CN109234183A - A kind of salt tolerant growth-promoting bacteria strain, screening technique and application - Google Patents
A kind of salt tolerant growth-promoting bacteria strain, screening technique and application Download PDFInfo
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Abstract
A kind of salt tolerant growth-promoting bacteria strain of the present invention, screening technique and application, salt tolerant growth-promoting bacteria strain is screened from alkaline land soil, the hereditary capacity and physio-biochemical characteristics of the bacterial strain are measured, determine that the bacterial strain is bacillus megaterium B7, on May 17th, 2018 is preserved in China typical culture collection center, and tolerance of salinity analysis is carried out to it, is tested the salt tolerant growth-promoting functions of cucumber in the Seed Germination in Cucumber and potted plant experiment under condition of salt stress.Salt tolerant growth-promoting bacteria strain, screening technique and application provided by the invention, the tolerance of salinity of bacillus megaterium B7 is 0~8%, have simultaneously and produces IAA, produce acc deaminase, produce a variety of growth-promoting characteristics such as ammonia, fixed nitrogen and phosphorus decomposing, cucumber salt tolerant growth-promoting ability can be significantly improved under salt stress using bacillus megaterium bacterium solution, slow down salt damage phenomenon, increase biomass, the use of chemical fertilizer is reduced, and can be used for preparing bacterium solution, for promoting crop germination and growth in salt stress.
Description
Technical field
The invention belongs to microorganism separation and applied technical fields, and in particular to a kind of salt tolerant growth-promoting bacteria strain, screening side
Method and application.
Background technique
Currently, the demand to cultivated land resource is also more and more as social development and population increase.The soil salinization is
The salinity of finger soil bottom or underground water, which rises to earth's surface after moisture evaporation with capillary, makes salt accumulation in topsoil
Process.Ease of solubility salinity upper soll layer accumulate the phenomenon that or process, also referred to as salinization of soil.Except in coastal region, due to by sea
Water retting influence and occur outside salinization of soil, arid and semi-arid lands be also because of industrialized agriculture caused by soil secondary salinization
Major reason.The salinization of soil is the main abiotic stress factor of current threat Global Agriculture production and crop yield
One of.The whole world has 50% to irrigate land all by different degrees of salt stress at present, point of Chinese salinized soil or saline-alkali soil
Cloth range is wide, area is big, type is more, about 100,000,000 hm of the gross area2, and area is threatened to increase year by year.How to improve and utilize and is big
Area salinization land and protection and to improve important industrial crops to resist salt stress be that current international community is urgently to be resolved
Scientific And Technical Problems.
Currently, primary biological modification method is to cultivate and plant saline-alkali tolerant crop, such as plant sorghum, beet, sunflower with
And the salt-tolerant plants such as Chinese tamarisk, Sha Pengcao, false indigo, in addition there are the researchs for being directed to salt tolerant genetically modified plants, but such method is logical
Often the consuming period is long, costly, and plant transgenic method is not also accepted extensively and approved by society.
Rhizosphere microorganism refers to the microorganisms such as growth and breeding bacterium, actinomyces, fungi within the scope of root system of plant soil.
Most of rhizosphere microorganisms have facilitation to non-phytotoxic and to plant growth, these rhizosphere microorganisms have fixed nitrogen, molten
Phosphorus, the function of generating auxin, siderophore and inducing plant resistance improve salt-soda soil using rhizosphere microorganism and make
The method of object salt tolerant growth-promoting has the characteristics that small investment, quick, high efficiency, the function in relation to rhizosphere microorganism salt tolerant growth-promoting
Activity research has been subjected to the extensive concern of correlative study person, but still not about microorganism separating and purifying technology in salt-soda soil
Enough maturations, stability are poor.
Summary of the invention
Goal of the invention: to solve problems of the prior art, the present invention provides a kind of salt tolerant growth-promoting bacteria strain, sieve
Choosing method and application can be used for improving salt tolerance of crops, while can be the clone and transgenic salt-tolerant wheat plant of resistant gene of salt
Building lays the foundation.
Scheme of the invention: to achieve the above object, the invention is realized by the following technical scheme:
A kind of salt tolerant growth-promoting bacteria strain, the classification naming of the salt tolerant growth-promoting bacteria strain are bacillus megaterium
B7Bacillus megaterium B7, deposit number are as follows: CCTCC NO:M 2018284 is preserved in Chinese Typical Representative culture
Collection, depositary institution's code: CCTCC, address: China, Wuhan, Wuhan University, Wuhan City, Hubei Province Wuchang District Bayi Road
Luo Jia Shan, preservation time are as follows: on May 17th, 2018.
Further, the 16S rDNA nucleotide sequence of the bacillus megaterium B7 is as shown in sequence table, huge gemma
Bacillus B7 is in Gram-positive, and indole reaction is positive, and bacterium colony is white, neat in edge, and median rise, surface is smooth wet, no
Transparent, diameter is 2~3mm, aerobic, chemoheterotrophy.
Further, comprising the following steps:
Step 1: the separation and screening of salt tolerant growth-promoting bacteria strain
Soil sample is acquired from the alkaline land soil of Jiangsu Yancheng beach, appropriate soil sample is weighed and is suspended in sterile water, it is dilute
After releasing certain multiple, equal amount soil dilution liquid is taken, is respectively coated on the NA culture medium flat plate of different NaCl concentrations and carries out
Step-sizing culture observes the condition of production of bacterial strain under different salinity in 29 DEG C~31 DEG C 2~3d of culture, and to filtering out
Well-grown bacterial strain carry out repeatedly plate streaking purify, obtain the salt tolerant growth-promoting bacteria strain of single bacterium colony；
Step 2: the identification of salt tolerant growth-promoting bacteria strain
By the extraction DNA of single bacterium colony salt tolerant growth-promoting bacteria strain made from step 1 and as template, 16S rDNA is general to be drawn
Object is primer, is expanded to 16S rDNA nucleotide fragments, the segment of amplification is directly carried out sequencing, to the bacterium
Strain carries out physiological and biochemical test, obtains the title of salt tolerant growth-promoting bacteria strain according to measurement result and is preserved in public collection；
Step 3: the Salt Tolerance Analysis of salt tolerant growth-promoting bacteria strain
By the salt tolerant growth-promoting bacteria strain bacterium solution of separated purifying transfer respectively equal amount to different salinity Liquid Culture
In base, shaking table culture for a period of time, is observed under different salinity under the conditions of 29~31 DEG C of temperature, 180~210r/min of revolving speed
The growing state of bacterial strain tests bacteria concentration, determines the tolerance of salinity of salt tolerant growth-promoting bacteria strain.
Further, in step 3, the preparation step of the salt tolerant growth-promoting bacteria strain bacterium solution of the separated purifying are as follows: will
The salt tolerant growth-promoting bacteria strain for the single bacterium colony that step 1 obtains is activated, and is subsequently placed into constant-temperature table culture for a period of time, vibration
It swings to shake and take out afterwards, adjust light absorption value to 0.6~0.8 with sterile water, the tolerance of salinity of the salt tolerant growth-promoting bacteria strain is 0-8%.
Further, a kind of bacterial strain bacterium solution of salt tolerant growth-promoting bacteria strain preparation.
Further, comprising the following steps: picking is in the well-grown salt tolerant growth-promoting bacteria strain single bacterium of LB solid plate
It falls, is inoculated in LB liquid medium, a timing is cultivated in 29~31 DEG C of temperature, the shaking flask of 180~210r/min of revolving speed
Between, it when strain growth to logarithmic growth phase, is put into sterile centrifugation tube and is centrifuged, abandon supernatant, collect bacterial strain and use sterile water
For several times, using sterile water to adjust the OD value of bacterial strain is 0.5~0.8 as bacterial strain bacterium solution for washing.
Further, the application that a kind of salt tolerant growth-promoting bacteria strain promotes crop to germinate and grow in salt stress.
Further, comprising the following steps:
1) cucumber seeds of unprocessed mistake are taken and is put into beaker the sterilizing that carries out disinfection in right amount, first do not had kind with ethyl alcohol
Son is soaked for a period of time, and then pours out ethanol solution, is added the mercury chloride now matched and is impregnated, pours out mercuric chloride solution after finishing,
Finally for several times with sterile water wash；
2) cleaned cucumber seeds are put into the salt tolerant growth-promoting bacteria strain bacterium solution prepared and are soaked for a period of time,
It is spare；
3) prepare appropriate culture dish, absorbent cotton is laid in culture dish bottom, sterilize after covering stand-by；
4) sodium chloride solution for configuring different salinity, pours into after sterilizing and does not cross absorbent cotton in the culture dish of step 3),
It marks, the cucumber seeds of step 2) is taken into equal amount, are sandwiched respectively with sterilizing tweezers in the culture dish of different salinity,
Culture dish is then put into 25~27 DEG C of culture a period of times in illumination box, dark culture and illumination cultivation are alternately, real
Germination percentage, fresh weight and the dry weight of cucumber seeds are recorded after testing and carry out interpretation of result.
Further, comprising the following steps: take appropriate be put into beaker to carry out disinfection the cucumber seeds of unprocessed mistake
Sterilizing, there first was not seed to be soaked for a period of time, and then poured out ethanol solution with ethyl alcohol, adds the mercury chloride now matched and impregnates, bubble
Mercuric chloride solution is poured out after complete, finally for several times with sterile water wash；Volume ratio is used to mix for 4:1~6:1 Nutrition Soil with vermiculite
Potting media is obtained, then in 120~122 DEG C of sterilizing a period of times, equivalent is filled in the basin of cave after cooling；Every basin kind enters equivalent
The cucumber seeds handled well are poured with the salt tolerant growth-promoting bacteria strain bacterium solution prepared, after emergence, calculate germination percentage and emergence rate,
Retain a strain number seedling, total root long, total projection area, total root surface area, average roots oxidizing and tip of a root number are measured after one month
Etc. the physical signs such as growth indexes and glucose, lysine content and carry out interpretation of result.
Further, in step 2), the preparation step of salt tolerant growth-promoting bacteria strain bacterium solution are as follows: the salt tolerant of single bacterium colony is taken to promote
Raw bacteria strain is activated, and is subsequently placed into constant-temperature table culture one day and is taken out, and adjusts light absorption value to 0.6~0.8 with sterile water.
Compared with prior art, the invention has the benefit that
The invention discloses a kind of salt tolerant growth-promoting bacteria strain, screening technique and applications, screen from alkaline land soil resistance to
Salt growth-promoting bacteria strain is measured the hereditary capacity and physio-biochemical characteristics of the bacterial strain, determines that the bacterial strain is huge gemma bar
Bacterium B7 (Bacillus megaterium), and tolerance of salinity analysis is carried out to it, to the Seed Germination in Cucumber under condition of salt stress
And the salt tolerant growth-promoting functions of cucumber are tested in potted plant experiment, the results showed that, which is 0~8%, most suitable
The tolerance of salinity is 4~6%, while having and producing IAA, produce acc deaminase, produce NH3, fixed nitrogen, phosphorus decomposing and produce a variety of growth-promotings such as thermophilic iron element
Characteristic can significantly improve cucumber salt tolerant growth-promoting energy using the bacterium solution of bacillus megaterium B7 preparation, bacterial strain bacterium solution under salt stress
Power slows down salt damage phenomenon, increase biomass helps to improve the salt tolerance of cucumber, subtract to promote the effect of cucumber growth
The use of few chemical fertilizer, the salt tolerant growth-promoting bacteria strain and its bacteria liquid has broad application prospect.
Detailed description of the invention
Fig. 1 is the plate culture picture of salt tolerant growth-promoting bacteria strain of the present invention；
Fig. 2 is the tolerance of salinity curve graph of salt tolerant growth-promoting bacteria strain of the present invention；
Fig. 3 A is that blank control of the invention is tested；
Fig. 3 B is the lab diagram of cucumber seeds of the invention under condition of salt stress；
Fig. 3 C is the lab diagram of cucumber seeds of the invention under bacterium solution and salt stress；
Fig. 4 is the phylogenetic tree of the salt tolerant growth-promoting bacteria strain constructed based on 16S rDNA gene order.
Specific embodiment
The technical scheme of the present invention will be explained in further detail With reference to embodiment.
Picture 1-4 and shown in nucleotides sequence list, a kind of salt tolerant growth-promoting bacteria strain, the classification life of salt tolerant growth-promoting bacteria strain
Entitled bacillus megaterium B7Bacillus megaterium B7, deposit number are as follows: CCTCC NO:M 2018284 is preserved in
China typical culture collection center, depositary institution's code: CCTCC, address: China, Wuhan, Wuhan University, Wuhan City, Hubei Province
City Wuchang District Bayi Road Luo Jia Shan, preservation time are as follows: on May 17th, 2018, bacillus megaterium bacterial strain B7 is in gram sun
Property, indole reaction is positive, and bacterium colony is white, neat in edge, and median rise, surface is smooth wet, opaque, and diameter is 2~
3mm, aerobic, chemoheterotrophy.
A kind of screening technique of salt tolerant growth-promoting bacteria strain, comprising the following steps:
Step 1: the separation and screening of salt tolerant growth-promoting bacteria strain
Soil sample is acquired from the alkaline land soil of Jiangsu Yancheng beach, appropriate soil sample is weighed and is suspended in sterile water, it is dilute
After releasing certain multiple, equal amount soil dilution liquid is taken, is respectively coated on different NaCl concentrations (2%, 4%, 6%, 8%)
Step-sizing culture is carried out on NA culture medium flat plate observes bacterial strain under different salinity in 29 DEG C~31 DEG C 2~3d of culture
The condition of production, and plate streaking is carried out repeatedly to the well-grown bacterial strain filtered out and is purified, the salt tolerant for obtaining single bacterium colony promotees
Raw bacteria strain；
Step 2: the identification of salt tolerant growth-promoting bacteria strain
By the extraction DNA of single bacterium colony salt tolerant growth-promoting bacteria strain made from step 1 and as template, 16S rDNA is general to be drawn
Object is primer, expands to 16S rDNA nucleotide fragments, is detailed in nucleotides sequence list, and the segment of amplification is directly carried out
Sequencing carries out physiological and biochemical test to the bacterial strain, title and the guarantor of salt tolerant growth-promoting bacteria strain is obtained according to measurement result
It is hidden in public collection；
Step 3: the Salt Tolerance Analysis of salt tolerant growth-promoting bacteria strain
By the salt tolerant growth-promoting bacteria strain bacterium solution of separated purifying transfer respectively equal amount to different salinity Liquid Culture
In base, shaking table culture for a period of time, is observed under different salinity under the conditions of 29~31 DEG C of temperature, 180~210r/min of revolving speed
The growing state of bacterial strain tests bacteria concentration, determines the tolerance of salinity of salt tolerant growth-promoting bacteria strain, obtain salt tolerant as shown in Figure 2 and write music
Line chart；The preparation step of the salt tolerant growth-promoting bacteria strain bacterium solution of separated purifying are as follows: the salt tolerant for the single bacterium colony for obtaining step 1
Growth-promoting bacteria strain is activated, and is subsequently placed into constant-temperature table culture for a period of time, and oscillation is shaken takes out afterwards, is adjusted with sterile water
For light absorption value to 0.6~0.8, the tolerance of salinity of salt tolerant growth-promoting bacteria strain is 0-8%.
The step of bacterial strain bacterium solution is prepared using salt tolerant growth-promoting bacteria strain B7 are as follows: picking is well-grown in LB solid plate
Salt tolerant growth-promoting bacteria strain single colonie, be inoculated in LB liquid medium, in 29~31 DEG C of temperature, 180~210r/min of revolving speed
Certain time is cultivated in shaking flask, when strain growth to logarithmic growth phase, is put into sterile centrifugation tube and is centrifuged, abandons supernatant, is collected
Bacterial strain simultaneously using sterile water washing for several times, using sterile water to adjust the OD value of bacterial strain is 0.5~0.8 as bacterial strain bacterium solution, with
In promoting in crop germination and growth application, when the bacterial strain bacterium solution of preparation adjusts light absorption value to 0.6~0.8 using sterile water, side
It can obtain the salt tolerant growth-promoting bacteria strain bacterium solution suitable for plant growth；Stabilizer can also be added, required microbial inoculum is made in conven-tional adjuvants
Carry out the application of salt tolerant growth-promoting.
A kind of application that salt tolerant growth-promoting bacteria strain promotes crop to germinate and grow in salt stress, is trained for cucumber seeds
Ware germination, comprising the following steps:
1) cucumber seeds of unprocessed mistake are taken and is put into beaker the sterilizing that carries out disinfection in right amount, first do not had kind with ethyl alcohol
Son is soaked for a period of time, and then pours out ethanol solution, is added the mercury chloride now matched and is impregnated, pours out mercuric chloride solution after finishing,
Finally for several times with sterile water wash；
2) cleaned cucumber seeds are put into the salt tolerant growth-promoting bacteria strain bacterium solution prepared and are soaked for a period of time,
It is spare；
3) prepare appropriate culture dish, absorbent cotton is laid in culture dish bottom, sterilize after covering stand-by；
4) sodium chloride solution for configuring different salinity, pours into after sterilizing and does not cross absorbent cotton in the culture dish of step 3),
It marks, the cucumber seeds of step 2) is taken into equal amount, are sandwiched respectively with sterilizing tweezers in the culture dish of different salinity,
Culture dish is then put into 25~27 DEG C of culture a period of times in illumination box, dark culture and illumination cultivation are alternately, real
Germination percentage, fresh weight and the dry weight of cucumber seeds are recorded after testing and carry out interpretation of result.
In step 2), the preparation step of salt tolerant growth-promoting bacteria strain bacterium solution are as follows: take the salt tolerant growth-promoting bacteria strain of single bacterium colony into
Row activation is subsequently placed into constant-temperature table culture one day and takes out, and adjusts light absorption value to 0.6~0.8 with sterile water.
A kind of application that salt tolerant growth-promoting bacteria strain promotes crop to germinate and grow in salt stress, it may also be used for potting training
It supports, comprising the following steps: the cucumber seeds of unprocessed mistake are taken and are put into beaker the sterilizing that carries out disinfection in right amount, first use ethyl alcohol
It does not cross seed to be soaked for a period of time, then pours out ethanol solution, add the mercury chloride now matched and impregnate, pour out chlorination after finishing
Mercury solution, finally for several times with sterile water wash；Volume ratio is used to be mixed to get potting base for 4:1~6:1 Nutrition Soil and vermiculite
Matter, then in 120~122 DEG C of sterilizing a period of times, equivalent is filled in the basin of cave after cooling；Every basin kind enters the Huang that equivalent is handled well
Melon seed is poured with the salt tolerant growth-promoting bacteria strain bacterium solution prepared, after emergence, is calculated germination percentage and emergence rate, is retained a strain number
Seedling measures total root long, total projection area, total root surface area, the average growth indexes such as roots oxidizing and tip of a root number after one month
And the physical signs such as glucose, lysine content and carry out interpretation of result.
The present invention is not limited to the growth-promoting applications of cucumber seeds, it may also be used for other same or related subjects crop
Growth-promoting application.
Embodiment 1
Step 1: the separation and screening of salt tolerant growth-promoting bacteria strain
On July 6th, 2017 acquires 15 parts of soil sample from the alkaline land soil of Jiangsu Yancheng beach, and each soil sampling 1g is outstanding
Float in 100ml sterile water, and be diluted to 105 times with this, use NaCl concentration for 2%, 4%, 6%, 8% NA culture medium
Step-sizing salt tolerant growth-promoting bacteria strain, specifically: take soil dilution liquid 0.1ml to be coated on culture medium flat plate respectively with pipettor
On, 29~31 DEG C are cultivated 2~3 days, are chosen at the good bacterium colony of 8% salinity growth, being crossed repeatedly, it is standby to save after purification
With, screen single bacterium colony, one plant of salt tolerant growth-promoting bacteria strain B7, as shown in Figure 1.
Step 2: the identification of salt tolerant growth-promoting bacteria strain
The identification of bacteria molecule genetic classification is carried out to the salt tolerant growth-promoting bacteria strain screened: DNA is extracted to bacterial strain B7,
Using the DNA as template, 16S rDNA universal primer is primer, is expanded to 16S rDNA segment, and the segment of amplification is direct
Sequencing is carried out, the blast program on the result input website NCBI of sequencing is compared, as the result is shown bacterial strain 16S
The 16s rDNA sequence of Bacillus megaterium in rDNA nucleotide sequence and GenBank gene pool in bacillus
Homologous degree highest is arranged, homology reaches 100%, as shown in Fig. 4 and sequence table；Have by DNAMAN6.0 in Genbank
Bacillus 16SrDNA sequence carry out phylogenetic analysis, the results show that salt tolerant growth-promoting bacteria strain B7 16S rDNA
It, therefore can be using the preliminary judgement bacterial strain as the huge gemma of bacillus with Bacillus megaterium homology highest
Bacillus B7.
With reference to " common bacteria system identification handbook ", by carrying out physiological and biochemical test progress to salt tolerant growth-promoting bacteria strain
Preliminary Identification, qualification result are shown: Gram-positive, and indole reaction is positive, can hydrolyze starch and glucose, can liquefy gelatin；
The general experiment reaction negative of Fu-, clark and Lubsreaction are negative, and hydrogen sulfide is negative, and citrate experiment is negative.
According to above-mentioned qualification result, salt tolerant growth-promoting bacteria strain B7 is accredited as bacillus megaterium B7.
Step 3: the Salt Tolerance Analysis of salt tolerant growth-promoting bacteria strain
Bacterial strain after activation is put into after constant-temperature table to cultivate one day and take out, with sterile water be transferred to light absorption value be 0.6~
0.8, the salt tolerant growth-promoting bacteria strain bacterium solution isolated and purified, the fluid nutrient medium of equal amount of transferring respectively to different salinity
In, 30 DEG C, cultivate in the shaking table of 180~210r/min, growing state is observed, surveys its bacteria concentration (OD600), afterwards for 24 hours with determination
The salt tolerant degree of the bacterial strain obtains tolerance of salinity curve graph as shown in Figure 2.
Salt tolerance experimental result is as shown in Figure 2, it is seen that salt tolerant growth-promoting bacteria strain B7 has wider adaptation model to sodium chloride
It encloses, in salt-free situation autolysis does not occur for bacterial strain, and higher thallus still can be kept when salinity reaches 6%
Concentration can be grown under 0~8% salinity, and most suitable salinity is 4%~6%.
Step 4: bacterial strain application experiment
As shown in Fig. 3 A, 3B and 3C, the application that salt tolerant growth-promoting bacteria strain promotes crop to germinate and grow in salt stress,
The following steps are included:
Cucumber seeds culture dish Seed germination: appropriate be put into beaker is taken to disappear the cucumber seeds of unprocessed mistake
Poison sterilizing, there first was not seed to impregnate 1min, and poured out ethanol solution after finishing, add 0.1% now matched with 75% ethyl alcohol
Mercury chloride impregnates 1min, mercuric chloride solution is poured out after finishing, finally three times with sterile water wash；Above-mentioned cleaned kind
Son, which is put into salt tolerant growth-promoting bacteria strain bacterium solution, impregnates 6h；Prepare suitable culture dish, absorbent cotton be laid in culture dish bottom,
It sterilizes after covering stand-by；The sodium chloride solution for configuring 200mM, different salinity, pours into after sterilizing and does not cross degreasing in culture dish
Cotton, and mark；The soaking cucumber seeds for crossing bacterium solution are sandwiched to the culture dish of different salinity with sterilized tweezers
In, 40 seeds are put in each culture dish, and the above-mentioned culture dish that all steps are completed is put into illumination box 25 DEG C
Cultivate 7d, first three days dark culture, rear four days 12h illumination cultivations, 12h dark culture；Using only plus sterile Aquaponic culture dish as
Blank control, the culture dish for only using equal parts salt tolerant growth-promoting bacteria strain bacterium solution, 200mM NaCl to cultivate respectively as to according to the facts
Test 1 and control experiment 2；Seed Germination in Cucumber rate, fresh weight and dry weight are recorded after experiment.As the result is shown: salt tolerant Promoting bacteria bacterium
Strain bacterium solution adds the germination percentage of salt stress processing group to be 1.25 times of salt stress group, and fresh weight is its 1.23 times, illustrates salt tolerant Promoting bacteria
Bacterial strain B7 has apparent salt tolerant growth-promoting functions to cucumber seeds, and salt tolerant growth-promoting bacteria strain as shown in Table 1 sends out cucumber seeds
The influence of bud and primary growth.
Table 1
Cucumber pot experiment: after cucumber seeds warm water soaked overnight, first do not had seed immersion with 75% ethyl alcohol
1min pours out ethanol solution after finishing, and adds 0.1% mercury chloride now matched and impregnates 1min, it is molten to pour out mercury chloride after finishing
Liquid, it is spare after finally being cleaned three times with sterilized distilled water；The Nutrition Soil and vermiculite of volume ratio 4:1 is mixed to get potting
Matrix is filled in the basin of cave, cave basin size then in 121 DEG C of sterilizing 1h after cooling are as follows: 10cm long × 10cm wide × 10cm high, often
Basin is packed into 200g；Blank control group (no salt stress), salt stress group and salt stress is respectively set and adds salt tolerant growth-promoting bacteria strain bacterium solution
Group, every group is done 5 repetitions；Every basin kind enters cucumber seeds 3 handled well, and salt adds the seed of bacterium solution group first to use salt tolerant Promoting bacteria
6h is impregnated in bacterial strain bacterium solution, the seed aqua sterilisa immersion 6h of blank group and salt stress group is then poured with different solutions, it may be assumed that
Blank group is poured with sterile water, and salt adds bacterium solution group salt tolerant growth-promoting bacteria strain bacterium solution mixing 200mM NaCl solution to pour, the salt side of body
Compel group to be poured with 200mM NaCl solution, pouring processing is primary weekly, remaining time all pours sterile water, after emergence, calculates hair
Bud rate and emergence rate, then every basin retains 1 strain number seedling, measured after one month total root long, total projection area, total root surface area,
The average physical signs such as the growth indexes such as roots oxidizing and tip of a root number and glucose, lysine content, as shown in table 2-3, table 2
For influence of the salt tolerant growth-promoting bacteria strain in potted plant experiment to cucumber seedling early growth, table 3 is salt tolerant growth-promoting bacteria strain in potting
To the influence of cucumber seedling physical signs in experiment, salt tolerant growth-promoting bacteria strain bacterium solution adds total root long of salt stress group, total projection face
Long-pending, total root surface area, average roots oxidizing and tip of a root number are significantly increased, and the physical signs such as glucose, lysine content obtain
It significantly improves, proves the effect of salt tolerant growth-promoting bacteria strain of the invention in terms of growth-promoting salt tolerant.
Table 2
Table 3
Embodiment 2
A kind of bacterial strain bacterium solution of salt tolerant growth-promoting bacteria strain preparation, comprising the following steps:
Picking grows 48h, well-grown salt tolerant growth-promoting bacteria strain B7 single colonie on LB solid plate, is inoculated in
In 250ml LB liquid medium, after cultivating 48h in 29~31 DEG C of temperature, the shaking flask of 180~210r/min of revolving speed, to bacterium
Body grows to logarithmic growth phase, is put into sterile centrifugation tube, and 10min is centrifuged under 12000rpm revolving speed, abandons supernatant, collects lower layer
Bacterial strain, with sterile water washing thalline twice after, using sterile water to adjust the OD value of thallus is 0.5~0.8 as bacterial strain bacterium solution,
For plant salt tolerance growth-promoting.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Sequence table
<110>Suzhou Agricultural Vocational College
<120>a kind of salt tolerant growth-promoting bacteria strain, screening technique and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1414
<212> DNA
<213>bacillus megaterium (Bacillus megaterium)
<400> 2
ctagctcctt acggttactc caccgacttc gggtgttaca aactctcgtg gtgtgacggg 60
cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg 120
attccagctt catgtaggcg agttgcagcc tacaatccga actgagaatg gttttatggg 180
attggcttga cctcgcggtc ttgcagccct ttgtaccatc cattgtagca cgtgtgtagc 240
ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg 300
gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt tgcgctcgtt 360
gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac 420
tctgtccccc gaaggggaac gctctatctc tagagttgtc agaggatgtc aagacctggt 480
aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540
caattccttt gagtttcagt cttgcgaccg tactccccag gcggagtgct taatgcgtta 600
gctgcagcac taaagggcgg aaaccctcta acacttagca ctcatcgttt acggcgtgga 660
ctaccagggt atctaatcct gtttgctccc cacgctttcg cgcctcagcg tcagttacag 720
accaaaaagc cgccttcgcc actggtgttc ctccacatct ctacgcattt caccgctaca 780
cgtggaattc cgcttttctc ttctgcactc aagttcccca gtttccaatg accctccacg 840
gttgagccgt gggctttcac atcagactta agaaaccgcc tgcgcgcgct ttacgcccaa 900
taattccgga taacgcttgc cacctacgta ttaccgcggc tgctggcacg tagttagccg 960
tggctttctg gttaggtacc gtcaaggtac aagcagttac tcttgtactt gttcttccct 1020
aacaacagag ttttacgacc cgaaagcctt catcactcac gcggcgttgc tccgtcagac 1080
tttcgtccat tgcggaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc 1140
agtcccagtg tggccgatca ccctctcagg tcggctatgc atcgttgcct tggtgagccg 1200
ttacctcacc aactagctaa tgcaccgcgg gcccatctgt aagtgatagc cgaaaccatc 1260
tttcaatcat ctcccatgaa ggagaagatc ctatccggta ttagcttcgg tttcccgaag 1320
ttatcccagt cttacaggca ggttgcccac gtgttactca cccgtccgcc gctaacgtca 1380
tagaagcaag cttctaatca gttcgctcga cttg 1414
Claims (10)
1. a kind of salt tolerant growth-promoting bacteria strain, which is characterized in that the salt tolerant growth-promoting bacteria strain is bacillus megaterium B7, is preserved in
China typical culture collection center, address: China, Wuhan, Wuhan University, Wuhan City, Hubei Province Wuchang District Bayi Road Luo Ka
Mountain, deposit number are as follows: CCTCC M 2018284, preservation time are as follows: on May 17th, 2018.
2. a kind of salt tolerant growth-promoting bacteria strain according to claim 1, which is characterized in that the bacillus megaterium B7 is in leather
Lan Shi is positive, and indole reaction is positive, and bacterium colony is white, neat in edge, and median rise, surface is smooth wet, and opaque, diameter is
2~3mm, aerobic, chemoheterotrophy.
3. a kind of screening technique of salt tolerant growth-promoting bacteria strain according to claim 1 or 2, which is characterized in that including following
Step:
Step 1: the separation and screening of salt tolerant growth-promoting bacteria strain
Soil sample is acquired from the alkaline land soil of Jiangsu Yancheng beach, appropriate soil sample is weighed and is suspended in sterile water, be diluted to
After certain multiple, equal amount soil dilution liquid is taken, is respectively coated on the NA culture medium flat plate of different NaCl concentrations and carries out step by step
Screening and culturing observes the condition of production of bacterial strain under different salinity, and the life to filtering out in 29 DEG C~31 DEG C 2~3d of culture
Long good bacterial strain carries out repeatedly plate streaking and purifies, and obtains single bacterium colony salt tolerant growth-promoting bacteria strain；
Step 2: the identification of salt tolerant growth-promoting bacteria strain
By the extraction DNA of single bacterium colony salt tolerant growth-promoting bacteria strain made from step 1 and as template, 16S rDNA universal primer is
Primer, expands 16S rDNA nucleotide fragments, and the segment of amplification is directly carried out sequencing, carries out to the bacterial strain
Physiological and biochemical test obtains the title of salt tolerant growth-promoting bacteria strain according to measurement result and is preserved in public collection；
Step 3: the Salt Tolerance Analysis of salt tolerant growth-promoting bacteria strain
The salt tolerant growth-promoting bacteria strain bacterium solution of separated purifying is transferred into equal amount into the fluid nutrient medium of different salinity respectively,
Shaking table culture for a period of time, observes bacterial strain under different salinity under the conditions of 29~31 DEG C of temperature, 180~210r/min of revolving speed
Growing state tests bacteria concentration, determines the tolerance of salinity of salt tolerant growth-promoting bacteria strain.
4. a kind of screening technique of salt tolerant growth-promoting bacteria strain according to right 3, which is characterized in that in step 3, it is described
The preparation step of the salt tolerant growth-promoting bacteria strain bacterium solution isolated and purified are as follows: the salt tolerant Promoting bacteria bacterium for the single bacterium colony for obtaining step 1
Strain is activated, and is subsequently placed into constant-temperature table culture for a period of time, and oscillation is shaken and taken out afterwards, with sterile water adjust light absorption value to
0.6~0.8, the tolerance of salinity of the salt tolerant growth-promoting bacteria strain is 0-8%.
5. using a kind of bacterial strain bacterium solution of salt tolerant growth-promoting bacteria strain preparation of any of claims 1 or 2.
6. a kind of bacterial strain bacterium solution of salt tolerant growth-promoting bacteria strain preparation according to claim 5, which is characterized in that including following
Step: picking is inoculated in LB liquid medium, in the well-grown salt tolerant growth-promoting bacteria strain single colonie of LB solid plate in temperature
Certain time is cultivated in 29~31 DEG C of degree, the shaking flask of 180~210r/min of revolving speed, when strain growth to logarithmic growth phase, is put
Enter in sterile centrifugation tube and be centrifuged, abandon supernatant, collect bacterial strain and use sterile water washing for several times, sterile water is used to adjust the OD of bacterial strain
Value is used as bacterial strain bacterium solution for 0.5~0.8.
7. a kind of salt tolerant growth-promoting bacteria strain according to claim 1 or 2 promotes crop to germinate and grow in salt stress
Using.
8. a kind of salt tolerant growth-promoting bacteria strain according to claim 7 promotes in salt stress, crop germinates and what is grown answers
With, which comprises the following steps:
The cucumber seeds of unprocessed mistake are taken and are put into beaker the sterilizing that carries out disinfection in right amount, did not first have seed to impregnate one with ethyl alcohol
The section time, ethanol solution is then poured out, adds the mercury chloride now matched and impregnate, mercuric chloride solution is poured out after finishing, finally uses nothing
Bacterium water cleans for several times；
Cleaned cucumber seeds are put into the salt tolerant growth-promoting bacteria strain bacterium solution prepared and are soaked for a period of time, it is spare；
Prepare appropriate culture dish, absorbent cotton is laid in culture dish bottom, sterilizes after covering stand-by；
The sodium chloride solution of different salinity is configured, is poured into after sterilizing and does not cross absorbent cotton in the culture dish of step 3), carry out mark
The cucumber seeds of step 2) are taken equal amount, are sandwiched respectively with sterilizing tweezers in the culture dish of different salinity by note, then will training
Feeding ware is put into illumination box 25~27 DEG C of cultures a period of times, dark culture and illumination cultivation alternately, after experiment
It records germination percentage, fresh weight and the dry weight of cucumber seeds and carries out interpretation of result.
9. a kind of salt tolerant growth-promoting bacteria strain according to claim 7 promotes in salt stress, crop germinates and what is grown answers
With, which comprises the following steps: the cucumber seeds of unprocessed mistake are taken to be put into carry out disinfection in beaker in right amount and are gone out
Bacterium did not first have seed to be soaked for a period of time with ethyl alcohol, then poured out ethanol solution, adds the mercury chloride now matched and impregnates, finishes
After pour out mercuric chloride solution, finally for several times with sterile water wash；Volume ratio is used to mix for 4:1~6:1 Nutrition Soil with vermiculite
To potting media, then in 120~122 DEG C of sterilizing a period of times, equivalent is filled in the basin of cave after cooling；Every basin kind enters equivalent processing
Good cucumber seeds are poured with the salt tolerant growth-promoting bacteria strain bacterium solution prepared, after emergence, are calculated germination percentage and emergence rate, are retained
One strain number seedling measures total root long, total projection area, total root surface area, the average life such as roots oxidizing and tip of a root number after one month
The physical signs such as long pointer and glucose, lysine content simultaneously carry out interpretation of result.
10. a kind of salt tolerant growth-promoting bacteria strain according to claim 8 promotes in salt stress, crop germinates and what is grown answers
With, which is characterized in that in step 2), the preparation step of salt tolerant growth-promoting bacteria strain bacterium solution are as follows: take the salt tolerant Promoting bacteria of single bacterium colony
Bacterial strain is activated, and is subsequently placed into constant-temperature table culture one day and is taken out, and adjusts light absorption value to 0.6~0.8 with sterile water.
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| CN115287212A (en) * | 2022-04-14 | 2022-11-04 | 国家林业和草原局竹子研究开发中心 | Salt-tolerant growth-promoting bacterium ACP81 and application thereof |
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