CN109439589A - A kind of rhizobium YZLH133 and its application - Google Patents
A kind of rhizobium YZLH133 and its application Download PDFInfo
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- CN109439589A CN109439589A CN201811478038.4A CN201811478038A CN109439589A CN 109439589 A CN109439589 A CN 109439589A CN 201811478038 A CN201811478038 A CN 201811478038A CN 109439589 A CN109439589 A CN 109439589A
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- rhizobium
- yzlh133
- peanut
- nitrogen
- tieback
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/41—Rhizobium
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
Abstract
The invention discloses a kind of rhizobium YZLH133 and its application, which is the rhizobium being captured from Fujian Province's Youxi Soils In The Region by dragon flower peanut varieties, and rhizobium are separated and purified from fresh root nodule, and to bacterial strain16sRDNA partial sequence is measured, by Phylogenetic Analysis, be determined as Bradyrhizobium (Bradyrhizobium) new strains system, and be named as good fortune agriculture 3.Rhizobium YZLH133 of the invention is tested by laboratory water planting tieback and crop field tieback is experiments have shown that its characteristic strong with efficient dross, nitrogen fixing capacity, being inoculated with the rhizobium can be improved peanut biomass and yield, it is applicable to South China Acidic Soil, one of the measure as peanut conventional cultivation.
Description
Technical field
The invention belongs to microorganism fields, and in particular to a kind of rhizobium YZLH133 and its application.
Background technique
Rhizobium (rhizobium) it is a kind of gramnegative bacterium being distributed widely in soil, pulse family plant can be infected
Root nodule (root nodule) or stem tumor (stem nodule) are formed behind object root or stem, in the form of homobium in fixed air
N 2The NH utilized as plant available4 +.Syntaxial system between leguminous plant and rhizobium is in entire biological nitrogen fixation
Occupy extremely important status.It is estimated that the nitrogen that earth surface is fixed from air every year is planted there are about 100,000,000 tons by pulse family
There are about fifty-five million tons for the nitrogen that object and rhizobium are fixed.Rhizobium and leguminous plant form Symbiotic association, in addition to meeting
The nitrogen of plant itself needs outer, and extra nitrogen can also be supplied to the non-leguminous plant of miscegenation therewith or kind, such as and standing grain
The non-leguminous plant of graminaceous plant mixed seeding can be with Shaoshi nitrogenous fertilizer；It utilizes15N tracer technique, the study found that with leguminous plant mixed seeding
Gramineae plant 30% the nitrogen fixed in leguminous plant of nitrogen source.The Symbiotic association of rhizobium and leguminous plant
Have the advantages that nitrogen fixing capacity is strong, amount of nitrogen fixation is big, high-output stress-resistance, soil fertility can be improved and provides protein battalion for people and animals
It supports.The symbiotic azotification for studying rhizobium and leguminous plant is advantageously implemented agricultural, environment and ecological sustainability and utilizes and hair
Exhibition.Rhizobium Inoculation is the important measures for improving legume production.Domestic and international a large number of experiments proves that Rhizobium Inoculation is in majority
Can be improved in soil legume yield and quality (Cao Li Li etc., 2000；Li Youguo etc., 2002).Bohlool is being summarized
Various crop points out that the yield of legume Rhizobium Inoculation is improved than not Rhizobium Inoculation in many years Rhizobium Inoculation effect
89.5%(Bohlool et al., 1973).
Peanut is important the main oil crops of legume and China.Rhizobium and Peanut Root System are capable of forming
Root nodule carries out symbiotic nitrogen fixation, provides nitrogen for peanut growth.It is inoculated with high-efficiency root-nodule bacteria, is to promote peanut symbiotic nitrogen fixation, improves flower
The important measures of raw nitrogen nutrition.However, also lacking mature effective high-efficiency peanut nitrogen-fixing root nodule microbial inoculum at present.This research and utilization
Peanut varieties from different vitellariums carry out potting, field experiment, separate from its root nodule, purify high-efficiency nitrogen-fixing rhizobium；
Then it is identified, and passed through using rhizobium of the round pcr to purifying16sRDNA technology carries out sequencing identification；In greenhouse benefit
With the method validation host specificity and nodule nitrogenase activities of tieback, finally screening in field experiment is suitable for acid soil in the south
The high-efficiency peanut rhizobium strains of growth, and high-efficiency peanut Rhizobium Inoculant is prepared according to soil nutrient characteristic.Result of study will
Actually active technical support is provided to the production of peanut high-yield and high-efficiency.
Summary of the invention
The purpose of the present invention is to provide a kind of Peanut Rhizobium, which is the new strains of Bradyrhizobium
System, has the characteristics that dross rate height and nitrogen fixing capacity are strong.
Another object of the present invention is to be applied using above-mentioned Rhizobium strains in leguminous plant peanut agricultural production
On, by improving the biological nitrogen fixation ability of peanut, growth is promoted to achieve the purpose that synergy of losing weight in peanut production.
Above-mentioned technical purpose of the invention is achieved through the following technical solutions:
A kind of rhizobium YZLH133 bacterial strain (i.e. good fortune agriculture 3), is preserved in China typical culture collection on April 19th, 2018
Center, deposit number are CCTCC NO:M 2018216, and the address of China typical culture collection center is China, and Wuhan is military
Chinese university.
Its16sRDNA sequence is as shown in SEQ ID NO.1.
Isolated rhizobium YZLH133 bacterial strain is on colonial morphology, round smaller, milky, semi-transparent for slow raw type
It is bright, cement is more.Diameter after growing 5~7 days on YMA plate is 0.8~2.0 mm
The present invention has the advantages that
(1) rhizobium of the invention have a dross rate height and the strong characteristic of nitrogen fixing capacity, tieback test discovery, and are not inoculated with root nodule
Bacterium is compared, and is inoculated with the rhizobium and is remarkably improved peanut fresh weight and dry weight, can be used for actual production；
(2) peanut is inoculated with rhizobium of the invention, improve extremely significantly total nitrogen content (P< 0.01), to reach promotion flower
Raw plant strain growth and the purpose for improving peanut yield；
(3) rhizobium of the invention are suitable for acid soil in the south area, can be used as one of the important measures of peanut conventional cultivation.
Detailed description of the invention
Fig. 1 is colonial morphology of the rhizobium YZLH133 on YMA culture medium.
Fig. 2 is rhizobium YZLH133's16s RDNA Analysis of Partial phylogenetic tree (is rhizobium in box
YZLH133)。
Fig. 3 is rhizobium YZLH133 water planting tieback effect picture.
Fig. 4 is nitrogenase activity and dross number.
Fig. 5 is influence of the water planting Rhizobium Inoculation YZLH133 to peanut growth.
Fig. 6 is influence of the Rhizobium Inoculation YZLH133 in crop field to peanut growth, yield.
Fig. 7 is impact effect figure of the Rhizobium Inoculation YZLH133 in crop field to peanut growth, yield.
Specific embodiment
The separation and purifying of 1 rhizobium YZLH133 of embodiment
(1) in the florescence of peanut normal growth, fresh entire peanut root is taken, takes peanut fresh, mature and a in root
Big full root nodule, is rinsed with water completely, blots surface moisture with filter paper.
(2) it is first put into the ethyl alcohol of volume fraction 95% and handles 3~5 minutes, take out and use aseptic water washing 5~6
It is secondary, place into 1g/L HgCl2Sterilizing 3~5 minutes is taken out and is used aseptic water washing 5~6 times.
(3) it cuts in half on the glass slide of flame sterilization, clamps half of tumor with aseptic nipper, notch is towards YMA (table
1) media surface is crossed, and is cultivated at 28 DEG C after inversion.
(4) after growing thallus, a small amount of rhizobium bacterium colony is scraped from plate with the toothpick of sterilizing, after being diluted with sterile water
Again in flat lining out culture, bacterium colony situation is observed after 3 days, observing 15 days always, (slow raw rhizobium need go out for 7~15 days
Existing bacterium colony).Such as occur without monoclonal colonies, then needs to purify in flat lining out repeatedly, until there are monoclonal colonies.
1 YMA(Yeast Mannitol Agar of table) culture medium prescription
Rhizobium can be tentatively judged whether it is according to following methods:
Colonial morphology: the bacterium colony of rhizobium is circle, milky, translucent, neat in edge, and cement is more.Culture 3~5 days
Colony diameter is fast-growing Rhizobium up to 2~4 mm, and 7~10 days colony diameters of culture are then to give birth to type root nodule slowly up to 1 mm
Bacterium.
The present embodiment bacterial strain YZLH133 is Slow_growing rhizobia, is round smaller, milky, half on colonial morphology
It is transparent, cement is more；Diameter after growing 5~7 days on YMA plate is 0.8~2.0 mm(attached drawing 1)；The most suitable growth
Condition are as follows: pH=7.0,28 DEG C of temperature, 180 r/min of revolving speed, using extensive carbon source and nitrogen source.
It with this isolation and purification method, is purified repeatedly through several generations, the scribing line culture in YMA solid medium (table 1),
More plants of pure bacterial strain systems are obtained, pure bacterial strain system isolates and purifies through its nodulation and nitrogen fixation ability of water planting tieback verification experimental verification, the present embodiment
Dross rate height and the strong bacterial strain system YZLH133 of nitrogen fixing capacity.
2 rhizobium YZLH133's of embodiment16srDNA sequence
PCR specific amplification is carried out to rhizobium monoclonal bacterium solution, primer is respectively V3-V4-F and V4-V5-R, forward direction
Primer V3-V4-F is 5 '-GWATTACCGCGGCKGCTG-3 '；Reverse primer V4-V5-R is 5 '-
CCGTCAATTCMTTTRAGTTT-3 ', enzyme select 2 × star Mix.PCR amplified production does electrophoretic image technology and is examined
It surveys, observes whether it has band, remaining PCR amplified production is used for sequencing.Sequencing result such as SEQ ID NO:1 institute
Show.Its PCR reaction system is as follows:
Table 216s rDNA 2 × starMix enzyme reaction system
12 reference strains sequences are obtained from NCBI (GenBank) database, with software BioEdit and MEGA6
To isolated strains and reference strains16s RDNA partial sequence is analyzed, the system for constructing isolated strains and reference strains
Development tree (attached drawing 2).So that it is determined that rhizobium YZLH133 be Bradyrhizobium (Bradyrhizobium) new strains
System, and it is named as good fortune agriculture 3.
The test of 3 tieback of embodiment
1, strain culturing:
The strains tested switching that -80 DEG C are saved is in YMA Liquid Culture, culture to logarithmic phase, the growing state of bacterial strain
It is detected with nucleic acid-protein analyzer, calculates bacteria containing amount using OD value；YMA (Yeast Mannnitol Agar) fluid nutrient medium
Ingredient are as follows: weigh 10 g of mannitol, MgSO4∙7H20.1 g of O 0.2g, NaCl, yeast powder 3 g, K2HPO40.25 g,
KH2PO40.25 g, CaCO33 g (are added) when preservation, and 15 g of agar is dissolved in 1 L pure water.
2, sterilization treatment
2.5L plastic barrel required by testing, beaker, tweezers, culture dish, filter paper, glass bar are wrapped, by vermiculite with fresh-keeping
Packed good, 121 DEG C, sterilize 30 min.YMA fluid nutrient medium, distilled water are installed with bottle, and 121 DEG C, sterilizing 20
min 。
The seed of peanut is put into 1g/L HgCl2Middle 3min carries out epidermis sterilizing, and dry with clear water repeated flushing
Only, the 2.5L plastic barrel equipped with vermiculite is put at artificial growth chamber vernalization 7 days, and growing 3-5cm to radicle can tieback
Water planting.
3, water planting and inoculation:
(1) peanut that vernalization is completed is placed in culture dish, is placed into the rhizobium (attached drawing 4) of purifying, peanut root is complete
30min in rhizobium, bacterial content 10 are submerged entirely9 ∙mL-1 。
(2) the good peanut of above-mentioned tieback is transplanted into 2.5L plastic tub to and is added 2L plant Poor nitrogen nutrition liquid,
And it is cultivated in artificial growth chamber.Growth room's (photoperiod are as follows: day night=16h/8h, temperature are as follows: day night=26 DEG C/24 DEG C)；
The plant Poor nitrogen nutrition formula of liquid are as follows: Ca (NO3)2∙4H2O 0.03 g, MgSO4·7H2O 0.12 g, CaCl2·2H2O
0.10 g, KH2PO40.10 g, Na2HPO4·12H2O 0.15 g, C6H5O7Fe·5H20.005 g of O, 1 mL of microelement,
Distilled water 1000 mL, pH 5.8-6.0.Trace element formula (g L-1): H2MoO40.02 g, MnSO41.81 g, H3BO3
2.86 g, CuSO4·5H2O 0.8 g, ZnSO4·7H2O 0.22 g。
Not to be inoculated with as control, 3 repetitions are done, inoculation is harvested after 30 days, contained with plant fresh weight, dry weight, dross number, plant
Nitrogen quantity and SPAD value are as the associativity of measurement rhizobium and the judge index of nitrogen fixing capacity.
The results show that blank control (CK) is without dross phenomenon, the average every peanut of tieback processing can be dross 17.67.It cuts
Root nodule discovery is opened, is red inside root nodule, primitive decision strains tested YZLH133 is the rhizobium (attached drawing with nitrogen-fixing efficiency
4) it, and to its nodule nitrogenase activities is measured, value is 3.57 umol (g h)-1。
By Fig. 3 and Fig. 5 it is found that compared with not Rhizobium Inoculation, Fujian spends No. 8 Rhizobium Inoculation YZLH133 can
Significantly improve its plant fresh weight, plant weights, SPAD value and plant nitrogen content (P<0.01).Illustrate the knot of rhizobium YZLH133
Conjunction property and nitrogen-fixing efficiency are higher.
The test of 4 Field inoculation of embodiment
1, strain culturing:
The strains tested switching that -80 °C are saved is in YMA Liquid Culture, culture to logarithmic phase, the growing state of bacterial strain
It is detected with nucleic acid-protein analyzer, calculates bacteria containing amount using OD value.
2, field experiment is implemented:
This test carries out in the Anxi County in Fujian Province village Long Juanxiang Ju Yuan Ju Yuan Tea Industry cooperative society tea place.Seedtime is 2018 3
The moon 28.This test is equipped with 2 processing: connecing rhizobium YZLH133 (good fortune agriculture 3) and does not connect nitragin processing, while pressing 5
Kg calcium magnesium phosphate/mu dose applies calcium magnesium phosphate, and calcium magnesium phosphate is disposably applied in a manner of base fertilizer according to unified standard
Enter.This test sets 3 repetitions.RANDOMIZED BLOCK DESIGN is taken in test, in the ranks plants in tea, and each tea row sows 1 row, thickness of sowing
For 200cm × 25cm (tea line-spacing × spacing in the rows).Sowing, fertilising, irrigation, weeds and pest and disease damage are executed by peanut Routine Management.
Field investigation carries out (on July 26th, 2018) in florescence (on June 26th, 2018), the productive phase of peanut, investigation
Individual nodule number, biomass, SPAD value, plant nitrogen content, single plant yield (result is shown in Fig. 6).
From Fig. 6 as it can be seen that Rhizobium Inoculation YZLH133 can promote peanut dross.Inoculation handles and is not inoculated with processing
Root nodule number there is significant differences.The biomass of individual tree for being inoculated with processing presents aobvious with the biomass of individual tree for not being inoculated with processing
Sex differernce is write, Rhizobium Inoculation may advantageously facilitate peanut growth (such as Fig. 7).The peanut overground part dry weight of inoculation processing obviously compares not
It is inoculated with the height of processing, increased amplitude is 38%.Rhizobium Inoculation YZLH133, which bears pods for peanut to count, significant impact,
Increasing degree is 72.4%.Inoculation peanut rhizobium YZLH133 has significant impact for peanut yield, and increasing degree is
54.6% 。
This experimental study shows in the case where not applied nitrogen there is apparent increasing using rhizobium YZLH133 merely
Produce effect, it is seen that the rhizobium YZLH133 developed by this research department can be used for the popularizing application of large area.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>a kind of rhizobium YZLH133 and its application
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 381
<212> DNA
<213>artificial sequence
<400> 1
cattgcgtct ccatgcggat gcttaagcgt tagctgcgcc actagtgagt aaacccacta 60
acggctggca ttcatcgttt acggcgtgga ctaccagggt atctaatcct gtttgctccc 120
cacgctttcg tgcctcagcg tcagtaccgg gccagtgagc cgccttcgcc actggtgttc 180
ttgcgaatat ctacgaattt cacctctaca ctcgcagttc cactcacctc tcccggactc 240
aagatcttca gtatcaaagg cagttctgga gttgagctcc aggatttcac ccctgactta 300
aaaacccgcc tacgcaccct ttacgcccag tgattccgag caacgctagc ccccttcgta 360
ttaccgcggg ggctggcacc a 381
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
gwattaccgc ggckgctg 18
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
ccgtcaattc mtttragttt 20
Claims (3)
1. a kind of rhizobium (RhizobiumSp.) YZLH133 is preserved in Chinese Typical Representative culture on April 19th, 2018
Collection, deposit number are CCTCC NO:M 2018216.
2. a kind of rhizobium YZLH133 bacterial strain according to claim 1, it is characterised in that its16sRDNA sequence such as SEQ ID
Shown in NO.1.
3. rhizobium YZLH133 bacterial strain as described in claim 1 is improving the application in peanut yield.
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| CN201811478038.4A CN109439589B (en) | 2018-12-05 | 2018-12-05 | Rhizobium YZLH133 and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
| CN103571770A (en) * | 2013-08-28 | 2014-02-12 | 董金皋 | Efficient peanut rhizobiumleguminosarum strain and application thereof |
| CN103627662A (en) * | 2013-12-03 | 2014-03-12 | 领先生物农业股份有限公司 | Peanut bradyrhizobium sp. and application thereof |
| CN105567615A (en) * | 2016-03-14 | 2016-05-11 | 中国科学院烟台海岸带研究所 | Bradyrhizobium sp. and application thereof |
-
2018
- 2018-12-05 CN CN201811478038.4A patent/CN109439589B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
| CN103571770A (en) * | 2013-08-28 | 2014-02-12 | 董金皋 | Efficient peanut rhizobiumleguminosarum strain and application thereof |
| CN103627662A (en) * | 2013-12-03 | 2014-03-12 | 领先生物农业股份有限公司 | Peanut bradyrhizobium sp. and application thereof |
| CN105567615A (en) * | 2016-03-14 | 2016-05-11 | 中国科学院烟台海岸带研究所 | Bradyrhizobium sp. and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 刘世旺，等: "高效土著花生根瘤菌筛选的初步研究", 《黄冈师范学院学报》 * |
| 刘瑞瑞，等: "高效固氮花生根瘤菌株的筛选", 《江苏农业科学》 * |
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