CN109097302A - A kind of bacillus is promoting the application in plant growth - Google Patents

A kind of bacillus is promoting the application in plant growth Download PDF

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CN109097302A
CN109097302A CN201810934535.4A CN201810934535A CN109097302A CN 109097302 A CN109097302 A CN 109097302A CN 201810934535 A CN201810934535 A CN 201810934535A CN 109097302 A CN109097302 A CN 109097302A
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plant
bacillus
bacterial strain
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plant growth
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陈杰
袁静
黄晓慧
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Zhejiang A&F University ZAFU
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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
    • 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
    • C12R2001/07—Bacillus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B13/00—Fertilisers produced by pyrogenic processes from phosphatic materials
    • C05B13/02—Fertilisers produced by pyrogenic processes from phosphatic materials from rock phosphates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
    • Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a kind of bacillus to promote the application in plant growth.Bacillus of the invention is Te Jila bacillus (Bacillus tequilensis), bacterial strain number: HTTA-X0189, deposit number CGMCC No.15770.HTTA-X0189 Biological Characteristics of Strain of the invention and fertilizer effect are stablized, cultural method is simple and feasible, production cost is low, industrialization easy to accomplish, and HTTA-X0189 bacterial strain of the invention can promote plant growth from many aspects, and can induce plant anti-salt, it is one plant of bacterial strain for having multi-functional facilitation to plant, fertilizer application can be reduced.

Description

A kind of bacillus is promoting the application in plant growth
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of bacillus is promoting the application in plant growth.
Background technique
Cultivated plant is guarantee for the survival of mankind, and China has a large population in a small area, and the use of fertilizer is to improve crop unit area The important measures of yield, although chemical fertilizer was once playing important role to increasing crop yield, long-term simple application chemistry fertilizer Material makes the physicochemical character in arable land gradually aggravate, and soil acidification is hardened, and normal configuration is destroyed, soil fertility decline, not only directly Crop yield and quality are influenced, and unused chemical fertilizer enters atmosphere and water body causes secondary pollution.Cellulose is on the earth Distribution is most wide, but other biologies include the resource that plant can not directly utilize, and only agricultural waste material just may be up to every year in China 0.7×109Ton, these raw materials are largely incinerated, and not only utilization rate is low, and brings immense pressure to environmental protection.Micro- life Object can degrade noxious material and cellulose, repair contaminated soil, promote straw-returning, with dual economy and The ecological value.
Nitrogen, phosphorus, potassium are referred to as three elements necessary to plant growth, and nitrogen is the main component for constituting protein, to axis The growth of leaf and the development of fruit play an important role, and phosphorus can promote bud differentiation root growth and improve fruit quality.Potassium can It adjusts inside plants cellular water and divides mobilization, plant stalk stalwartness is promoted to improve fruit quality, enhance plant anti-adversity ability. Any element lacks, and the metabolism and photosynthesis that will lead to plant are interfered and inhibit, and causes the short and small blade of plant Yellow, low output poor quality.Microbial manure is that the enzyme that microbial life activity generates and benefit materials play a role, fixed nitrogen Microorganism synthesizes fixed nitrogen reductase for the N in air by gene nifH coding2It is reduced to directly be absorbed and utilized by plant NH4.There are a large amount of dissolubilities are poor in soil, it is difficult to it is absorbed by plants the potassium of Phos and silicate mineral form, it is micro- Biology encodes the phytase that synthesizes by phy gene, and the unavailable phytic acid of plant can be converted to plant is utilizable to be had Imitate P elements.
Plant hormone yields positive results with important adjusting control action, without these auxin to plant growth and development Participation plant be unable to reach the standard eaten for the mankind, heteroauxin (IAA) is a kind of plant endogenous auxin, is participated in thin Intracellular growth and the adjusting and control for forming a variety of physiological and biochemical procedures such as split layer, can not lack, one slightly in plant growth The α of α-transacetylase and yhcX gene chemical synthesis that biology is encoded by ysnE gene-acetonitrile hydrolase control generates IAA, to plant Object growth has good supplementary function.
Most of ferro element exists in nature with insoluble state, and plant is difficult to be utilized, and siderophore is a kind of with very The small molecule compound of strong special chelating Fe3+, many plant rhizosphere probiotic microorganisms can be absorbed by synthesizing this substance Iron in environment fights for iron with pathogen and extra iron is supplied to plant utilization, reaches and promote plant growth and control disease Harmful double action, siderophore can also the metal ions such as Chelated Manganese, lead, mercury, chromium, cadmium, to soil environment reparation, food safety Equal fields are of great significance.
Plough existing more than 0.2 hundred million hm in China2The salt-soda soil of area, and have increased trend year by year, because what is had a large population and a few land National conditions, the productivity for improving salt-affected soil are constantly subjected to promotion energetically from country to local government, and microbial manure is micro- life Object vicinity of plants metabolism work, be mostly active bacteria formulation, the microorganism from land because salt-affected soil salt content compared with Height influences its growth and cannot normally play a role, and the unique geographical location in ocean especially polar region and bitter cold, with high salt etc. are extreme Environment creates a variety of adaptable existings of microorganism, can preferably play a role in salt lick, promotes plant growth, induction Plant anti-salt.
Summary of the invention
The object of the present invention is to provide a kind of bacillus to promote the application in plant growth.
Te Jila bacillus (Bacillus tequilensis) of the invention is preserved in Chinese microorganism strain preservation Administration committee's common micro-organisms center (CGMCC), preservation date on May 17th, 2018, preservation address: BeiJing, China, bacterial strain Number: HTTA-X0189, deposit number CGMCC No.15770.
To achieve the goals above, the invention provides the following technical scheme:
A kind of bacillus is promoting the application in plant growth, the Te Jila bacillus (Bacillus Tequilensis), bacterial strain number: HTTA-X0189, deposit number CGMCC No.15770.
Further, the application includes degradation phosphorus, potassium, phytic acid.
Further, the application includes by N in air2It is reduced to NH4。
Further, the application includes generating heteroauxin.
Further, the application includes generating iron.
Further, the application includes induction plant stress-resistance or raising plant K+ content.
A kind of chemical fertilizer includes above-mentioned bacillus.
There are phy genes and nifH gene, respectively 610bp and 632bp in bacterial strain functional genome, can synthesize fixed nitrogen Reductase has N in air2It is reduced to NH4Nitrogen fixing capacity, can be by the unavailable phytic acid of plant, being converted to can be with The effective P elements utilized.The biology test of bacterial strain also further confirms that genetic test result is correct, HTTA-X0189 bacterial strain Has the function of good fixed nitrogen and degradation phosphorus potassium
Amplification is to ysnE and yhcX genetic fragment from bacillus HTTA-X0189 strain gene group, respectively 1260bp and 500bp, bacterial strain can be by ysnE, and yhcX gene control synthesis relevant enzyme, catalysis, which generates, promotes plant growth indoles Acetic acid IAA, through L-Trp R2A culture medium culture, Salkowski colorimetric is positive, and further illustrates that bacterial strain can produce indoles Acetic acid IAA.
Bacterial strain well-grown on surveying siderophore CAS occurs as soon as apparent orange-yellow haloing for 24 hours, when with culture Between extension, orange-yellow haloing becomes larger, and haloing diameter reaches 25mm after 48 hours, and bacterial strain produces siderophore activity and reaches at this time To 68.7%, there is stronger production siderophore ability.
Detect that bacillus HTTA-X0189 bacterial strain is related with induction plant stress-resistance there are 3 through gene magnification AlsD, alsS, alsR gene, respectively 480bp, 870bp and 500bp, deducibility bacterial strain have the energy of induction plant stress-resistance Power, plant living body experiment further confirm that bacterial strain has the ability of good induction plant anti-salt, bacterium solution treated tomato Seedling plant height balanced growth 50.1%, root long increase by 137.5%, and fresh weight increases 97.1%, promotes growth result significant, tomato The chlorophyll content of seedling increases second day after processing, is continued until the 15th day, and average content is increased than control 57.4%.
After the processing of HTTA-X0189 bacterium, compared with the control, the accumulation of Na+ content reduces 39.6%- in tomato root, leaf 54.5%, K in root and blade+Content improves 32.6%-40.2%.Illustrate that bacterial strain can reduce Na+In the intracorporal product of plant It is poly-, improve K in plant+Content, to mitigate or alleviate to some extent salt injury.
Application effect of the invention is: HTTA-X0189 bacterial strain can promote plant growth from many aspects, and can induce plant Object salt resistance is one plant of bacterial strain for having multi-functional facilitation to plant, and artificial cultivation and fermentation is at low cost, can reduce fertilizer application.
Detailed description of the invention
Fig. 1 Te Jila bacillus HTTA-X0189 bacterial strain affiliation branch tree.
Specific embodiment
Following specific embodiments are the further explanations to method provided by the invention and technical solution, but be should not be understood At limitation of the present invention.
Embodiment 1: the taxonomic identification of Bacillus strain
Bacillus HTTA-X0189 bacterial strain, in solid protein peptone culture medium: peptone 4g, yeast powder 2g, NaCl, 15- 25g, KCl 0.2-0.4g, MgCl2·7H2O 1.5-2.5g, water 1000ml, pH 7.0, agar 16-19g, 25 DEG C of -27 DEG C of trainings It supports 2-3 days, observes and records the variation such as colonial morphology, color, micro- sem observation thalli morphology, the test of Bacteria Identification conventional method Its biochemical reactions.Bacterial strain utilizes the universal primer of bacterial 16 S rDNA gene, conventional method PCR through above method culture Amplified production sequencing, and is compared by the blast program of NCBI, analytical sequence homology and does affiliation branch tree (see Fig. 1) closer edge type.
The results show that growing, normal Te Jila bacillus HTTA-X0189 bacterium colony rough surface is opaque, and culture is just Phase bacterium colony is white, and the later period switchs to brown, and bacterium colony is big and tiles, and opaque, there is fold on surface, and edge is not rounded, gram dye Color is positive, microscope hypothallus direct rod shape or nearly direct rod shape, peritrichous, there is oval gemma, raw inclined side in gemma, thallus It can movement.Amphimicrobian, catalase reacting positive, arginine dihydrolase reacting positive, V-P reaction negative, indole reaction, H2S reaction negative can produce acid using glucose, cannot make nitrate reduction nitrite.
The biochemical reactions of 1 Te Jila bacillus HTTA-X0189 bacterial strain of table
In conjunction with colonial morphology, biochemical reactions and molecular biology information, classify with reference to domestic and international related Bacillus It identifies monograph, identifies bacterial strain are as follows: Te Jila bacillus tequilensis.
Embodiment 2: the fixed nitrogen of bacterial strain and the relevant gene of degradation phosphorus potassium and effect determine
According to the principle that the various functions of microorganism are all controlled by gene, first passes through NCBI and find fixed nitrogen reductase NifH gene universal primer and phytase phy gene universal primer, using the genomic DNA of bacterial strain as template, conventional method PCR Amplification, whether detection bacterial strain has fixed nitrogen and the organic phosphorus ability of macromolecular of degrading, while passing through reported test microbes The culture medium that nitrogen fixation culture medium (ACC culture medium), degradation potassium effect culture medium (MYK culture medium), degradation Phos act on (PKO culture medium) etc., which cultivates bacterial strain and observe its growing state cooperation genetic test, determines its function.
The results show that the nifH genetic fragment size for passing through PCR amplification to bacterial strain is 610bp, i.e. bacterial strain can synthesize fixed nitrogen Reductase has N in air2It is reduced to NH4Nitrogen fixing capacity.Specific primer also detects that be existed in strain gene group Phy genetic fragment, phy genetic fragment size are 632bp, and the phytase of this gene coding synthesis can cannot utilize plant Phytic acid be converted to utilizable effective P elements.Bacterial strain can grow more apparent bacterium colony on ACC nitrogen-free agar, Colony diameter reaches 16mm after cultivating 5d, it was demonstrated that genetic test result is correct, and Te Jila bacillus HTTA-X0189 bacterial strain has Good nitrogen fixation.
Te Jila bacillus HTTA-X0189 bacterial strain is in test degradation potassium effect culture medium (MYK culture medium), drop simultaneously All well-grown on the culture medium (PKO culture medium) of Phos effect is solved, can produce obvious and biggish transparent circle, this shows Phos and inorganic potassium in periphery of bacterial colonies culture medium is by strains for degrading, and degradation is fine
Embodiment 3: generation siderophore, hormone and the relevant gene of bacterial strain
By primer amplified method same as Example 1, first check that HTTA-X0189 bacterial strain synthesis plant swashs YsnE the and yhcX gene of plain heteroauxin IAA and alsS relevant to stress resistance of plant is promoted, alsD, alsR gene, then lead to The biological methods verifying such as culture test is crossed, whether reported test microbes generate the L-Trp of heteroauxin IAA R2A culture medium, if produce the CAS culture medium of siderophore, observe growth conditions, bacterium solution processing measurement light absorption value (630nm) As (0.2 < As < 0.8), and siderophore units activity is calculated, siderophore activity %=[(Ar-As)/Ar] × 100%.3-1
The results show that expanding from Te Jila bacillus HTTA-X0189 strain gene group to ysnE and yhcX gene Segment, size are respectively ysnE the and yhcX genetic fragment of 1260bp and 500bp control synthesis, and bacterial strain can be by genome YsnE, yhcX control synthesis relevant enzyme, the α-transacetylase encoded by ysnE gene, yhcX gene chemical synthesis α-acetonitrile water Enzyme is solved, catalysis, which generates, promotes plant growth heteroauxin IAA.
R through L-Trp2It after A culture medium fermented and cultured 4d, is found by Salkowski colorimetric, bacterial strain reaction is in sun Property, further illustrate that bacterial strain can produce heteroauxin IAA.Bacterial strain well-grown on surveying siderophore CAS, occurs as soon as 24 hours Apparent orange-yellow haloing, with the extension of incubation time, orange-yellow haloing is become larger, and haloing diameter reaches after 48 hours 25mm, bacterial strain production siderophore activity has reached 68.7% at this time, has stronger production siderophore ability.
Embodiment 4: bacterial strain inducing plant anti-salt ability
First detecting thallus with 1 same procedure of embodiment whether there is alsS, alsD related with induction plant stress-resistance, AlsR gene, then plant living body detects its effect, and tomato seedling is planted after bacterium solution is handled in artificial salinized soil (NaCl concentration 0.4%), to be compareed in common plantation soil with salinized soil tomato seedling 2 that sporeless bacterium is handled if planting, after processing 15 days, Conventional method tests plant height and root long, plant fresh weight and tomato seedling chlorophyll content, while also measuring the Na of the root of blade sum+、 K+Content, data are analyzed (t- is examined, α=0.05) with SPSS19.0.
Detect that Te Jila bacillus HTTA-X0189 bacterial strain has there are 3 with induction plant stress-resistance through gene magnification The alsS of pass, alsD, alsR gene, alsD genetic fragment size are 480bp, alsS genetic fragment size 870bp, alsR base It, can ability of the initial guess bacterial strain with induction plant stress-resistance because clip size is 500bp.
Plant living body experiment further confirms that bacterial strain has the ability of good induction plant anti-salt, compares tomato seedling salt Evil performance is obvious, and leaves water loss dries up, and area is larger, and the tomato seedling handled through bacterial strain, salt damage symptom is substantially reduced, plant height, root Long and fresh weight is all greatly improved than control, increase of liquid treated the tomato seedling plant height average increasing amount than control 50.1%, for root long average increasing amount than the growth by 137.5% of control, mean fresh incrementss, which compare to compare, increases 97.1%, promotees Into significant effect, the chlorophyll content of tomato seedling increases second day after processing, is continued until the 15th day, chlorophyll Average content increases 57.4% than control.
Table 2 handles the influence to Na+, K+ content in tomato root leaf through bacillus
After the processing of HTTA-X0189 bacterium, compared with the control, Na in tomato root, leaf+Content has different degrees of drop It is low, compared with the control, Na+Accumulation in root and leaf reduces 39.6%-54.5%, K in root and blade+Content increases, root, K in leaf+Content improves 32.6%-40.2%.Illustrate that bacterial strain can reduce Na+In the intracorporal accumulation of plant, plant is improved Interior K+Content further demonstrates the accuracy of genetic test to mitigate or alleviate to some extent salt injury.
The method of the present invention that the above embodiments are only used to help understand and its core concept.It should be pointed out that for For those skilled in the art, without departing from the principle of the present invention, if can also be carried out to the present invention Dry improvement and modification, these improvement and modification are also fallen into the claims in the present invention protection scope.

Claims (7)

1. a kind of bacillus is promoting the application in plant growth, which is characterized in that the Te Jila bacillus (Bacillus tequilensis), bacterial strain number: HTTA-X0189, deposit number CGMCC No.15770.
2. a kind of bacillus according to claim 1 is promoting the application in plant growth, which is characterized in that described to answer With including degradation phosphorus, potassium, phytic acid.
3. a kind of bacillus according to claim 1 is promoting the application in plant growth, which is characterized in that described to answer With including by N in air2It is reduced to NH4。
4. a kind of bacillus according to claim 1 is promoting the application in plant growth, which is characterized in that described to answer With including generating heteroauxin.
5. a kind of bacillus according to claim 1 is promoting the application in plant growth, which is characterized in that described to answer With including generating iron.
6. a kind of bacillus according to claim 1 is promoting the application in plant growth, which is characterized in that described to answer With including induction plant stress-resistance or raising plant K+Content.
7. a kind of chemical fertilizer includes bacillus as claimed in any one of claims 1 to 6.
CN201810934535.4A 2018-08-16 2018-08-16 A kind of bacillus is promoting the application in plant growth Pending CN109097302A (en)

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CN114480165A (en) * 2021-12-22 2022-05-13 沣田宝农业科技有限公司 High-efficiency potassium-decomposing azotobacter and application thereof
US20220264892A1 (en) * 2020-06-17 2022-08-25 Bioconsortia, Inc. Agriculturally beneficial microbes, microbial compositions, and consortia
CN115873746A (en) * 2022-08-08 2023-03-31 兰州交通大学 Application of Bacillus tequilensis SY89 in promoting plant growth
CN117586925A (en) * 2024-01-18 2024-02-23 哈尔滨长升吉时农业发展有限公司 Bacillus tertageus, microbial inoculum, plant growth promoter, biological fertilizer and application

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Publication number Priority date Publication date Assignee Title
CN109761656A (en) * 2019-03-11 2019-05-17 云南省农业科学院生物技术与种质资源研究所 A kind of microbial-bacterial fertilizer combines the method for its fermentation liquid raising potassium content of tobacco leaf
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CN114480165A (en) * 2021-12-22 2022-05-13 沣田宝农业科技有限公司 High-efficiency potassium-decomposing azotobacter and application thereof
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CN117586925A (en) * 2024-01-18 2024-02-23 哈尔滨长升吉时农业发展有限公司 Bacillus tertageus, microbial inoculum, plant growth promoter, biological fertilizer and application
CN117586925B (en) * 2024-01-18 2024-04-12 哈尔滨长升吉时农业发展有限公司 Bacillus tertageus, microbial inoculum, plant growth promoter, biological fertilizer and application

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