CN104017750B - A kind of salt tolerance Methylotrophic Bacillus strain LJ and application thereof - Google Patents
A kind of salt tolerance Methylotrophic Bacillus strain LJ and application thereof Download PDFInfo
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Abstract
The invention provides a kind of salt tolerance Methylotrophic Bacillus strain LJ and application thereof, does is its Classification And Nomenclature Methylotrophic genus bacillus <i>Bacillus? methylotrophicus</i>, does is its deposit number: CGMCC? NO.7570.The present invention carries out tolerance of salinity analysis to it, tests pathogenic bacterium inhibition the pathogenic test of farm crop and bacterial strain, result shows, this bacterial strain tolerance of salinity is 25%-30%, to farm crop no pathogenicity, bacterial strain has comparatively ideal inhibition to tobacco bacterial disease, Disease, fungal disease.This bacterial strain can carry out composite with macroelement, long preservative period, and effective viable bacteria rate is high.This bacterial strain can use in saltings, has improvement soil, and opposing disease, improves the good results such as output.The invention solves microbial preparation and play positively effect for plant disease-proof and growth-promoting etc. in particular surroundings, this microbial inoculum has the effect of salt tolerant Promoting bacteria and disease prevention growth-promoting bacterium, is applicable to the improvement of salt marsh, alkaline earth and acid soil.
Description
Technical field
The invention belongs to microbial technology field, particularly relate to a kind of salt tolerance Methylotrophic Bacillus strain LJ and application thereof.
Background technology
Arid and semi-arid lands because of long-term drip irrigation cultivate the crops seedling stages such as the soil secondary salinization, fertilizer application excess poor efficiency and the soil-borne disease that cause be serious produce in outstanding problem, and there is the problems such as viable count is low, function singleness, long-lasting and poor stability in agricultural microbial agent in production and application, therefore be necessary using the microorganism in particular surroundings and plant mutually as plant salt tolerance, diseases prevention and growth-promoting approach, system research is carried out to the excavating resource of salt tolerant Promoting bacteria and disease prevention growth-promoting bacterium in different coenocorrelation and arrangement, biological characteristics and effect machine.In the face of day by day serious soil salinization phenomenon, be separated resistant gene of salt, build transgenic salt-tolerant wheat plant, developing saltings is the research direction that countries in the world are paid close attention to, Facultative Halophiles is also referred to as facultative halophilic bacterium, can have salt environment and without salt environment in growth and breeding, the tolerance of salinity can up to 20%, its topmost feature is facultative addicted to salt, the environmental field that can grow is wide, be the abundant source of the gene of Facultative Halophiles, although growth some Natrinema altunense sp tolerance of salinitys are in extreme circumstances higher, really can not grow under less salt or salt-free condition.So be separated the Facultative Halophiles obtained in normal circumstances should have more general biological significance.
Water-soluble salt resistant function bacterium is applicable to the improvement of salt marsh, alkaline earth and acid soil.Water-soluble salt resistant function bacterium can decomposite multiple meta-bolites, by improving the activity of plant materials endoenzyme, promote crop growth, stimulate division and the growth of root system meristematic cell, make seedling root of hair fast, secondary root increases, and the quantity of root increases, root elongation, thus can strengthen Crop ability greatly.Highly active amylase and multiple promotive factor can be discharged during water-soluble salt resistant function bacterium propagation, improve Fertilizer application rate, promote plant absorption nutrient; Dissolve unavailable phosphorus in soil, improve fertilizer efficiency; The poly-Vetsin produced has stronger preserve moisture and fertility ability.As Arizona, USA university have collected thousands of kinds of halophytess, find wherein to have severally to be expected for food crop; By sulfuric acid and the quick sweetening of the soil alkalescence of ferric sulfate.As the U.S., in western part, many areas adopt ammonium sulfate to reduce pH, to improve soil water permeability; Acid soil is planted acidproof perennial grass or liming stone, silicate and acidic phosphorus mine powder etc.
Water-soluble salt resistant function bacterium is applicable to coordinating bio-feritlizer, fertilizer fertilizer, bio-bacterial manure etc. to use together, water-soluble salt resistant function bacterium is more suitable for adding in water soluble fertilizer uses, as sprinkling irrigation, drip irrigation, have and add punching fertilising, foliage fertilizer, composite fertilizer, compound organic and inorganic fertilizer, macroelement Water soluble fertilizer, moderate-element Water soluble fertilizer and trace element water-soluble fertilizer etc. to.
China saltings is large in number and widely distributed, and transformation is administered and these resources of rational exploitation and utilization are one of important channels of China's agricultural sustainable development, and also to improving the ecological environment, thrust zone economy, society and Ecological Sustainable Development have particularly important meaning.Wherein biological modification has important practical significance, and mainly comprises plantation salt-tolerant plant, uses microbial-bacterial fertilizer etc.
Summary of the invention
The invention provides a kind of salt tolerance Methylotrophic Bacillus strain LJ and application thereof, the present invention is from the bacterial strain of the higher tolerance of salinity of separation screening the ecological conditions such as varieties in saline-alkali areas and saltern mud, the structure of the clone and transgenic salt-tolerant wheat plant that can be resistant gene of salt lays the foundation, and more may be used for the research and development of Salt-tolerant microbial agent.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
The present invention screens a kind of salt tolerance Methylotrophic Bacillus strain LJ, its Classification And Nomenclature is Methylotrophic bacillus methylotrophicus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCCNO.7570.
The tolerance of salinity of the resistance to NaCl of described bacterial strain LJ is 25%-30%.Described salt tolerance Methylotrophic Bacillus strain LJ bacterium colony is in white, and surface irregularity, edge is irregular, and LJ bacterium colony size is 2-4mm, easily provokes, flat, and without projection, opaque, thalline rod-short, tool mobility, can form gemma.
The invention provides described salt tolerance Methylotrophic Bacillus strain LJ and preparing the application in fungistat, described Methylotrophic Bacillus strain LJ has bacteriostatic action to black shank pathogenic bacteria, Alternaria alternate pathogenic bacteria, fusarium tabacinum, tobacco bacterial wilt pathogenic bacteria and tobacco mosaic viruses.
Described Methylotrophic Bacillus strain LJ is to the equal no pathogenicity of wheat, tomato, capsicum and cucumber.
The invention provides described salt tolerance Methylotrophic Bacillus strain LJ and preparing the application in microbial fertilizer, the water-soluble fertilizer of described bacterial strain LJ and macroelement is composite, and composite rear viable count reaches 400,000,000/more than g.
Present invention also offers the described application of salt tolerance Methylotrophic Bacillus strain LJ in the microbiobacterial agent for the preparation of biological restoration saltings, wherein said Methylotrophic genus bacillus bacteria containing amount is 30,000,000,000/more than g, and microbial inoculum usage quantity is 2kg/ mu.
Compared with prior art, advantage of the present invention and positively effect are: the present invention screens salt tolerance Methylotrophic Bacillus strain from saltings, genetic analysis test and bio-chemical characteristics are carried out to this bacterial strain, determines that this bacterial strain is Methylotrophic genus bacillus; And it is carried out tolerance of salinity analysis, is tested pathogenic bacterium inhibition the pathogenic test of farm crop and bacterial strain, result shows, this bacterial strain tolerance of salinity is 25-30%, described bacterial strain LJ is to farm crop no pathogenicity, and bacterial strain has comparatively ideal inhibition to tobacco bacterial disease, Disease, fungal disease.This bacterial strain can carry out composite with macroelement, long preservative period, and effective viable bacteria rate is high.This bacterial strain can use in saltings, has improvement soil, and opposing disease, improves the good results such as output.The invention solves microbial preparation and play positively effect for plant disease-proof and growth-promoting etc. in particular surroundings, this microbial inoculum has the effect of salt tolerant Promoting bacteria and disease prevention growth-promoting bacterium, is applicable to the improvement of salt marsh, alkaline earth and acid soil.
Accompanying drawing explanation
Fig. 1 is the slat chain conveyor figure of Methylotrophic Bacillus strain LJ of the present invention.
Fig. 2 is the salt tolerance experimental result of Methylotrophic Bacillus strain LJ of the present invention.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1
One, the separation of salt-enduring strain and screening
On July 4th, 2012 gathers soil sample 24 parts from Binzhou, Shandong vega cigarette strain rhizosphere soil, and each soil sampling 1g is suspended in 100ml sterilized water, and is diluted to 10 with this
5doubly.Adopt Gibbons improved culture medium or beef-protein medium to screen, Gibbons improved culture medium is filled a prescription: casein hydrolyzate 5.0g, Trisodium Citrate 3.0g, yeast leaching powder 10.0g, KCl2.0g, peptone, 5.0g, MgSO
47H
2o, 20gNaCl100g, pH7.0.Utilize the NaCl step-sizing salt-enduring strain of 1%, 4%, 7%, 10%.Coat on two kinds of culture medium flat plates with the pipettor earth diluent 0.1ml that fetches earth respectively, cultivate 2 ~ 3 days, be chosen at the bacterium colony that 10% salinity growth is good, save backup after purifying of repeatedly ruling, screen a strain salt-enduring strain LJ for 30 DEG C.
Two, the qualification of salt-enduring strain LJ
The bacterial strain LJ screened is carried out slat chain conveyor, and visible colonies is in white, and surface irregularity, edge is irregular, and LJ bacterium colony size is 2-4mm, easily provokes, flat, and without projection, opaque, thalline rod-short, tool mobility, can form gemma.The slat chain conveyor picture of bacterial strain as shown in Figure 1.
The qualification of bacteria molecule genetic classification is carried out to the salt-enduring strain LJ screened: extract DNA to bacterial strain LJ, with this DNA for template, 16SrDNA universal primer is primer, increases to 16SrDNA fragment.The fragment of amplification is directly carried out sequencing.Blast program on the result of order-checking input NCBI website is compared, the 16srDNA sequence homology degree that result shows the Bacillusmethylotrophicus in this bacterial strain 16SrDNA sequence and GenBank gene pool in bacillus is the highest, and homology reaches 99%.Phylogenetic analysis is carried out by the 16SrDNA sequence of DNAMAN6.0 to bacillus existing in Genbank, result shows, bacterial strain LJ16SrDNA and Bacillusmethylotrophicus homology the highest, can this bacterial strain of preliminary judgement be therefore the Methylotrophic genus bacillus of bacillus.
With reference to " common bacteria system identification handbook ", preliminary evaluation is carried out: Gram-positive by carrying out physiological and biochemical test to salt-enduring strain, V-P reaction, clark and Lubsreaction are positive, indole-positive, can anaerobic growth, energy hydrolyzed starch, hydrogen sulfide is negative, can utilize Citrate trianion, liquefy gelatin, nitrate reduction, catalase, catalase are positive, can hydrolyzed casein.
According to above-mentioned qualification result, this bacterial strain LJ is accredited as Methylotrophic bacillus methylotrophicus.
The bacterial strain LJ screened is carried out culture presevation, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC); Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on May 8th, 2013; Deposit number is: Bacillusmethylotrophicus is numbered CGMCCNo.7570.
Three, the Salt Tolerance Analysis of Methylotrophic Bacillus strain LJ
By the salt-enduring strain LJ bacteria suspension of separation and purification, concentrate, 37 DEG C in identical amount of transferring respectively to the liquid nutrient medium of different salt concn, 200r/min shaking table is cultivated, and observes growing state, surveys its bacteria concentration (OD at set intervals
600), to determine bacterium salt tolerant degree.
Salt tolerance experimental result as shown in Figure 2, visible bacterial strain LJ has wider subject range to sodium-chlor, bacterial strain is not autolyze phenomenon in salt-free situation, still higher cell concentration can be kept under salt concn reaches 30% situation, all can grow under the salt concn of 0-35%, the suitableeest salt concn is 25%-30%.
Four, Methylotrophic Bacillus strain LJ is to the pathogenic test experiments of farm crop
With asepsis injector the suspension of salt-enduring strain LJ is expelled to the seedling basal part of stem of wheat, tomato, capsicum and cucumber, every seedling 0.5ml, repeats 12 times, moisturizing 24h, starts to observe seedling basal part of stem and whether occur scab after 3 days.
Test-results shows, by Facultative Halophiles suspension inoculation to wheat, tobacco, corn and watermelon seedlings basal part of stem, cultivates and all occurs without obvious scab afterwards for 3 days.Therefore, this strain Facultative Halophiles can be confirmed to above-mentioned 4 kinds of crops without potential pathogenic.
Five, Methylotrophic Bacillus strain LJ is to the restraining effect of fungoid, bacterial disease, Disease
Dull and stereotyped face-off method is adopted to test described bacterial strain LJ to the bacteriostatic activity of indicator, 9cmPDA dull and stereotyped central authorities inoculation indicator black shank pathogenic bacteria, at distance indicator bacterium block 2.8cm place inoculation single bacterium colony to be tested, opposite side does not connect bacterium, for the blank indicator radius of measurement, 25 DEG C of constant temperature culture 5d, observe indicator mycelial growth situation, measure antibacterial result.
Repeat above-mentioned steps, bacterial strain LJ is used for suppress in the experiment of tobacco alternaric bacteria (red-star like disease pathogenic bacteria), black shank pathogenic bacteria, pinch outs oronoco.Experimental result is as shown in table 1, and this salt-enduring strain LJ all can reach more than 60% to the inhibiting rate of disease.
Adopt double-layer agar technique, carry out bacterial strain LJ and the antagonistic action of tobacco bacterial wilt is tested.First in culture dish, pour bottom solid medium into, after solidifying, pour the semisolid medium of the fermented liquid containing Stalk Rot and certain extent of dilution bacterial strain to be detected again into.After cultivating for some time, observe transparent circle size, measure antibacterial result, this bacterial strain has stronger restraining effect to bacterial wilt.
Adopt local lesion's method, the TMV tobacco tender leaf sterilizing mortar grinder of fully falling ill is become homogenate, with phosphoric acid buffer (PH7.0) the 1:40 dilution proportion after sterilizing.Zuo Banye inoculation NB substratum and viral equal-volume mix liquid and compare, right half leaf inoculating active strain fermentation supernatant liquor and viral equal-volume mix liquid and deal with, blade face is sprayed with water immediately after inoculation, add up withered spot number order after 3d, inhibiting rate method of calculation are as follows: inhibiting rate (%)=[(contrast withered spot number-process withered spot number)/contrast withered spot number] × 100%.The inhibition of bacterial strain to TMV reaches 72%, and this bacterial strain is also very outstanding to the inhibition of other diseases, can as broad spectrum type disease resistance biocontrol strain.
Table 1 bacterial strain LJ is to the Inhibition test result of various disease pathogenic bacterium
Six, Methylotrophic Bacillus strain LJ and macroelement water-soluble fertilizer is composite
Preparation LJ bacterial strain seed liquor, is then inoculated in fermention medium by conventional inoculative proportion, optimizes mixed fermentation condition, make the total number of bacterial colony of fermented liquid reach 8 × 10 by adjustment fermention medium carbon nitrogen source, pH value, leavening temperature, inoculative proportion
9cfu/ml.
Mix with certain proportion with carrier, spraying dry, form bacterium powder.Bacterium powder living bacteria count is 30,000,000,000/g.With urea, ammonium sulfate, saltpetre, potassium primary phosphate, prepare n-p-k ratio and be classified as 10-3-15, and containing medium trace element boron, manganese, iron, magnesium, copper, zinc etc., living bacteria count >=400,000,000/g high-efficiency composite biological macroelement water-soluble fertilizer.This product is at room temperature placed, at set intervals plate count is carried out to preparation, detect the stability of viable count.
Active degradation rule under table 2LJ bacteria preparation room temperature
| Time | 0d | 30d | 60d | 90d | 120d | 180d | 240d | 360d |
| Effective bacterium number (hundred million/g) | 5.26 | 5.24 | 5.01 | 5.03 | 4.96 | 4.52 | 4.39 | 4.25 |
Found out by table 2, this product is at room temperature preserved, along with the prolongation of time, bacterium number slightly declines, and preserves 12 months, and bacterium number reduces to 4.25 hundred million/g by 5.26 original hundred million/g, illustrate that macroelement water-soluble fertilizer containing salt tolerant active bacterial strain at room temperature, effective viable bacteria shelf time is long, not perishable.Analyze its reason mainly this bacterial strain contain resistant gene of salt, still can long-term surviving under the environment of high salt; In addition, this bacterial strain is genus bacillus, forms gemma and is easy to storage and transport, have the potential quality being developed as biological prevention and control agent.
Use the microbial fertilizer or bio-feritlizer that with the addition of described bacterial strain LJ, obviously can reduce the saltiness of salt-affected soil, increase the quantity of organic content and Soil Microorganism monoid, good action is played for improvement soil physics, chemistry and biological property.Meanwhile, after the microbial fertilizer using different sorts and concentration, the upgrowth situation of plant is all improved, and growth result is all better than blank group, and concentration and effect relation remarkable.Bacterial strain LJ contains the abundant and active bacteria of high value, and have phosphorus decomposing, release potassium, promote decomposition of soil organic matter, improve Soil Microorganism group, the function of improvement Soil structure, significantly improves the utilization ratio of fertilizer.Retention capacity of soil can be improved simultaneously.
Seven, saltings biological restoration
Experiment is mainly carried out in saltings.Field test adopts randomized block design.Take tobacco as experimental subjects.Experiment establishes two process, the process of LJ microbiobacterial agent: Methylotrophic gemma bacillus agent 30,000,000,000/g+ conventional fertilizer application, and microbial inoculum usage quantity is each 2kg/ mu, and transplanting and group's phase spread manuer in holes.Control treatment (CK): conventional fertilizer application.Each process 5 row, often row 36 cigarette strains, spacing in the rows 0.5m, line-spacing 1m.Each process arrange 3 parallel.Other cultivation management measures are consistent with land for growing field crops.Investigation discovery is used microbiobacterial agent and can be improved soil physical properties, soil porosity and pore porosity increase, water permeability improves, can the soil organism be increased, increase soil fertility, promote the eubiosis of terrestrial space, suppress the earth's surface accumulation of salt in the surface soil, guarantee that crop normal growth is grown, significantly, amount of increase in production reaches more than 30% for disease and insect resistance and somatotrophic ability.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (9)
1. a salt tolerance Methylotrophic Bacillus strain LJ, it is characterized in that: its Classification And Nomenclature is Methylotrophic genus bacillus (Bacillusmethylotrophicus), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCCNO.7570.
2. salt tolerance Methylotrophic Bacillus strain LJ according to claim 1, is characterized in that: the tolerance of salinity of the resistance to NaCl of described bacterial strain LJ is 25%-30%.
3. salt tolerance Methylotrophic Bacillus strain LJ according to claim 1, is characterized in that: bacterium colony is white, surface irregularity, and edge is irregular, LJ bacterium colony size is 2-4mm, easily provokes, flat, without projection, opaque, thalline rod-short, tool mobility, can form gemma.
4. salt tolerance Methylotrophic Bacillus strain LJ according to claim 1 is preparing the application in fungistat, it is characterized in that: described Methylotrophic Bacillus strain LJ has bacteriostatic action to black shank pathogenic bacteria, Alternaria alternate pathogenic bacteria, fusarium tabacinum, tobacco bacterial wilt pathogenic bacteria and tobacco mosaic viruses.
5. salt tolerance Methylotrophic Bacillus strain LJ according to claim 4 is preparing the application in fungistat, it is characterized in that: described Methylotrophic Bacillus strain LJ is to the equal no pathogenicity of wheat, tomato, capsicum and cucumber.
6. salt tolerance Methylotrophic Bacillus strain LJ according to claim 1 is preparing the application in microbial fertilizer.
7. salt tolerance Methylotrophic Bacillus strain LJ according to claim 6 is preparing the application in microbial fertilizer, it is characterized in that: the water-soluble fertilizer of described bacterial strain LJ and macroelement is composite, composite rear viable count 400,000,000/more than g.
8. the application of salt tolerance Methylotrophic Bacillus strain LJ according to claim 1 in the microbiobacterial agent for the preparation of biological restoration saltings.
9. the application of salt tolerance Methylotrophic Bacillus strain LJ according to claim 8 in the microbiobacterial agent for the preparation of biological restoration saltings, it is characterized in that: described Methylotrophic genus bacillus bacteria containing amount is 30,000,000,000/more than g, and microbial inoculum usage quantity is 2kg/ mu.
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| CN110408567B (en) * | 2019-08-01 | 2020-12-29 | 山东佐田氏生物科技有限公司 | Saline-alkali-tolerant methylotrophic bacillus and viable bacteria preparation and application thereof |
| CN112175885A (en) * | 2020-10-30 | 2021-01-05 | 江西顺泉生物科技有限公司 | Preparation and application of bacillus amyloliquefaciens capable of preventing and treating tobacco bacterial wilt |
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Address after: 266000 No. 127 Huizhiqiao Road, Qingdao High-tech Zone, Shandong Province Patentee after: Qingdao Haida biological Group Co.,Ltd. Address before: 266003 Shandong province Qingdao City fish Road No. 5 Patentee before: QINGDAO SEAWIN BIOTECH GROUP Co.,Ltd. |