformis according to claim 1 and other bacterial strain and auxiliary material.
3. microbiobacterial agent according to claim 2, is characterized in that: relative to the microbiobacterial agent of 1g, and the content of described Bacillus licheniformis is 10
6-10
11cFU, is preferably 10
8-10
9cFU.
4. the microbiobacterial agent according to Claims 2 or 3, is characterized in that: this microbiobacterial agent is also containing subtilis, and the deposit number of described subtilis is CCTCCNO.M2013496; And described Bacillus licheniformis is 1:(0.1-0.3 with the CFU ratio of described subtilis).
5. according to the microbiobacterial agent in claim 2-4 described in any one, it is characterized in that: described auxiliary material contains at least one in dispersion agent, nutrition agent and inert support.
6. microbiobacterial agent according to claim 5, is characterized in that: described dispersion agent comprises at least one in polycarboxylate, sodium lignosulfonate, calcium lignin sulphonate, dialkyl sodium sulfosuccinate, sodium alkylarysulfonate, polyoxyethylene alkyl phenyl ether, tripoly phosphate sodium STPP, polyxyethylated aryl phosphide, polyoxyethylene alkylaryl ether, polyxyethylated aryl polymer, polyalkylene glycol alkyl phenyl ether polyoxyethylene nonylphenol ether, sulfonation formaldehyde sodium naphthalene, Triton X-100 and tween 80; Described nutrition agent comprises at least one in the peat composed of rotten mosses, soyflour, rice, wheat, dextrin, collagen, grape sugar and starch; Described inert support comprises at least one in calcium carbonate, loess, diatomite, wilkinite, talcum powder, mica powder, kaolin and calcite in powder.
7. according to the microbiobacterial agent in claim 2-4 described in any one, it is characterized in that: so-called microbiobacterial agent is granule, and described auxiliary material contains the peat composed of rotten mosses, collagen, tripoly phosphate sodium STPP, calcium carbonate and starch; Relative to 10
9the described Bacillus licheniformis of CFU, the content of the peat composed of rotten mosses is 0.3-3g, and the content of collagen is 0.01-0.1g, and the content of tripoly phosphate sodium STPP is the content of calcium carbonate is 0.1-0.5g, and the content of starch is 0.1-1g.
8. the application of microbiobacterial agent described in any one in control plant continuous cropping disease in Bacillus licheniformis according to claim 1 or claim 2-7.
9. application according to claim 8, is characterized in that: described plant is at least one in plant of Solanaceae, cucurbitaceous plant, Malvaceae plant, rosaceous plant and vitaceae.
10. application according to claim 9, is characterized in that: described plant of Solanaceae comprises at least one in tomato, capsicum and eggplant; Described cucurbitaceous plant comprises at least one in cucumber, watermelon and muskmelon; Described Malvaceae plant comprises at least one in cotton, piemarker and hibiscus cannabinus; Described rosaceous plant comprises at least one in apple, Chinese pear-leaved crabapple, Malus spectabilis, pears, peach, Lee, apricot, plum, cherry, loquat, hawthorn, strawberry and raspberry; Described vitaceae comprises grape.
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