CN107058458A - A kind of method for detecting plant rhizosphere growth-promoting bacterium in root colonization - Google Patents
A kind of method for detecting plant rhizosphere growth-promoting bacterium in root colonization Download PDFInfo
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- CN107058458A CN107058458A CN201710332234.XA CN201710332234A CN107058458A CN 107058458 A CN107058458 A CN 107058458A CN 201710332234 A CN201710332234 A CN 201710332234A CN 107058458 A CN107058458 A CN 107058458A
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Abstract
The present invention relates to a kind of detection plant rhizosphere growth-promoting bacterium in the method for root colonization, it comprises the following steps：1）Wheat is transferred and Liquid Culture：The wheat seedling cultivated in solid medium is transferred in liquid MS medium, under 25 28 DEG C of daytime, the h of illumination 16,15 20 DEG C of night, dark condition, 24 48 h are cultivated in 20 50 rmp on horizontal shaker, then 13 ml bacteria suspensions are added, are cultivated for 5 10 days；2）Wheat root colonizes bacterial number counting：Wheat seedling is taken out, the culture medium of root is wiped with aseptic filter paper, weighed；It is subsequently placed in tissue grinder, adds 21 × PBSs of ml, be ground to pulpous state, gradient dilution plate count is carried out using LB solid mediums, calculates the colony forming single-digit of every gram of fresh weight wheat root.The inventive method liquid concussion and cultivate in an aseptic environment, condition of culture is stably and controllable, and sampling method standard is unified.
Description
Technical field
Determination techniques field is colonized the invention belongs to root system of plant, and in particular to one kind detection plant rhizosphere growth-promoting bacterium exists
The method of root colonization.
Background technology
Plant growth-promoting rhizosphere bacteria（Plant growth-promoting rhizobacteria, PGPR）It is to plant
Thing rhizosphere colonization, giving property, suppress the growth of pathogen or producing some special growth-promoting materials for raising plant nutrient, promotes
Plant seed germination and plant growth, a class bacterium of development.Numerous studies show, using PGPR bacterial manure, can reduce chemical fertilizer
Consumption 20%-30%, reduces pesticide dosage more than 30%, and increasing crop yield 10%-80%, the effect in Developing Sustainable Agriculture is got over
To be more taken seriously.
PGPR is detected in the method for rhizosphere colonization, conventional is solid state substrate method.With the solid-state base such as soil, turf or vermiculite
Matter（Sterilizing does not sterilize）Cultivated plant, is inoculated with microbial inoculum to be measured, after culture a period of time, takes root system of plant, plate count or aobvious
It is microcosmic to examine bacterium and colonize situation in rhizosphere.Solid state substrate method detection PGPR is more in the influence factor of rhizosphere colonization, specific bag
Matrix used in including different experiments room differs greatly, the influence of other microorganisms in matrix and air, and water content of substrate is difficult to stabilization
Control, the leaching loss of thalline can be caused by watering, and Rhizosphere Soil sampling method is difficult unification etc., cause the repeatability of experiment poor, different
The data obtained between laboratory are difficult to compare.It would therefore be highly desirable to study new detection plant rhizosphere growth-promoting bacterium in root colonization
Method.
The content of the invention
Present invention aims to overcome that prior art defect detects plant rhizosphere growth-promoting bacterium in root colonization there is provided one kind
Method, this method liquid concussion and cultivate in an aseptic environment, condition of culture is stably and controllable, and sampling method standard is unified.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of detection plant rhizosphere growth-promoting bacterium is in the method for root colonization, and it comprises the following steps：
1）Wheat is transferred and Liquid Culture：The wheat seedling cultivated in solid medium is transferred in liquid MS medium,
Under 25-28 DEG C of daytime, the h of illumination 16,15-20 DEG C of night, dark condition, 24-48h is cultivated in 20-50 rmp on horizontal shaker,
Then 1-3 ml bacteria suspensions are added, are cultivated for 5-10 days；
2）Wheat root colonizes bacterial number counting：Wheat seedling is taken out, the culture medium of root is wiped with aseptic filter paper, weighed；
It is subsequently placed in tissue grinder, adds 21 × PBSs of ml, be ground to pulpous state, ladder is carried out using LB solid mediums
Spend dilution plate to count, calculate the colony forming single-digit of every gram of fresh weight wheat root.
Specifically, step 1）In, the wheat seedling cultivated in solid medium is obtained through following step：Solidified MS media
In the tissue culture flasks for being sub-packed in 100-500 ml, every bottle of packing 20-100 ml, 110-121 DEG C of autoclaving 10-30 min；
In superclean bench, the wheat seed after vernalization is seeded in solidified MS media surface, 5-10 seeds of every bottle of sowing；So
Tissue culture flasks after planting are cultivated to small under conditions of 25-28 DEG C of daytime, the h of illumination 16,15-20 DEG C of night, dark afterwards
Wheat plant height 5-8 cm（About need culture 5-10 days）.
Specifically, step 1）In, bacteria suspension is obtained through following step：TSB fluid nutrient mediums are filled in the test tube of 30 ml specifications
5-10ml, 110-121 DEG C of sterilizing 10-30 min；Access 1 ring pseudomonas putida UW4 lawns, 28-37 DEG C, 200-260 rmp
12-24 h are cultivated, thalline is collected by centrifugation, thalline is washed with liquid MS medium, is then suspended and be adjusted to liquid MS medium
OD600It is worth and is produced for 1.
Compared to the prior art, beneficial effects of the present invention：
The inventive method uses liquid concussion and cultivate plant, and typical PGPR microbial inoculums are inoculated with nutrient solution, and culture terminates to use
Colonization amount of the colony counting method detection bacterium in rhizosphere.Compared with solid state substrate method, liquid concussion and cultivate method can be in asepsis ring
Cultivated under border, condition of culture is stably controlled, sampling method standard is unified.
Embodiment
Technical scheme is further discussed in detail with reference to embodiments, but protection scope of the present invention
It is not limited thereto.
Embodiment
Materials and methods.
Plant and bacterial strain
Wheat seed is that Commercial wheat is short by anti-58；
Bacterium bacterial strain is pseudomonas putida UW4（Pseudomonas putida UW4）：In american agriculture research culture presevation
Heart preservation（Deposit number is NRRL B-50193, and preservation day is on June 9th, 2008）.American agriculture studies DSMZ
Positioned at Illinois Pi Qiliya, the DSMZ for the government's property supported by Agricultural Research Center,
English full name Agrieultutal Research Service Culture Colleetion, abbreviation NRRL.UW4 can be produced
1-Aminocyclopropane-1-carboxylate deaminase, can be trained in the culture medium such as ADF using 1- amino-cyclopropane -1- carboxylic acids as only nitrogen source
Support in base and grow.
Culture medium
（1）Liquid MS medium（L）：KNO3 1900mg, NH4NO31650 mg, KH2PO4170 mg, MgSO4·7H2O 370
Mg, CaCl2·2H2O 440 mg, KI 0.83 mg, H3BO36.2 mg, MnSO4·4H2O 22.3 mg, ZnSO4·7 H2O
8.6 mg, Na2MoO4·2H2O 0.25 mg, CuSO4·5 H2O 0.025 mg, CoCl2·6H2O 0.025 mg, Na2-
EDTA 37.25 mg, FeSO4·7H2The mg of O 27.85, the mg of inositol 100, the mg of glycine 2, the mg of thiamine hydrochloride 0.1, hydrochloric acid
The mg of pyridoxol 0.5, nicotinic acid 0.5 mg, sucrose 30g；
（2）Solidified MS media（L）：Agar 7g is added in liquid MS medium；
（3）TSB fluid nutrient mediums：The g of soy peptone 3, the g of tryptone 17, the g of glucose 2.5, the g of sodium chloride 5, phosphoric acid hydrogen
The g of dipotassium 2.5, plus distilled water 1000 ml, pH 7.1-7.5；
（4）LB solid mediums：The g of yeast extract 5, tryptone 10 g, NaCl 10 g, the g of agar 20, deionized water
1000 mL；
（5）ADF culture mediums：
A liquid（Trace element）：MnSO4·H2O 11.19 mg, H3BO3 10 mg, CuSO4·5H2O 78.22 mg, ZnSO4·
7H2O 124.6 mg, MoO310 mg, are dissolved in 100 mL ultra-pure waters（It is sterile）In, -4 DEG C of preservations；
B liquid（FeSO4）：FeSO4·7H2The mg of O 100 are dissolved in 10mL ultra-pure waters（It is sterile）In, need matching while using；
A, B solution respectively take 0.1 mL, sequentially add Na2HPO46.0 g, KH2PO44.0 g, 1- amino-cyclopropane -1- carboxylic acids
2.0 g, MgSO4·7H2The g of O 0.2, the g of gluconic acid 2.0, the g of glucose 2.0, the g of citric acid 2.0, the g of agar 20, ultra-pure water
1000 mL are settled to, 7.2,121 DEG C of 20 min of sterilizing of pH are adjusted to.
Liquid concussion and cultivate.
1.3.1 wheat seed is pre-processed
The complete wheat seed of full seed, plumule is selected in clean flat board.The alcohol of addition 95% makes seed soak completely
After bubble, 30 s of processing, with sterile water washing three times, with autoclaved filter paper adsorption surface moisture.With the 0.1% of filtration sterilization
Mercuric chloride soaks 5 min, then with sterile water wash 5 times, is air-dried in superclean bench.Tiled in sterile flat board one big
Small suitable aseptic filter paper, the wheat seed after air-drying is tiled onto filter paper, and 10 mL sterilized waters are added into flat board.Stand
In 25 DEG C of h of constant incubator vernalization 24.
1.3.2 solid medium nursery
Solidified MS media is sub-packed in 300 ml tissue culture flasks, every bottle of 50 ml of packing.121 DEG C of min of autoclaving 20.
In superclean bench, the wheat seed after vernalization is seeded in solidified MS media surface, 5 seeds of every bottle of sowing.It will broadcast
Tissue culture flasks after kind are under 25-28 DEG C of daytime, the h of illumination 16,15-20 DEG C of night, dark condition, culture to Plant Height in Wheat
5-8 cm, incubation time 8-10 days.
1.3.3 prepared by bacteria suspension
TSB fluid nutrient mediums 5 ml, 121 DEG C of 30 min of sterilizing are filled in the test tube of 30 ml specifications.Access 1 ring pseudomonas putida
UW4 lawns, 28 DEG C, 220 rmp cultures, 12 h.8000 rpm, 5 min of centrifugation collect thalline, and thalline is washed with liquid MS medium
Wash 3 times, suspended with liquid MS medium and be adjusted to OD600It is worth for 1.
1.3.4 wheat is transferred and Liquid Culture
300 every bottle of ml tissue culture flasks dispense the ml of liquid MS medium 50, a built-in drift plate with holes, 121 DEG C of autoclavings
20 min.The wheat seedling cultivated in solid medium is transferred in liquid MS medium, 3 are connect in every bottle of fluid nutrient medium
Seedling.Tissue culture flasks are under conditions of 25-28 DEG C of daytime, the h of illumination 16,15-20 DEG C of night, dark on horizontal shaker 40
Rmp cultivates 24h.1 ml bacteria suspensions are added in superclean bench, are cultivated for 7 days.Simultaneously 1 ml sterilized waters are connect to be used as pair
According to.
1.3.5 wheat root colonizes bacterial number counting
Wheat seedling is taken out from liquid MS medium, the culture medium of root is gently wiped with aseptic filter paper, analysis is placed in
The weight of root is determined in balance.Take the root of 3 wheats to be placed in together in tissue grinder in each tissue culture flasks, add 2 ml
1 × PBS, is ground to pulpous state.Gradient dilution plate count is carried out using LB solid mediums, every gram of fresh weight wheat is calculated
The colony forming single-digit of root.
Solid culture.
1.4.1 seed disinfection
The complete wheat seed of full seed, plumule is selected in clean flat board.The alcohol of addition 95% makes seed soak completely
After bubble, 30 s of processing, with sterile water washing three times, with autoclaved filter paper adsorption surface moisture.With the 0.1% of filtration sterilization
Mercuric chloride soaks 5 min, then with sterile water wash 5 times, is air-dried in superclean bench.
1.4.2 microbionation
The bacteria suspension for taking 1.3.3 to prepare, 4 DEG C of 8000 rpm centrifuges 5min, and thalline is collected by centrifugation, and with sterile water washing 2 times, hangs
Float in sterilized water, regulation bacterium number is 109CFU/ml.Wheat seed is soaked into 3-4 h in bacteria suspension, using sterilized water as pair
According to.Make the abundant imbibition of seed, then filter out bacteria suspension, covered with the gauze of infiltration sterilized water in the surface of the seed, keep seed
Wet environment, is placed in 25-28 DEG C of constant incubators, until seed shows money or valuables one carries unintentionally.
1.4.3 vermiculite is potted plant
Vermiculite compares 1 with turfy soil with dry weight:1 mixes, and pours after moisture, 121 DEG C of sterilizing 3h.It is sub-packed in 72 cave polyethylene
In seedlings nursing plate, treated seed is sowed, per 1, cave, the cm of seed level 1.Plant is placed in 22-25 DEG C of daytime, illumination 16
H, 15-20 DEG C of night.Pour 5ml sterilized waters daily.4th d, 8 d and 12 d inoculation bacteria suspensions 5 mL.
Wheat root colonizes bacterial number counting
Potted plant 15-21 d, carefully all dig out plant root.Shake off the grogs adhered on root system, be placed in assay balance and survey
Determine the weight of root.Then, it is placed in tissue grinder, adds 21 × PBSs of ml, be ground to pulpous state.Using LB solids
Culture medium carries out gradient dilution plate count, calculates the colony forming single-digit of every gram of fresh weight wheat root（Total bacteria count）.It is solid with ADF
Body culture medium carries out gradient dilution plate count, calculates the colony forming single-digit of every gram of fresh weight wheat root（1- amino-cyclopropanes-
1- carboxylic acid deaminase producing strains bacterium numbers）.
As a result
2.1 liquid concussion and cultivate wheat roots colonize bacterial number
As seen from Table 1, the sterilized water control group of bacterium is not connect, the bacterium for not detecting to colonize in wheat rhizosphere shows whole reality
The process of testing can maintain aseptic condition well.Pseudomonas putida UW4 is inoculated with, it is thin that wheat rhizosphere can detect to colonize
The coefficient of variation in bacterium, 3 tissue culture flasks between the bacterial population of wheat rhizosphere colonization is less than 15%, and experimental error is smaller, table
The PGPR of prescribed liquid concussion and cultivate plnat monitoring rhizosphere colonization is feasible.
The liquid concussion and cultivate wheat rhizosphere of table 1 colonizes bacterial population
2.2 solid culture wheat roots colonize bacterial number
Although from table 2 and table 3 as can be seen that the coefficient of variation of experimental result is smaller, due to that can not accomplish in aseptic condition
Lower operation, the check plant root system pollution of bacterium is not connect 1-Aminocyclopropane-1-carboxylate deaminase producing strains and other bacteriums, shadow
Ring the authenticity of experimental result.
The solid culture wheat rhizosphere of table 2 colonizes total bacteria
The solid culture wheat rhizosphere of table 3 colonizes 1-Aminocyclopropane-1-carboxylate deaminase producing strains bacterium number
Claims (3)
1. a kind of method for detecting plant rhizosphere growth-promoting bacterium in root colonization, it is characterised in that comprise the following steps：
1）Wheat is transferred and Liquid Culture：The wheat seedling cultivated in solid medium is transferred in liquid MS medium,
Under 25-28 DEG C of daytime, the h of illumination 16,15-20 DEG C of night, dark condition, 24-48h is cultivated in 20-50 rmp on horizontal shaker,
Then 1-3 ml bacteria suspensions are added, are cultivated for 5-10 days；
2）Wheat root colonizes bacterial number counting：Wheat seedling is taken out, the culture medium of root is wiped with aseptic filter paper, weighed；
It is subsequently placed in tissue grinder, adds 21 × PBSs of ml, be ground to pulpous state, ladder is carried out using LB solid mediums
Spend dilution plate to count, calculate the colony forming single-digit of every gram of fresh weight wheat root.
2. method of the plant rhizosphere growth-promoting bacterium in root colonization is detected as claimed in claim 1, it is characterised in that step 1）
In, the wheat seedling cultivated in solid medium is obtained through following step：In superclean bench, by the wheat seed after vernalization
It is seeded in solidified MS media surface；Then in 25-28 DEG C of daytime, the h of illumination 16, trained under conditions of 15-20 DEG C of night, dark
Support to Plant Height in Wheat 5-8 cm.
3. method of the plant rhizosphere growth-promoting bacterium in root colonization is detected as claimed in claim 1, it is characterised in that step 1）
In, bacteria suspension is obtained through following step：TSB fluid nutrient medium 5-10 ml, 110-121 DEG C of sterilizing 10-30 min；Access stench
Pseudomonad UW4 lawns, 28-37 DEG C, 200-260 rmp culture 12-24 h, are collected by centrifugation thalline, thalline is cultivated with liquid MS
Base is washed, and is then suspended with liquid MS medium and is adjusted to OD600It is worth and is produced for 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114875058A (en) * | 2022-04-28 | 2022-08-09 | 河南农业大学 | Engineering bacterium transformation method for preventing and treating root rot of planted root tips |
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| CN114875058A (en) * | 2022-04-28 | 2022-08-09 | 河南农业大学 | Engineering bacterium transformation method for preventing and treating root rot of planted root tips |
| CN114875058B (en) * | 2022-04-28 | 2023-07-07 | 河南农业大学 | Engineering bacterium transformation method for preventing and treating root rot of field planting root tip |
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