CN110205273A - A kind of bacillus amyloliquefaciens and its application with growth promotion and resistant effect - Google Patents
A kind of bacillus amyloliquefaciens and its application with growth promotion and resistant effect Download PDFInfo
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Abstract
The disclosure belongs to microorganisms technical field, and in particular to a kind of bacillus amyloliquefaciens and its application with growth promotion and resistant effect.It is poor in the presence of certain use defect, such as field colonization ability that it is directed to the relevant microorganism formulation of bacillus amyloliquefaciens in the prior art, pre- disease prevention single effect etc..Present disclose provides a kind of bacillus amyloliquefaciens (B.amyloliquefaciens) 44, which has the function of inhibiting various plants pathogen, promotes plant growth, applied widely.In addition, present invention also provides a kind of composite bacteria agents, it is made of bacillus amyloliquefaciens (B.amyloliquefaciens) 44, rahnella aquatilis (R.aquatilis) 27 and bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619, in addition to good antibacterial effect and growth-promoting ability, also there is good colonization ability.
Description
Technical field
This disclosure relates to microorganisms technical field, and in particular to a kind of solution starch gemma with growth promotion and resistant effect
Bacillus (Bacillus amyloliquefaciens) 44 and its growth promotion and disease-resistant application.
Background technique
The information for disclosing the background technology part is merely intended to increase the understanding to the general background of the disclosure, without certainty
It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art
Art.
Root system of plant has the function of absorption, synthesis, transporting moisture and fixed nutrient, can be directly using in external environment
Mineral nutrient, the effect that the root system of some plants also reserves nutrient and breeds.The root activity of enhancing plant is conducive to add
The photosynthesis at fast Plant aboveground position, accelerates the metabolism of crop, promotes plant growth to reach, delays plant leaf blade
The effects of aging.The importance of root system of plant is more and more paid attention to by people.But recently as fertilizer and pesticide usage amount
Continuous increase, bring negative effect it is increasingly apparent.Soil texture and microbial community diversity by destroy, soil it is dirty
The a series of problem such as dye, soil hardening is severely impacted root system of plant flora, root system of plant bacterial micro-ecological balance
It being destroyed, root protection barrier is weak, and it is poor to the resistance of soil-borne disease pathogenic bacteria, and then influence the normal life of plant root
It is long, influence plant health.
Plant growth-promoting effect bacterium (plant growth-promoting rhizobacteria, PGPB) is can to promote to plant
A kind of beneficial bacteria of object growth, can colonize in plant root, and obtain itself growth and breeding by the ablation substance of root
Required nutriment.Plant growth-promoting effect bacterium can be protected by site competition, the generation mechanism such as antibiotic and inducible system resistance
Plant is protected from the harm of phytopathogen and reduces its extent of injury, promotes plant growth indirectly.Bacillus
(Bacillus spp), many bacterial strains, which have, inhibits phytopathogen, can partially promote plant growth.The gemma studied at present
Mainly having with bacteriostatic activity in bacillus: bacillus subtilis (B.subtilis), bacillus amyloliquefaciens
(B.amyloliquefaciens), atrophy bacillus (B.atropheaus), etc..Wherein, bacillus amyloliquefaciens
(B.amyloliquefaciens) in its growth course, the extracellular metabolin with antibacterial activity can be secreted, it can be well
Inhibit various plants pathogen, these extracellular substances play an important role in controlling plant diseases, some bacterial strains are in addition to energy
Controlling disease can also promote plant growth.Bacillus amyloliquefaciens are distributed extensively in nature, be can be easily separated, are isolated from plant mostly
It is environmentally friendly and be not easy to produce resistance near object root soil, thus receive the extensive concern of domestic and international researcher.
It has been recognised by the inventors that for the correlation of bacillus amyloliquefaciens (B.amyloliquefaciens) biological prevention and control agent exploitation
Study numerous, biological prevention and control probiotics product category is various on the market, but there are still certain use defects, including biological and ecological methods to prevent plant disease, pests, and erosion
Effect is unstable, and field colonization ability is poor；Single microbial inoculum, pre- disease prevention is single, and comprehensive protection effect is poor；Probiotics produce
Product by store, transport, the conditions such as environment are influenced, and viable count difference is larger etc..
Summary of the invention
Background in view of the above technology, inventor think that developing one kind has wide spectrum inhibitory effect, energy for phytopathogen
Enough bacterial strains to multiple kinds of crops with growth-promoting functions, advantageously reduce the use of pesticide and fertilizer during crop planting
Amount reduces chemical reagent to the harmful effect of soil.In order to realize the above technical purpose, the disclosure the following technical schemes are provided:
The disclosure is in a first aspect, provide a bacillus amyloliquefaciens (B.amyloliquefaciens) 44, the bacterial strain is
China typical culture collection center, abbreviation CCTCC, address were preserved on August 23rd, 2018 are as follows: Wuhan City, Hubei Province Wuchang
Luo Jia Shan, biological deposits number are as follows: CCTCC M 2018564.
Bacillus amyloliquefaciens described in first aspect have 16S rDNA sequence shown in SEQ ID NO:1.
The strain isolation from the pedotheque of Ningyang County of Shandong Province, the sequence and other known bacillus of the same race
16S rDNA sequence has 99% similitude, and comprehensive sequencing is determined as bacillus amyloliquefaciens with biochemical reactions result, fixed
Entitled bacillus amyloliquefaciens (B.amyloliquefaciens) 44.
The morphological feature of the bacillus amyloliquefaciens (B.amyloliquefaciens) 44 is as follows:
Thallus feature: gram-positive bacteria, bacteriform straight-bar, size are 0.6 × 1.0-1.4 μm, have circular distal,
Single or grouping in pairs, forms endospore；Spore is ellipse, has center or sub- top positioning；Sporangium does not have swelling.
Colony characteristics: bacterium colony is smooth after NA Solid media for plates culture 48h, opaque, irregularly, grey, diameter 2-
4mm has toothed edge and flat profile, is raised slightly at center, quality is sticky.
The physiological and biochemical property of the bacillus amyloliquefaciens (B.amyloliquefaciens) 44 is as follows:
Oxidizing ferment, catalase, gelatin hydrolysis experiment, the test of Preece's Cauer, nitrate reduction test, malonate
It is tested using test, Starch Hydrolysis test, casein hydrolysis positive；Indoles detection, H2S test, NH3Test, urea decompose examination
It tests, the utilization experiment of citrate, methylation test, lecithinase, phenylalanine feminine gender.
Preferably, the culture medium of above-mentioned bacillus amyloliquefaciens (B.amyloliquefaciens) 44 includes soluble forms sediment
Powder/molasses 2.0%-3.5%, peptone/dregs of beans/Dried Corn Steep Liquor Powder 1.5%-3.0%, K2HPO4×3H2O0.5-1.0%,
KH2PO40.1-0.5%, (NH4)2SO40.1-0.3%, sodium citrate × 2H2O 0.01-0.05%, MgSO4×7H2O
0.01%, surplus is water.
Preferably, the culture medium of above-mentioned bacillus amyloliquefaciens (B.amyloliquefaciens) 44 is with the molasses of 1-3%
As carbon source, using the dregs of beans of 1-4% as nitrogen source.
It is further preferred that the molasses culture medium of the bacillus amyloliquefaciens (B.amyloliquefaciens) 44 is
Molasses 2.5~3.5%, dregs of beans 1.5~2.5%, K2HPO4×3H2O 0.5-1.0%, KH2PO40.1-0.5%, (NH4)2SO4
0.1-0.3%, sodium citrate × 2H2O 0.01-0.05%, MgSO4×7H2O 0.01%, surplus are water, pH 6.6-7.0.
Through the disclosure research shows that using: in the case that carbon source and nitrogen source ratio are different in culture medium, will affect culture medium
The viable count and gemma number of middle bacterium, as the accounting of nitrogen source in the medium increases, the ratio between viable count, gemma number and the two
Maximum viable count and bud are help to obtain through research discovery when using the culture medium of aforementioned proportion in unordered variation
Spore number.
Above-mentioned culture medium cultivates above-mentioned bacillus amyloliquefaciens (B.amyloliquefaciens) 44, is conducive to
Obtain maximum viable count and gemma number.
Preferably, the cultivation temperature of above-mentioned bacillus amyloliquefaciens (B.amyloliquefaciens) 44 is 10~40 DEG C,
Further preferred is 30~40 DEG C；Cultivating pH value is 3.5~8.5, is further preferably 6.5~7.5.
Disclosure second aspect, provides a kind of microbial inoculum, and the microbial inoculum includes bacillus amyloliquefaciens described in first aspect
(B.amyloliquefaciens) 44 and/or bacterium culture.
The disclosure third aspect, provides a kind of composite bacteria agent, and the composite bacteria agent includes solution starch described in first aspect
Bacillus (B.amyloliquefaciens) 44, rahnella aquatilis (Rahnella aquatilis) 27 and solution starch bud
Spore bacillus (B.amyloliquefaciens) B-1619 microbial inoculum and its respective culture, wherein rahnella aquatilis
(R.aquatilis) 27 it was preserved in Wuhan University's China typical culture collection center on August 23rd, 2018, abbreviation CCTCC,
Address are as follows: Luojiashan, Wuchang, Wuhan City, Hubei Province, deposit number: CCTCC M 2018566；The bacillus amyloliquefaciens
(B.amyloliquefaciens) B-1619 is purchased from China General Microbiological Culture Collection Center.
Preferably, in the composite bacteria agent, the bacillus amyloliquefaciens (B.amyloliquefaciens) 44: aquatic
Draw engler bacterium (R.aquatilis) 27: the ratio of bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 is 0.8-
5.2:0.8-3:0.8-2。
It is further preferred that the ratio of three kinds of bacterium is 1:1:1 in the composite bacteria agent.
It is further preferred that in the composite bacteria agent, living bacteria count >=1,000,000,000/mL.
When different strains are in common environment, since bacterial strain mostly causes to go out between bacterial strain with mucilage secretion
Now a variety of influencing factors of collaboration or antagonism.When the variation of the ratio of bacterial strain, these influencing factors are also among variation, finally
It may show mutually to inhibit, mutually promote, a variety of effects such as viable count increases, gemma number increases, certain secretion content increases
Fruit has certain Unpredictability.The disclosure research shows that above-mentioned three kinds of bacterium be in the combination product under certain proportion tool
There are good growth-promoting, protection effect, can effectively reduce the dosage of pesticide during crop planting, chemical fertilizer, there is significant economy
Benefit.
Preferably, the culture medium of the rahnella aquatilis (R.aquatilis) 27 is cornstarch 3.0%, peptone
2.0%, K2HPO4×3H2O 0.7%, KH2PO40.3%, (NH4)2SO40.15%, sodium citrate × 2H2O 0.05%,
MgSO4×7H2O 0.01%；Surplus is water.And/or fermentation condition is р Н 6.8；37 DEG C, stirring rate 150rpm/min, culture
48 hours, obtain rahnella aquatilis (R.aquatilis) 27 fermentation liquid.
Preferably, the culture medium of bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 is glucose
5%, tryptone 5%, yeast extract 5%, surplus is water.And/or fermentation condition is pH 7.0；28 DEG C, stirring rate 180rpm/
Min cultivates 48 hours acquisition bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 fermentation liquids.
Preferably, the dosage form of composite bacteria agent described in microbial inoculum or the third aspect described in second aspect is liquid bacterial agent, pulvis
Or granule；It is further aqueous suspension agent, oil-suspending agent, wettable powder or water dispersion granule can be dispersed.
Preferably, further including that can be connect in Pesticide Science in composite bacteria agent described in microbial inoculum or the third aspect described in second aspect
The auxiliary material received, acceptable auxiliary material is selected from dispersing agent, wetting agent, disintegrating agent, binder, defoaming agent, freeze proof in the Pesticide Science
One of agent, thickener, filler and solvent are a variety of.The disclosure there is not the source etc. of acceptable auxiliary material in the Pesticide Science
Have it is specifically limited, generally use commercial product.
Disclosure fourth aspect provides bacterial strain described in above-mentioned first aspect, microbial inoculum and/or the third aspect described in second aspect
Application of the composite bacteria agent in following either side:
(1) inhibit phytopathogen, including plant epiphyte pathogen and vegetative bacteria pathogen；
(2) promote plant growth；
Preferably, the plant epiphyte pathogen include but is not limited to Fusarium oxysporum, Rhizoctonia solani Kuhn, rod method,
Sclerotinite, root rotof flax pathogen；
Preferably, the vegetative bacteria pathogen includes but is not limited to Irving's formula bacillus, Agrobacterium tumefaciens, yellow unit cell
Bacterium, pseudomonas syringae, bacterial canker of tomato, blakleg of potato bacterium；
Preferably, the promotion plant growth, which is embodied in, promotes plant seedlings to increase and/or increase weight；The plant include but
It is not limited to garlic, tomato, radish, romaine lettuce, cucumber and wheat.
Compared with prior art, the beneficial effect of the disclosure is:
1. 44 pairs of the bacillus amyloliquefaciens (B.amyloliquefaciens) that the disclosure provides cause to cause crops disease
The various plants pathogen of disease has good inhibiting effect, to the inhibition zone point of plant epiphyte cause of disease and vegetative bacteria pathogen
Not Wei 13-23mm and 20-31mm, facilitation can be realized to the growth of the various crops such as tomato, radish, cucumber, wheat.
2. the disclosure additionally provides a kind of bacillus amyloliquefaciens (B.amyloliquefaciens) 44, aquatic drawing engler
Bacterium (R.aquatilis) 27, bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 composite bacteria agent, it is above-mentioned multiple
Combined bacteria agent shows good facilitation to the growth of plant under certain ratio, effectively improves percentage of seedgermination, seedling
Weight and length.
3. in addition to this, which also has good Biocontrol Effect, in laboratory conditions by inoculated plant disease
The radish seed of opportunistic pathogen confirms that the composite bacteria agent can be effectively reduced the pathogenetic probability of disease after co-culturing with composite bacteria agent；?
Under the conditions of greenhouse, using the composite bacteria agent as the plantation for being used for tomato, after applying for four weeks, using composite bacteria agent to tomato plant
Facilitation be significantly higher than control group, it was demonstrated that the composite bacteria agent also have it is good colonize effect, can adapt to crop field environment,
It is expected to be expected to substitution part fertilizer promotion crop growth as being administered for crop growth.
Detailed description of the invention
The Figure of description for constituting a part of this disclosure is used to provide further understanding of the disclosure, and the disclosure is shown
Meaning property embodiment and its explanation do not constitute the improper restriction to the disclosure for explaining the disclosure.
Fig. 1 is the shape of 44 thallus of bacillus amyloliquefaciens (B.amyloliquefaciens) under the microscope in embodiment 1
State figure；
Fig. 2 is 44 colonial morphology figure of bacillus amyloliquefaciens (B.amyloliquefaciens) in embodiment 1；
Fig. 3 is 1 bacillus amyloliquefaciens of embodiment (B.amyloliquefaciens), 44 pairs of pseudomonas syringaes and sharp spore
The antagonism figure of sickle-like bacteria；
Fig. 4 is compound formulation group and control group garlic bolt root growth situation map in embodiment 1；
Fig. 5 is compound formulation group and control group tomato plant root growth situation map in embodiment 1.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the disclosure.Unless another
It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As background technique is introduced, for bacillus amyloliquefaciens (B.amyloliquefaciens) biological prevention and control agent
Correlative study it is more, but existing biological prevention and control agent is there is also certain deficiency, as field colonization ability is poor, pre- disease prevention effect
Single, comprehensive protection effect difference etc..In order to solve technical problem as above, present disclose provides a kind of bacillus amyloliquefaciens
(B.amyloliquefaciens) 44, which has effects that good inhibition phytopathogen and promotes plant growth；Separately
Outside, the disclosure additionally provides a kind of composite bacteria agent, including bacillus amyloliquefaciens (B.amyloliquefaciens) 44, aquatic
Draw engler bacterium (R.aquatilis) 27 and bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619, the composite bacteria agent
Equally have the function of good inhibition phytopathogen, promote plant strain growth.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment and comparative example of body.
Ordinary culture medium involved in experimentation and its ingredient are as follows:
NB culture medium: peptone -1%, powdered beef -0.3%, sodium chloride -0.5%；Agar -2% is NA culture medium.
PDA culture medium: potato -2%, glucose -0.2%, agar -2%.
Embodiment 1
One, the screening and purifying of bacterial strain
Pedotheque is collected from Ningyang County of Shandong Province, weighs in 1g sample access 100ml sterile water, is shaken in 30 DEG C of insulating boxs
Culture 5-7d is swung, 10 are carried out to soil sample liquid5-106Multiple dilution, dilution are coated on the NA solid medium containing 2% agar
Plate in, 30 DEG C of overnight incubations.It isolated strains and is purified from the plate for grow bacterium colony, access NA slant medium saves.
Two, bacterial strain is identified
The strain characteristics (Fig. 1, Fig. 2) of microscopically observation after purification: bacteriform straight-bar, size are 0.6 × 1.0-1.4
μm, there is circular distal, single or grouping in pairs forms endospore；Spore is ellipse, has center or sub- top fixed
Position；Sporangium does not have swelling.Its bacterium colony is smooth after cultivating 48h, opaque, and irregularly, grey, diameter 2-4mm has dentation fringe
Edge and flat profile are raised slightly at center, and quality is sticky.
The further verifying purpose bacterial strain of bio-chemical characteristics is carried out with reference to primary Jie Shi systematic bacteriology handbook identification method,
It reacts as follows:
The physio-biochemical characteristics of table 1, bacterial strain
Picking single colonie is seeded in NB fluid nutrient medium, and 37 DEG C, 180rpm shake culture for 24 hours, takes 1-5ml bacterium solution to utilize
Bacterial genomes DNA extraction kit extracts strain gene group DNA.16S rRNA universal primer 8f (5'-
Agagtttgatcctggctcag-3') strain gene group DNA is expanded with 1492r (5'-ggttaccttgttacgactt-3')
Increase and send to Shanghai Sheng Gong biotech firm and is sequenced.The identity that BLAST software compares the analysis bacterial strain and following bacterial strain is reachable
99%:Bacillus amyloliquefaciens subsp.plantarum FZB42 (99%), Bacillus subtilis
168 (99%), Bacillus vallismortis IAM 12118 (99%)；MALDI-TOF mass spectral analysis its with
The accurate property coefficient of B.amyloliquefaciens is 2.294.
Comprehensive sequencing determines that the bacterial strain belongs to bacillus amyloliquefaciens with biochemical reactions result, and it is big to be sent to Wuhan
It learns China typical culture collection center and carries out preservation, deposit number is CCTCC M 2018564.
Three, the optimization of culture medium and condition of culture
In order to optimize the composition of the Major Nutrient culture medium for cultivating bacterial strain 44, selecting cane molasses respectively, (1-3% is dense
Degree) and dregs of beans (1-4%) respectively as carbon source and nitrogen source (table 2).The multiple dilution method of counting counts bacterium solution.
The viable bacteria of table 2, bacillus amyloliquefaciens (B.amyloliquefaciens) on the nutrient medium of different component
Number
Table note:
1, the constituent of 1# culture medium: cane molasses 3.0%；K2HPO40.7%；KH2PO40.3%；(NH)2SO4
0.15%；Sodium citrate 0.05%；MgSO4·7H2O 0.01%, surplus are water；pH 7.0-7.2.
2,1# culture medium+dregs of beans 1.0%
3,1# culture medium+dregs of beans 1.5%
4,1# culture medium+dregs of beans 1.5%, omits (NH)2SO4
5,1# culture medium+dregs of beans 2.0%
6,1# culture medium+dregs of beans 2.0%, omits (NH)2SO4
It is analyzed according to viable count and gemma Number synthesis, finally determines medium component are as follows: molasses 3.0%, dregs of beans 2.0%,
K2HPO4×3H2O 0.7%, KH2PO40.3%, (NH4)2SO40.15%, sodium citrate × 2H2O0.05%, MgSO4×
7H2O 0.01%, surplus are water.
The culture medium for being inoculated with the bacterial strain is placed in shaking table culture 48h under different temperatures, according to the muddy degree of bacterium solution, microscopy
Without miscellaneous bacteria, it can confirm that the bacterial strain in 10~40 DEG C of environment can be grown, and the strain growth under 30~40 DEG C of environment is most fast.It will
The culture medium for being inoculated with the bacterial strain is placed in shaking table culture 48h under different initial pH value environment, the ring for being 3.5~8.5 in pH value range
Bacterial strain can be grown in border, and be grown most fastly in the environment that pH value is 6.5~7.5.
Be placed in 37 DEG C, pH value be shaking table culture under conditions of 6.8,120rpm, 150rpm, 180rpm is respectively set in revolving speed,
Culture solution is taken to count in the 12nd, 24,36,48,60.Such as table 3, table 4:
The optimization 1 of table 3,44 condition of culture of bacillus amyloliquefaciens (B.amyloliquefaciens)
The optimization 2 of table 4,44 condition of culture of bacillus amyloliquefaciens (B.amyloliquefaciens)
It is final to determine bacillus amyloliquefaciens (B.amyloliquefaciens) 44 optimal culture condition are as follows: р Н 6.8；Temperature
Degree is 37 DEG C, mixing speed 180r/min, incubation time 48h.
Four, 44 bacterial strain growth promotion of bacillus amyloliquefaciens is tested
44 bacterium of bacillus amyloliquefaciens (B.amyloliquefaciens) is tested under laboratory condition in accordance with the following methods
The growth promoting function (table 5) of strain:
1. seed impregnates two minutes in 70% ethyl alcohol
2. being rinsed with aseptic water
3. aseptic seed is impregnated 30 minutes in bacteria preparation, then under conditions of test tube, training is placed them in
It supports in the sterile moist chamber on the surface in ware.For every kind of variant, ten seeds, a formula three are placed in each moist chamber
Part.Seed in control group is impregnated with tap water.Seedling is cultivated 7-10 days in the environment of 28 DEG C.
After 7-10 days, the length and weight of seedling are measured.
Growth stimulating activity level (PGP, %): it is calculated as follows
PGP=(A-B)/A*100
A is the arithmetic mean of instantaneous value for compareing the length (quality) of seedling；
B is the arithmetic mean of instantaneous value of the length (quality) of seedling in experiment.
Growth of seedling situation (being shown in Table 5) is estimated in 2 weeks: applying 2% bacillus amyloliquefaciens
(B.amyloliquefaciens) 44 culture solutions make the length of seedling and weight increase separately 20.19% and 26.7%, preliminary to demonstrate,prove
Bright bacillus amyloliquefaciens (B.amyloliquefaciens) 44 have certain growth promoting function.
The growth promoting function of table 5,44 bacterial strain of bacillus amyloliquefaciens
Five, bacteriostatic experiment
Using 44 pairs of Beating holes method measurement bacillus amyloliquefaciens (B.amyloliquefaciens) agriculture principal causatives
The inhibiting effect of bacterium and fungi, lower layer's culture medium are 2%NA solid medium, and 1ml pathogen and 5ml 1.2% are taken after solidification
NA culture medium (or 1.2%PDA culture medium), which mixes well, makees upper layer culture medium, and punch punches after solidification, and 100 μ l Xie Dian are added
44 bacterium solution of afnyloliquefaciens (B.amyloliquefaciens), in standing 2h in refrigerator, 37 DEG C of (bacterium)/28 DEG C (fungi) are just
Set culture 48 hours.By measuring antibacterial circle diameter, the bacteriostasis of bacterium to be measured is detected.Experimental result is as shown in table 6 and table 7:
Table 6, bacillus amyloliquefaciens (B.amyloliquefaciens) 44 pairs of plant epiphyte pathogens inhibiting effect
Table 7, bacillus amyloliquefaciens (B.amyloliquefaciens) 44 pairs of vegetative bacteria pathogens inhibiting effect
Bacillus amyloliquefaciens (B.amyloliquefaciens) 44 pairs of plant epiphyte pathogens (Fusarium oxysporums, rice
Sheath blight fungus, rod method, sclerotinite, root rotof flax pathogen) and vegetative bacteria pathogen (Irving's formula bacillus, crown gall soil
Bacillus, Xanthomonas campestris, pseudomonas syringae, bacterial canker of tomato, blakleg of potato bacterium) there is good inhibiting effect, press down
Bacterium circle is respectively 13-23mm and 20-31mm.
Six, compound formulation growth promotion is tested
Two plants of growth-promoting roots are filtered out from the existing strain library in laboratory acts on preferable bacterial strain rahnella aquatilis
(R.aquatilis) 27, bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619, with bacillus amyloliquefaciens
(B.amyloliquefaciens) 44 carry out compounding probiotics are made.
27 culture presevation unit of rahnella aquatilis (R.aquatilis) is in Wuhan University's China typical culture collection
The heart, deposit number: CCTCC M 2018566.27 its culture medium of rahnella aquatilis (R.aquatilis) and fermentation condition are as follows: beautiful
Rice starch 3.0%, peptone 2.0%, K2HPO4×3H2O 0.7%, KH2PO40.3%, (NH4)2SO40.15%, citric acid
Sodium × 2H2O 0.05%, MgSO4×7H2O 0.01%；Surplus is water.рН6.8；37 DEG C, stirring rate 150rpm/min, training
It supports 48 hours, obtains rahnella aquatilis (R.aquatilis) 27 fermentation liquid.
Bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 bacterial strain is protected purchased from China General Microbiological strain
Administrative center is hidden, bacterium solution preparation method: glucose 5%, tryptone 5%, yeast extract 5%, surplus are water；pH 7.0；28
DEG C, stirring rate 180rpm/min is cultivated 48 hours, obtains bacillus amyloliquefaciens (B.amyloliquefaciens) B-
1619 fermentation liquids.
Formula one: a kind of compound formulation, by bacillus amyloliquefaciens (B.amyloliquefaciens) 44: aquatic drawing grace
Salmonella (R.aquatilis) 27: bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619=1:1:1 is obtained.
Formula two: a kind of compound formulation, by bacillus amyloliquefaciens (B.amyloliquefaciens) 44: aquatic drawing grace
Salmonella (R.aquatilis) 27: bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619=5:3:2 is obtained.
1, potted plant experiment is carried out under laboratory condition.
To investigate the composite bacteria agent to the growth-promoting ability of plant, in the present embodiment, using garlic as the representative of plant, choose
Health garlic planting of the same size, is arranged experimental group and control group, every group of 3 repetitions.Experimental group is probiotics, experiment
Group is poured with 300-500 times of dilution of product, and control group pours clear water, and a water is poured within 3-5 days after garlic planting.It is right after 15 days
The overall length of garlic, root long, root measure again, and data are shown in Table 8.
Table 8, different solutions pour Garlic Growth situation comparing result
Table note: 1.: 2. control group-clear water is formulated two, dilutes 500 times of uses；3. being formulated two, 300 times of uses are diluted；4. matching
Fang Yi dilutes 500 times of uses；5. being formulated one, 300 times of dilution use
It is better than in terms of root long and weight by the garlic that each group of data in comparison table can be seen that one group of application formula
Other experimental groups, one experiment effect of formula are good compared with the experiment effect of formula two, with control group data comparison the results show that formula one
The Garlic Growth situation that product pours is preferable, and overall length increases 13.56%, and root long increases 17.64%, and root dry weight increases
28.75%.The result, which demonstrates compound formulation, has good growth-promoting root effect (Fig. 4) to plant.To sum up result is three plants selected
The adding proportion of bacterium is 1:1:1, and usage amount is 300 times of dilution.
Obtain compound formulation, it is 2% that content, which is made, in 50 times of dilution after three plants of fermented liquids being mixed according to 1:1:1 ratio
Compound formulation carries out growth promotion experiment on tomato and romaine lettuce seed.According to " bacillus amyloliquefaciens
(B.amyloliquefaciens) experimental method in 44 growth promoting function " handles seed, and growth of seedling feelings are counted after 2 weeks
23% and 24% has been respectively increased after the processing of the germination percentage of tomato and romaine lettuce seed as the result is shown in condition, and the weight of seedling mentions respectively
High by 22% and 28%, 29% and 27% has been respectively increased in seedling length.(table 9, table 10).
Table 9, compound formulation influence to the germination percentage of tomato seeds and growth of seedling
Table 10, compound formulation influence to the germination percentage of romaine lettuce seed and growth of seedling
2, the biocontrol effect under compound formulation laboratory condition
In the present embodiment, using radish seed as the representative of plant, to investigate inhibition of the composite bacteria agent to phytopathogen
Effect, selects to examine using Fusarium oxysporum and pseudomonas syringae as the representative of plant epiphyte pathogen and bacterial disease opportunistic pathogen respectively
Examine the fungistatic effect of the compound formulation.
Choose full radish seed, aseptic water washing 3 times, 28 DEG C of growth cabinet vernalization, sowing when bud grows to 1cm
In plastic seeding culturing hole tray (6cm × 6cm × high 5.5cm), every cave 10-15 seeds.Spraying concentration is 2% to answer after plantation 21d
Preparation is closed, every 4 cave sprays 15mL, and clear water processing is as control.It is inoculated with Fusarium oxysporum afterwards for 24 hours and pseudomonas syringae suspends
Liquid, bacteria concentration are 1 × 106CFU/mL.Disease is investigated after 7d, and a situation arises, calculates disease index and preventive effect (table 11).The state of an illness point
Grade standard: 0 grade, whole plant is disease-free；1 grade, plant cauline leaf has seldom scab, accounts for 1/5 or less the gross area；2 grades, plant cauline leaf has few
Number scab, accounts for the 1/5-1/3 of the gross area；3 grades, there are many scabs for plant cauline leaf, account for gross area 1/3-2/3, blade face turns yellow；4 grades,
Whole plant morbidity, blade face is withered.Compound formulation is respectively 50.94% to the preventive effect of pseudomonas syringae and Fusarium oxysporum
With 68.75%.
Table 11, compound formulation are to the preventive effect of pseudomonad and Fusarium oxysporum
3, greenhouse is tested
It leases greenhouse and plants tomato, test 3 groups of setting, every group of tomato seedling several 300, experimental group 1 is formula one
Product, experimental group 2 are two products of formula, and two experimental groups begin to use this product from before seedling replanting, and Sui Shuichong is applied, usage amount
It is 2%, control group rushes to fertilize, normal spraying pesticide.Remaining unified management in addition to variable of three groups.A data are measured weekly
(table 12).Since after plantation the 4th week, experimental group tomato stem length was apparently higher than control group, illustrated that the flora in product has been opened
Beginning colonizes and plays a role, and 1 effect of experimental group is better than experimental group 2.
Table 12, tomato plant stem length (cm)
In tomato seedling plantation after start count tomato plant situation to fruit maturation of catching an illness before (table 13), severity Scaling mark
Quasi-: 0 grade, whole plant is disease-free；1 grade, plant cauline leaf has seldom scab, accounts for 1/3 or less the gross area；2 grades, plant cauline leaf has a small number of diseases
Spot accounts for the 1/3-2/3 of the gross area；3 grades, the ill spot of plant cauline leaf accounts for 2/3 of the gross area or more, by analysis of statistical results, experiment
The situation of catching an illness of group 1 is better than control group, illustrates that the use of compound formulation is slightly better than the using effect of control group pesticide, can be with again
Close the pesticide sprayed during preparation substitution tomato plant strain growth.
Table 13, tomato in plastic greenhouse disease event
Compared to laboratory condition and potted plant experiment, by greenhouse the results showed that the composite bacteria agent is in plastic shed soil item
It good can be colonized in soil under part, it was demonstrated that the composite bacteria agent is can be applied to conscientiously in practical planting process.Application is multiple
The tomato plant disease condition of combined bacteria agent is less, and plant growth condition is due to control group and uses pesticide group and fertilizer group, it was demonstrated that
Composite bacteria agent in the application has effects that good growth-promoting and diseases prevention, is applied to actual production, pesticide can be effectively reduced
And the dosage of chemical fertilizer, reduce injury of the chemical reagent to soil.
The foregoing is merely preferred embodiment of the present disclosure, are not limited to the disclosure, for the skill of this field
For art personnel, the disclosure can have various modifications and variations.It is all within the spirit and principle of the disclosure, it is made any to repair
Change, equivalent replacement, improvement etc., should be included within the protection scope of the disclosure.
SEQUENCE LISTING
<110>the dark blue Biotechnology Co., Ltd in Shandong
<120>a kind of bacillus amyloliquefaciens and its application with growth promotion and resistant effect
<130> 2010
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1454
<212> DNA
<213>44 16S rDNA of bacillus amyloliquefaciens
<400> 1
tgaggtgggt gctaatacat gcaagtcgag cggacagatg ggagcttgct ccctgatgtt 60
agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120
aaaccggggc taataccgga tggttgtctg aaccgcatgg ttcagacata aaaggtggct 180
tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatagcgtag ccgacctgag aaggtgatcg gccacactgg aactgagaaa 300
cggcccagac tcctacggga ggcaggcagt aaggaatctt ccgcaatgga cgaaagtctg 360
accgagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420
agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg ggaattattg 540
ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg 600
ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt 660
agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg 720
taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780
acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa 840
cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 900
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960
aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac 1020
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg 1140
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200
acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac 1260
aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta 1320
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380
cacaccacga gagtttgtaa cacccgaagt cggtgaggta acctttagga gccagccgcc 1440
gaagtgacag atgg 1454
Claims (10)
1. a bacillus amyloliquefaciens (B.amyloliquefaciens) 44, which is characterized in that the bacterial strain is in 2018
It is preserved in China typical culture collection center, abbreviation CCTCC, address within August 23 days are as follows: Luojiashan, Wuchang, Wuhan City, Hubei Province,
Its biological deposits number are as follows: CCTCC M 2018564.
2. bacillus amyloliquefaciens 44 as described in claim 1, which is characterized in that the culture medium of the bacterial strain includes solubility
Starch/molasses 2.0%-3.5%, peptone/dregs of beans/Dried Corn Steep Liquor Powder 1.5%-3.0%, K2HPO4×3H2O 0.5-1.0%,
KH2PO40.1-0.5%, (NH4)2SO40.1-0.3%, sodium citrate × 2H2O 0.01-0.05%, MgSO4×7H2O
0.01%, surplus is water；Preferably, the culture medium is made using the molasses of 1-3% as carbon source with the bean extract of 1-4%
For nitrogen source.
3. bacillus amyloliquefaciens 44 as claimed in claim 2, which is characterized in that the culture medium is molasses 2.5-3.5%,
Dregs of beans 1.5-2.5%, K2HPO4×3H2O 0.5-1.0%, KH2PO40.1-0.5%, (NH4)2SO40.1-0.3%, citric acid
Sodium × 2H2O 0.01-0.05%, MgSO4×7H2O 0.01%, surplus are water, pH 6.6-7.0.
4. a kind of microbial inoculum, which is characterized in that the described in any item bacillus amyloliquefaciens of claim 1-3
(B.amyloliquefaciens) 44 and/or its culture.
5. a kind of composite bacteria agent, which is characterized in that the composite bacteria agent includes the described in any item solution starch buds of claim 1-3
Spore bacillus (B.amyloliquefaciens) 44, rahnella aquatilis (Rahnella aquatilis) 27 and solution starch gemma
Bacillus (B.amyloliquefaciens) B-1619 microbial inoculum and its respective culture, wherein rahnella aquatilis 27
(R.aquatilis) Wuhan University's China typical culture collection center, abbreviation CCTCC, ground were preserved on August 23rd, 2018
Location are as follows: Luojiashan, Wuchang, Wuhan City, Hubei Province, deposit number: CCTCC M 2018566；The bacillus amyloliquefaciens
(B.amyloliquefaciens) B-1619 is purchased from China General Microbiological Culture Collection Center.
6. composite bacteria agent as claimed in claim 5, which is characterized in that in the composite bacteria agent, the bacillus amyloliquefaciens
(B.amyloliquefaciens) 44: rahnella aquatilis (R.aquatilis) 27: bacillus amyloliquefaciens
(B.amyloliquefaciens) ratio of B-1619 is 0.8-5.2:0.8-3:0.8-2；Preferably, the solution starch gemma
Bacillus (B.amyloliquefaciens) 44: rahnella aquatilis 27 (R.aquatilis): bacillus amyloliquefaciens
(B.amyloliquefaciens) ratio of B-1619 is 1:1:1；Or in the composite bacteria agent, living bacteria count >=1,000,000,000/
mL。
7. microbial inoculum as claimed in claim 4 or composite bacteria agent described in claim 5 or 6, which is characterized in that the microbial inoculum or
The dosage form of composite bacteria agent is liquid bacterial agent, pulvis or granule；It preferably, is aqueous suspension agent, dispersible oil-suspending agent, wettable
Pulvis or water dispersion granule；It preferably, further include acceptable auxiliary material in Pesticide Science in the microbial inoculum or composite bacteria agent, it is described
Acceptable auxiliary material is selected from dispersing agent, wetting agent, disintegrating agent, binder, defoaming agent, antifreeze, thickener, filler in Pesticide Science
With one of solvent or a variety of.
44, claim 4 the described in any item bacillus amyloliquefaciens of claim 1-3 8. (B.amyloliquefaciens)
Application of the microbial inoculum and the described in any item composite bacteria agents of claim 5-7 in following either side:
(1) inhibit plant epiphyte pathogen and phytopathy bacterial disease opportunistic pathogen；
(2) promote plant growth；
9. application as claimed in claim 8, which is characterized in that the plant epiphyte pathogen includes but is not limited to sharp spore reaping hook
Bacterium, Rhizoctonia solani Kuhn, rod method, sclerotinite, root rotof flax pathogen；Preferably, the vegetative bacteria pathogen includes
But it is not limited to Irving's formula bacillus, Agrobacterium tumefaciens, Xanthomonas campestris, pseudomonas syringae, bacterial canker of tomato, the black shin of potato
Germ
10. application as claimed in claim 9, which is characterized in that the promotion plant growth, which is embodied in, promotes plant seedlings to increase
Long and/or weight gain；The plant includes but is not limited to garlic, tomato, radish, romaine lettuce, cucumber and wheat.
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