CN105274030B - A kind of rhizobium and application thereof - Google Patents
A kind of rhizobium and application thereof Download PDFInfo
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- CN105274030B CN105274030B CN201510771053.8A CN201510771053A CN105274030B CN 105274030 B CN105274030 B CN 105274030B CN 201510771053 A CN201510771053 A CN 201510771053A CN 105274030 B CN105274030 B CN 105274030B
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Abstract
The present invention relates to a kind of slow raw rhizobium (Bradyrhizobium sp.) LX JX01 and application thereof.Slowly raw rhizobium LX JX01 bacterial preparation process includes that prepared by liquid D M culture mediums, prepared by solid DM culture mediums, bacterial strain activates and rhizobium culture to the present invention.The bacterial strain can be with dalbergia odorifera dross symbiosis.When planting dalbergia odorifera seedling using dalbergia odorifera rhizobium LX JX01 microbial inoculums of the present invention, its root nodule dross number improves 3823% than control, root nodule fresh weight increases by 1038%, above-ground plant parts dry weight increases by 47.22%, 1030% and 42.3% is slightly respectively increased in seedling height and plant diameter, therefore, microbial inoculum of the invention can promote the growth of dalbergia odorifera.Therefore, microbial inoculum of the invention has broad application prospects in the plantation of dalbergia odorifera artificial forest.
Description
【Technical field】
The invention belongs to biotechnologies.More particularly it relates to which a kind of rhizobium, further relate to the rhizobium
Purposes.
【Background technology】
Rhizobium can be converted into ammonia with legume nodulation symbiotic nitrogen fixation, rhizobium by the nitrogen in fixed air,
To provide nitrogen nutrition for legume, and legume provides nutrient for rhizobium, and the two forms syntaxial system.Due to root
Tumor bacterium is infected to legume with specificity and validity, it is therefore desirable to which selection and breeding are suitble to the rhizobium of legume that could reach
To symbiosis effect.Rhizobium Inoculation is generally acknowledged in the world biological nitrogen fixation technology, and biological nitrogen fixation will not cause environment any dirt
Dye, while improving soil, promote plant growth, is the important measures of sustainable agriculture development.
Dalbergia odorifera is legume, is national two level Top-rated protected wild plants, and timber is wooden hard, texture drift
It is bright, it is the superior material for making classic hardwood furniture, root heartwood and trunk heartwood can be good for dalbergia wood, hyoscine
Analgestic.In addition, being alternatively arranged as greening-tree on Fujian, Guizhou and other places.Forest department of China is at research " dalbergia odorifera " for many years
Nursery work in put into a large amount of human and material resources, financial resources, though being able to success, survival rate is relatively low, by using rhizobium
Agent can form Symbiotic association with dalbergia odorifera, improve the survival rate of dalbergia odorifera and promote plant growing way, to dropping from now on
The artificial growth cultivation of fragrant yellow wingceltis is of great significance.
【Invention content】
[technical problems to be solved]
The object of the present invention is to provide a kind of rhizobium (Rhizobium sp.) LX-JX01.
It is a further object to provide the preparation methods of the Rhizobium Inoculant.
It is a further object to provide the purposes of the Rhizobium Inoculant.
[technical solution]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of rhizobium (Rhizobium sp.) LX-JX01, which exists in August in 2015 21 days
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, preserving number CGMCCNo.11265.
The invention further relates to a kind of rhizobium LX-JX01 bacterial preparation process.
The step of preparation method, is as follows：
A, prepared by liquid D M culture mediums
By 1~3 gram of yeast extract, 8~12 grams of sucrose, 1~3 gram of potassium nitrate, 0.4~0.6 gram of dipotassium hydrogen phosphate, 0.4~0.6
Gram potassium dihydrogen phosphate and 0.1~0.2 gram of sodium chloride are dissolved in distilled water, are used in combination distilled water constant volume to 1000ml；It reuses inorganic
The pH value of its solution is adjusted to 6.8~7.2 by acid or inorganic base, obtains the liquid D M culture mediums；
B, prepared by solid DM culture mediums
It is 18~20 grams of agar according to every liter of liquid D M culture medium, the liquid D M culture mediums being prepared toward step A are added
Agar, being heated to 100 DEG C makes its agar dissolve, and then carries out cooling its DM culture medium being made to become solid-like at 40 DEG C of temperature
State then obtains the solid DM culture mediums；
C, bacterial strain activates
The bacterial strain CGMCCNo.11265 for being stored in 4 DEG C of refrigerators of temperature in solid DM medium slants is transferred to newly
On the solid DM culture medium flat plates of fresh preparation, then single bacterium colony of crossing out preserves for use in the refrigerator of 4 DEG C of temperature；
D, rhizobium are cultivated
With disinfection inoculation ring from two ring single bacterium colony of picking on the solid DM culture medium flat plates of step C, it is inoculated into 150ml sterilizings
In liquid D M culture mediums, in shaking table under conditions of 160~200 revs/min of 26~30 DEG C of temperature and rotating speed shaken cultivation 70
It~75 hours, is determined by conventional microscopy and just obtains the liquid Rhizobium Inoculant without miscellaneous bacteria.
A preferred embodiment of the invention, the inorganic acid are selected from sulfuric acid, hydrochloric acid or phosphoric acid；The nothing
Machine alkali is selected from sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate.
According to another preferred method of implementation of the present invention, the liquid D M culture mediums are by 1.8~2.2 grams of yeast
Cream, 9~11 grams of sucrose, 1.8~2.2 grams of potassium nitrate, 0.45~0.55 gram of dipotassium hydrogen phosphate, 0.45~0.55 gram of potassium dihydrogen phosphate
It is prepared with 0.14~0.16 gram of sodium chloride.
According to another preferred method of implementation of the present invention, the liquid D M culture mediums are by 2 grams of yeast extracts, 10 grams of sugarcanes
What sugar, 2 grams of potassium nitrate, 0.5 gram of dipotassium hydrogen phosphate, 0.5 gram of potassium dihydrogen phosphate and 0.15 gram of sodium chloride were prepared.
The invention further relates to the rhizobium LX-JX01 microbial inoculums being prepared using the preparation method.
The invention further relates to purposes of the rhizobium LX-JX01 microbial inoculums in promoting dalbergia odorifera growth.
The invention further relates to purposes of the rhizobium LX-JX01 microbial inoculums in dalbergia odorifera nursery.
The present invention is described in more detail below.
The present invention relates to a kind of rhizobium (Rhizobium sp.) LX-JX01, which exists in August in 2015 21 days
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, preserving number CGMCCNo.11265.
The present inventor acquires yellow earth soil sample in May, 2010 from the hot woods central nursery in Pingxiang City In Guangxi Province, southern China forest-science academy of China
Product, therefrom isolated one plant of dalbergia odorifera rhizobium.
One, the acquisition of rhizobium
By the dalbergia odorifera seed of conventional surface disinfection after impregnating, drying processing in plantation to the yellow soil,
Plant nodulation and nitrogen fixation after 60 days.Root nodule is removed, surface sterilization is carried out using conventional method, then using conventional rhizobium point
From culture medium (document：Xu Yuerong, " Selective agar mediums of separation rhizobium ",《Soil》, 03 phase in 1988) using dilution apply
Cloth method therefrom isolated rhizobium.It verifies, is determined one through tablet purifying and the identification of subsequent fermented and cultured, tieback dross
The Strain Designation that strain is grown in solid separation culture medium is LX-JX01, which has symbiotic nodulation and nitrogen fixation with dalbergia odorifera
Effect, primarily determines that the bacterial strain is dalbergia odorifera rhizobium.
Two, the identification of rhizobium
1, conventional microscopy
Isolated dalbergia odorifera rhizobium are feminine gender by Gram's stain coloration result.It is solid in usual mannitol
It is cultivated 7 days under conditions of 28 DEG C of constant temperature in body plating medium, can grow that the surfaces macroscopic diameter about 2mm are smooth, side
The neat white circular colonies of edge；3d is cultivated in the liquid D M culture mediums in shaking flask, using conventional microscopy, form
One hollow not colored, and other parts are the lightpinks of uniform coloring, can determine that it belongs to slow according to morphological feature and habit
Raw type rhizobium.
2,16S rDNA sequencings：
It isolated dalbergia odorifera rhizobium is sent to Beijing three wins Radix Polygalae biotechnology Co., Ltd and survey
Sequence.By comparing, the similarity of the bacterial strain and rihizobium japonicum identified is 98%, so concluding that this bacterial strain is rhizobium
Belong to.
Rhizobium CGMCC No.11265 bacterial strain 16S rRNA gene sequencing results are shown in annex 1.
The bacterial strain identified and rihizobium japonicum (Rhizobiumjaponicum) homology highest.
3, Physiology and biochemistry is identified
1. beef extract-peptone detection
By in rhizobium LX-JX01 inoculations to beef extract-peptone culture medium in insulating box at 28 DEG C of temperature
Culture 5 days.Culture solution is clarified, and thalline has no growth, identical as the control of non-inoculating strain, this shows that the bacterial strain cannot be in beef
It is grown on cream-peptone culture medium.
Beef extract-peptone culture medium composition is as follows：5g beef extracts, 10g peptones, 5g sodium chloride, 1000ml distilled water,
pH7.2。
The constant incubator used in the step and subsequent step is current market sales of product.
2. BTB reacts
Rhizobium LX-JX01 bacterial strains are cultivated 7 days in insulating box at 28 DEG C of temperature on BTB tablets.Tablet color
Become blue, illustrate that the bacterial strain produces alkali on BTB tablets, belongs to slow raw type bacterial strain.
BTB reaction culture mediums composition is as follows：10g mannitol, 0.8g yeast extracts powder, 0.25g dipotassium hydrogen phosphates, 0.25g
Potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.1g sodium chloride, 15-20g agar, 1000ml distilled water, 1ml 0.25% bromines by weight
Bromothymol blue, pH6.8-7.0.
3. litmus milk reacts
By in rhizobium LX-JX-01 inoculations to litmus milk enrichment, cultivated 7 days at 28 DEG C of temperature, culture medium
Color becomes blue, and forms whey ring, shows that the bacterial strain produces alkali, belongs to slow raw type bacterial strain.
Litmus milk enrichment composition is as follows：100ml skim milks, 4ml2.5% reindeer moss solution, low pressure sterilizing.
4. 3- ketone group lactose reacts
The reaction of rhizobium LX-JX01 bacterial strains is negative, and periphery of bacterial colonies does not occur tan precipitate, shows that the bacterial strain is non-
Agrobacterium.
3- ketone group lactose mediums composition is as follows：10g lactose, 1g yeast extracts powder, 1000ml distilled water, 15-20g fine jades
Fat, pH7.2.
In summary qualification result shows that rhizobium strains CGMCC No11265 are soybean Slow_growing rhizobia
(Rhizobium japonicum)。
The present invention relates to a kind of rhizobium LX-JX01 bacterial preparation process.
The step of preparation method of the microbial inoculum, is as follows：
A, prepared by liquid D M culture mediums
By 1~3 gram of yeast extract, 8~12 grams of sucrose, 1~3 gram of potassium nitrate, 0.4~0.6 gram of dipotassium hydrogen phosphate, 0.4~0.6
Gram potassium dihydrogen phosphate and 0.1~0.2 gram of sodium chloride are dissolved in distilled water, are used in combination distilled water constant volume to 1000ml；It reuses inorganic
The pH value of its solution is adjusted to 6.8~7.2 by acid or inorganic base, obtains the liquid D M culture mediums.
Culture medium for rhizobium routine culture is YMA (Yeast-Mannitol-Agar), i.e. yeast powder-mannitol-
Agar medium.The DM culture mediums that the present invention uses are from YMA medium the difference is that carbon source and nitrogen source type are different, in DM trainings
It supports in base using sucrose as carbon source, using mannitol as carbon source in YMA medium.
The inorganic acid is selected from sulfuric acid, hydrochloric acid or phosphoric acid；The inorganic base is selected from sodium hydroxide, potassium hydroxide, carbon
Sour sodium or sodium bicarbonate.
Inorganic acid as used in the present invention is inorganic acid aqueous solution, and inorganic base is inorganic base aqueous solution.The nothing
The concentration of machine acid or inorganic base aqueous solution is typically 0.1-2.0N.
Preferably, the liquid D M culture mediums be by 1.8~2.2 grams of yeast extracts, 9~11 grams of sucrose, 1.8~2.2 grams
It is prepared by potassium nitrate, 0.45~0.55 gram of dipotassium hydrogen phosphate, 0.45~0.55 gram of potassium dihydrogen phosphate and 0.14~0.16 gram of sodium chloride
It obtains.
It is highly preferred that the liquid D M culture mediums are by 2 grams of yeast extracts, 10 grams of sucrose, 2 grams of potassium nitrate, 0.5 gram of phosphoric acid
What hydrogen dipotassium, 0.5 gram of potassium dihydrogen phosphate and 0.15 gram of sodium chloride were prepared.
The liquid D M culture mediums can be used for its strain culturing after using conventional sterilant processing.
B, prepared by solid DM culture mediums
It is 18~20 grams of agar according to every liter of liquid D M culture medium, the liquid D M culture mediums being prepared toward step A are added
Agar, being heated to 100 DEG C makes its agar dissolve, and is then cooled down at 40 DEG C of temperature, its DM culture medium is made to become solid-like
State then obtains the solid DM culture mediums.
The correlation circumstance of solid DM culture mediums is identical as liquid D M culture mediums, and details are not described herein.
C, bacterial strain activates
The bacterial strain CGMCCNo.11265 for being stored in 4 DEG C of refrigerators of temperature in solid DM medium slants is transferred to newly
On the solid DM culture medium flat plates of fresh preparation, then single bacterium colony of crossing out preserves for use in the refrigerator of 4 DEG C of temperature.
D, rhizobium are cultivated
With disinfection inoculation ring from two ring single bacterium colony of picking on the solid DM culture medium flat plates of step C, it is inoculated into 150ml sterilizings
In liquid D M culture mediums, in shaking table under conditions of 160~200 revs/min of 26~30 DEG C of temperature and rotating speed shaken cultivation 70
It~75 hours, is determined by conventional microscopy and just obtains the liquid rhizobium LX-JX01 microbial inoculums without miscellaneous bacteria.
It is current market sales of product in the shaking table that the step uses, such as limited by the permanent instrument and equipment of Shanghai conjunction
Company " closes permanent " product of sale with trade name.
The rhizobium LX-JX01 contents of acquired liquid rhizobium LX-JX01 microbial inoculums are using conventional in the art
Viable plate count method.
It measures and 100~20,000,000,000/ml liquid rhizobium is contained using the liquid Rhizobium Inoculant that the method for the present invention obtains
LX-JX01。
The invention further relates to the rhizobium LX-JX01 microbial inoculums being prepared using the preparation method.
The invention further relates to purposes of the rhizobium LX-JX01 microbial inoculums in promoting dalbergia odorifera growth.
The invention further relates to purposes of the rhizobium LX-JX01 microbial inoculums in dalbergia odorifera nursery.
The method and step that dalbergia odorifera nursery is carried out using microbial inoculum of the present invention is as follows：
(1) dalbergia odorifera seed is impregnated 24 hours with clear water, in seeding in nursery bed after drying；
(2) nursery applies the nitragin of the present invention toward its root after height of seedling 4-5cm grows true leaf；
(3) seedling at seedling stage, rhizobium dross on dalbergia odorifera root system forms dross seedling；
(4) seedling field transplanting；
(5) daily management method, it is ensured that plant survives.
For the present inventor by researchs such as the separation, culture, identification of rhizobium, the dalbergia wood for finding plant height effect nodulation and nitrogen fixation is yellow
Wingceltis Slow_growing rhizobia.The result of experiment is listed in Fig. 1-5, these test results show using after nitragin of the present invention, can
The survival rate of dalbergia odorifera is significantly improved up to 99% and dross rate 100%, ensures to obtain more nitrogen nutrition in growth,
The healthy growth of dalbergia odorifera in the natural environment is promoted, is conducive to reach recovery and protecting ecology during artificial growth
The purpose of environment.
[advantageous effect]
The beneficial effects of the invention are as follows：The rhizobium LX-JX01 of the present invention can improve the dross rate of dalbergia odorifera, ensure
More nitrogen nutrition are obtained in growth, promote the healthy growth of dalbergia odorifera in the natural environment.Indoor and outdoor test
Result show using after nitragin of the present invention, being remarkably improved the survival rate and dross rate of dalbergia odorifera, and promote to drop
The growth of fragrant yellow wingceltis, compared with compareing seedling, the root nodule dross number of dalbergia odorifera rhizobium LX-JX01 of the invention improves
3823%, root nodule fresh weight increases by 1038%, and above-ground plant parts dry weight increases by 47.22%, and seedling height and plant diameter are thick
1030% and 42.3% is respectively increased, promotes the growth of dalbergia odorifera.
Rhizobium (Rhizobium sp.) LX-JX01, the bacterial strain is in August in 2015 21 days in Chaoyang District, Beijing City north
In the institute 3 of occasion West Road 1 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms
Heart preservation, preserving number are CGMCC No.11265.
【Description of the drawings】
Fig. 1 is the average root nodule number statistical result of its plant after dalbergia odorifera seed is sowed 60 days；
Fig. 2 is the average root nodule fresh weight statistical result of its plant after dalbergia odorifera seed is sowed 60 days；
Fig. 3 is the average aerial part dry weight statistical result of its plant after dalbergia odorifera seed is sowed 60 days；
Fig. 4 is the average high statistical result of overground part plant division of its plant after dalbergia odorifera seed is sowed 60 days；
Fig. 5 is the thick statistical result of average diameter of its plant after dalbergia odorifera seed is sowed 60 days.
【Specific implementation mode】
The present invention is will be better understood that by following embodiments.
Embodiment 1：It is prepared by rhizobium LX-JX01 microbial inoculums
The implementation steps of the embodiment are as follows：
A, prepared by liquid D M culture mediums
By 1 gram of yeast extract, 10 grams of sucrose, 2 grams of potassium nitrate, 0.4 gram of dipotassium hydrogen phosphate, 0.4 gram of potassium dihydrogen phosphate and 0.15
Gram sodium chloride is dissolved in distilled water, is used in combination distilled water constant volume to 1000ml；Reuse 1.0N sulfuric acid or 1.0N sodium hydroxides by its
The pH value of solution is adjusted to 6.8, obtains the liquid D M culture mediums；
B, prepared by solid DM culture mediums
It is 18 grams of agar according to every liter of liquid D M culture medium, agar is added in the liquid D M culture mediums being prepared toward step A,
Being heated to 100 DEG C makes its agar dissolve, and is then cooled down at 40 DEG C of temperature, its DM culture medium is made to become solid state, in
It is to obtain the solid DM culture mediums；
C, bacterial strain activates
The bacterial strain CGMCC No.11265 for being stored in 4 DEG C of refrigerators of temperature in solid DM medium slants are transferred to newly
On the solid DM culture medium flat plates of fresh preparation, then single bacterium colony of crossing out preserves for use in the refrigerator of 4 DEG C of temperature；
D, rhizobium are cultivated
With disinfection inoculation ring from two ring single bacterium colony of picking on the solid DM culture medium flat plates of step C, it is inoculated into 150ml sterilizings
In liquid D M culture mediums, in by Wuxi Jiu Ping Instrument Ltd. with the shaking table of trade name " long flat " sale 26 DEG C of temperature with
Shaken cultivation 70 hours under conditions of 180 revs/min of rotating speed is determined by conventional microscopy and just obtains the liquid root without miscellaneous bacteria
Tumor bacteria agent.
It is measured using viable plate counts described in this specification, liquid rhizobium LX- manufactured in the present embodiment
Every milliliter of JX01 microbial inoculums contain 10,000,000,000 rhizobium LX-JX01.
Embodiment 2：It is prepared by rhizobium LX-JX01 microbial inoculums
The implementation steps of the embodiment are as follows：
A, prepared by liquid D M culture mediums
By 3 grams of yeast extracts, 8 grams of sucrose, 1 gram of potassium nitrate, 0.6 gram of dipotassium hydrogen phosphate, 0.5 gram of potassium dihydrogen phosphate and 0.1 gram
Sodium chloride is dissolved in distilled water, is used in combination distilled water constant volume to 1000ml；It reuses 0.1N sulfuric acid or 0.1N sodium hydroxides is molten by its
The pH value of liquid is adjusted to 7.0, obtains the liquid D M culture mediums；
B, prepared by solid DM culture mediums
It is 20 grams of agar according to every liter of liquid D M culture medium, agar is added in the liquid D M culture mediums being prepared toward step A,
Being heated to 100 DEG C makes its agar dissolve, and is then cooled down at 40 DEG C of temperature, its DM culture medium is made to become solid state, in
It is to obtain the solid DM culture mediums；
C, bacterial strain activates
The bacterial strain CGMCCNo.11265 for being stored in 4 DEG C of refrigerators of temperature in solid DM medium slants is transferred to newly
On the solid DM culture medium flat plates of fresh preparation, then single bacterium colony of crossing out preserves for use in the refrigerator of 4 DEG C of temperature；
D, rhizobium are cultivated
With disinfection inoculation ring from two ring single bacterium colony of picking on the solid DM culture medium flat plates of step C, it is inoculated into 150ml sterilizings
In liquid D M culture mediums, in 30 DEG C of temperature in the shaking table sold with trade name " three Hes " by Jintan City three and Instrument Ltd.
With shaken cultivation under conditions of 160 revs/min of rotating speed 75 hours, is determined by conventional microscopy and just obtain the liquid without miscellaneous bacteria
Rhizobium Inoculant.
It is measured using viable plate counts described in this specification, liquid rhizobium LX- manufactured in the present embodiment
Every milliliter of JX01 microbial inoculums contain 15,000,000,000 rhizobium LX-JX01.
Embodiment 3：It is prepared by rhizobium LX-JX01 microbial inoculums
The implementation steps of the embodiment are as follows：
A, prepared by liquid D M culture mediums
By 2 grams of yeast extracts, 12 grams of sucrose, 3 grams of potassium nitrate, 0.5 gram of dipotassium hydrogen phosphate, 0.6 gram of potassium dihydrogen phosphate and 0.2 gram
Sodium chloride is dissolved in distilled water, is used in combination distilled water constant volume to 1000ml；It reuses 2.0N sulfuric acid or 2.0N sodium hydroxides is molten by its
The pH value of liquid is adjusted to 7.2, obtains the liquid D M culture mediums；
B, prepared by solid DM culture mediums
It is 19 grams of agar according to every liter of liquid D M culture medium, agar is added in the liquid D M culture mediums being prepared toward step A,
Being heated to 100 DEG C makes its agar dissolve, and is then cooled down at 40 DEG C of temperature, its DM culture medium is made to become solid state, in
It is to obtain the solid DM culture mediums；
C, bacterial strain activates
The bacterial strain CGMCCNo.11265 for being stored in 4 DEG C of refrigerators of temperature in solid DM medium slants is transferred to newly
On the solid DM culture medium flat plates of fresh preparation, then single bacterium colony of crossing out preserves for use in the refrigerator of 4 DEG C of temperature；
D, rhizobium are cultivated
With disinfection inoculation ring from two ring single bacterium colony of picking on the solid DM culture medium flat plates of step C, it is inoculated into 150ml sterilizings
In liquid D M culture mediums, by Changzhou Run Hua Electrical Appliances Co., Ltd in the shaking table of trade name " profit China " sale, in 28 DEG C of temperature
With shaken cultivation under conditions of 200 revs/min of rotating speed 72 hours, is determined by conventional microscopy and just obtain the liquid without miscellaneous bacteria
Rhizobium Inoculant.
It is measured using viable plate counts described in this specification, liquid rhizobium LX- manufactured in the present embodiment
Every milliliter of JX01 microbial inoculums contain 20,000,000,000 rhizobium LX-JX01.
Test example 1：Fluid present invention rhizobium LX-JX01 microbial inoculum application dalbergia odorifera seedling growth tests
The implementation steps of the test example are as follows：
Test period：On June 5th, 2015 was to August 5 days.
Test site：Sun glasshouse of Leading Bio-agricultural Co., Ltd..
Test method and material：
A, seed treatment and vernalization
1. dalbergia odorifera seed impregnates 24 hours in sterile water, then the seed of processing is pulled out and is dried in the air by seed expansion
It is dry, it is placed in sterilizing vermiculite seedbed and (adds sterile water moisturizing), germinate within about 20 days.It waits for that seedling grows up to 4-5 centimetres, grows true leaf time shift
It plants in nutrient bag.
2. nutrient bag prepares
Test group nutrient bag：It is that 10% yellow soil below, will be under sieve by 10 mesh sieve by weight by water content
Yellow soil is fitted into nutrient bag, and the amount of yellow soil is the 1/3 of nutrient bag volume.
Control group nutrient bag：The vermiculite power of 0.75~1.0mm of diameter is fitted into nutrient bag, the amount of vermiculite power is nutrient bag
The 1/3 of volume.
B, sprigging
Yellow soil nutrient bag：
First test group (yellow soil nutrient bag)：Close dalbergia odorifera seedling is grown from 1. pipetting, with distilled water by root
It cleans up, then plants in test group nutrient bag, per 1 plant of dalbergia odorifera seedling of plastic bag cultivation.Then the liquid of every bag of injection 1mL present invention
Body rhizobium LX-JX01 microbial inoculums, the bacteria suspension concentration are 120CUF/mL.The nutrient bag installed is uniformly put in seedling bed.
First control group (yellow soil nutrient bag is not added with liquid rhizobium LX-JX01 microbial inoculums)：From 1. pipetting, growth is close
Root is cleaned up with distilled water, then planted in test group nutrient bag by dalbergia odorifera seedling, per 1 plant of dalbergia odorifera seedling of plastic bag cultivation.
The nutrient bag installed is uniformly put in seedling bed.
Vermiculite nutrient bag：
Second group of test group (vermiculite nutrient bag)：Close dalbergia odorifera seedling is grown from 1. pipetting, with distilled water by root
It cleans up, then plants in test group nutrient bag, per 1 plant of dalbergia odorifera seedling of plastic bag cultivation.Then the liquid of every bag of injection 1mL present invention
Body rhizobium LX-JX01 microbial inoculums, the bacteria suspension concentration are 120CUF/mL.The nutrient bag installed is uniformly put in seedling bed.
Second group of control group (vermiculite nutrient bag is not added with liquid rhizobium LX-JX01 microbial inoculums)：From 1. pipetting, growth is close
Root is cleaned up with distilled water, then planted in test group nutrient bag by dalbergia odorifera seedling, per 1 plant of dalbergia odorifera seedling of plastic bag cultivation.
The nutrient bag installed is uniformly put in seedling bed.
Experiment uses single factor test RANDOMIZED BLOCK DESIGN, each handles 20 repetitions.Inoculation observes dalbergia odorifera seedling after 60 days
Dross and its growing state.
3, nursery
It is after planting watered in due course according to weather condition, seedling raise period 60 days, the seed for being inoculated with processing then forms dross seedling.Knot
After tumor seedling is formed, according to the conventional survey standard test dross number of root nodule, root nodule fresh weight, aerial part dry weight, overground part
Plant division height and the thick index of diameter, 20 plants of plant of every group of statistics, the results are shown in attached drawing.
After nursery 60 days, average dross number (a) measurement result of dalbergia odorifera plant is listed in attached drawing 1, these knots
When fruit is shown using yellow soil or vermiculite as matrix, after applying rhizobium LX-JX01 microbial inoculums of the present invention, dalbergia odorifera plant is put down
Equal dross number improves 3823% obviously higher than the control treatment for not applying Rhizobium Inoculant.
After nursery 60 days, dalbergia odorifera plant root nodule fresh weight (g) measurement result that is averaged is listed in attached drawing 2.These results
When showing using yellow soil and vermiculite as matrix, after applying rhizobium LX-JX01 microbial inoculums of the present invention, dalbergia odorifera plant is averaged
Root nodule fresh weight pole is apparently higher than the control treatment for not applying Rhizobium Inoculant, increases by 1038%.
After nursery 60 days, average aerial part dry weight (g) measurement result of dalbergia odorifera plant is listed in Fig. 3.These
The result shows that when using yellow soil and vermiculite as matrix, after applying rhizobium LX-JX01 microbial inoculums of the present invention, dalbergia odorifera plant
Average aerial part dry weight increases by 47.22% obviously higher than the control treatment for not applying Rhizobium Inoculant.
After nursery 60 days, average overground part plant division high (cm) and thick (cm) measurement result of plant diameter of dalbergia odorifera plant
It is listed in Fig. 4 and Fig. 5, these results indicate that when using yellow soil and vermiculite as matrix, applies rhizobium LX-JX01 bacterium of the present invention
After agent, the average overground part plant division height of plant, slightly obviously higher than the control treatment for not applying Rhizobium Inoculant, divides with plant diameter
Indescribably high 1030% and 42.3%.
4, emergence plant and management
Nutrient bag seedling grows to 50 centimetres or so should be transplanted to reproducing area as early as possible.Bag film is first torn when plantation, is then placed in
In plant hole, plant hole specification can be 50cm × 50cm × 50cm, last backfill.The density of plantation is 50-60 plants per acre.It is wanted after transplanting
According to circumstances water, loosen the soil in time, weeding, expanding cave and trimming, to be prevented in time if plant is fallen ill, with ensure plant at
It is living.
Claims (7)
1. a kind of rhizobium（Rhizobium sp.）LX-JX01, the bacterial strain is in August in 2015 21 days in Chaoyang District, Beijing City
The institute 3 of North Star West Road 1 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms
Center preservation, preserving number are CGMCC No.11265.
2. a kind of rhizobium LX-JX01 bacterial preparation process, it is characterised in that the step of this method is as follows：
A, prepared by liquid D M culture mediums
By 1~3 gram of yeast extract, 8~12 grams of sucrose, 1~3 gram of potassium nitrate, 0.4~0.6 gram of dipotassium hydrogen phosphate, 0.4~0.6 gram of phosphorus
Acid dihydride potassium and 0.1~0.2 gram of sodium chloride are dissolved in distilled water, are used in combination distilled water constant volume to 1000ml；Reuse inorganic acid or
The pH value of its solution is adjusted to 6.8~7.2 by inorganic base, obtains the liquid D M culture mediums；The inorganic acid is selected from sulphur
Acid, hydrochloric acid or phosphoric acid；The inorganic base is selected from sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate；
B, prepared by solid DM culture mediums
It is 18~20 grams of agar according to every liter of liquid D M culture medium, agar is added in the liquid D M culture mediums being prepared toward step A,
Being heated to 100 DEG C makes its agar dissolve, then carried out at 40 DEG C of temperature it is cooling so that its DM culture medium is become solid state, then
Obtain the solid DM culture mediums；
C, bacterial strain activates
The bacterial strain CGMCC No.11265 for being stored in 4 DEG C of refrigerators of temperature in solid DM medium slants are transferred to fresh match
On the solid DM culture medium flat plates of system, then single bacterium colony of crossing out preserves for use in the refrigerator of 4 DEG C of temperature；
D, rhizobium are cultivated
With disinfection inoculation ring from two ring single bacterium colony of picking on the solid DM culture medium flat plates of step C, it is inoculated into 150ml sterilized liquids
In DM culture mediums, in shaking table under conditions of 160~200 revs/min of 26~30 DEG C of temperature and rotating speed shaken cultivation 70~75
Hour, it is determined by conventional microscopy and just obtains the liquid Rhizobium Inoculant without miscellaneous bacteria.
3. preparation method according to claim 2, it is characterised in that the liquid D M culture mediums are by 1.8~2.2 grams
Yeast extract, 9~11 grams of sucrose, 1.8~2.2 grams of potassium nitrate, 0.45~0.55 gram of dipotassium hydrogen phosphate, 0.45~0.55 gram of di(2-ethylhexyl)phosphate
What hydrogen potassium and 0.14~0.16 gram of sodium chloride were prepared.
4. preparation method according to claim 2, it is characterised in that the liquid D M culture mediums be by 2 grams of yeast extracts,
What 10 grams of sucrose, 2 grams of potassium nitrate, 0.5 gram of dipotassium hydrogen phosphate, 0.5 gram of potassium dihydrogen phosphate and 0.15 gram of sodium chloride were prepared.
5. the rhizobium LX-JX01 microbial inoculums being prepared according to preparation method described in any one of claim 2-4 claims.
6. purposes of the rhizobium LX-JX01 microbial inoculums according to claim 5 in promoting dalbergia odorifera growth.
7. purposes of the rhizobium LX-JX01 microbial inoculums according to claim 5 in dalbergia odorifera nursery.
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| CN108676722B (en) * | 2018-07-13 | 2019-07-02 | 浙江初农生物科技有限公司 | Improve the store method of nitragin survival rate |
| CN109370953B (en) * | 2018-12-05 | 2020-06-16 | 福建农林大学 | Juemina rhizobium JYN6 and application thereof |
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| CN110713953B (en) * | 2019-11-15 | 2020-06-02 | 中国科学院南京土壤研究所 | Mesorhizobium strain with phosphate solubilizing property and application thereof |
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