CN105961441A - Application of bacillus JC65 and volatile substance thereof to plant growth promotion - Google Patents
Application of bacillus JC65 and volatile substance thereof to plant growth promotion Download PDFInfo
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- CN105961441A CN105961441A CN201610440616.XA CN201610440616A CN105961441A CN 105961441 A CN105961441 A CN 105961441A CN 201610440616 A CN201610440616 A CN 201610440616A CN 105961441 A CN105961441 A CN 105961441A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention discloses application of bacillus JC65 and a volatile substance thereof to plant growth promotion. An existing bacterial strain is developed again and screened, the bacillus JC65 is obtained, the volatile substance generated by the bacillus JC65 can efficiently promote growth of plants in a broad spectrum mode and especially can promote growth of crops such as vegetables and grain and oil crops, the growth promotion effect exceeds 21.55% and can reach 829.14% at the highest level, and quality of the crops is improved. The problem that chemical fertilizer or other methods are poor in effect or residues exist is solved, the environment is protected, sustainable development of agriculture is promoted, and the bacillus JC65 and the volatile substance thereof have good application prospect.
Description
Technical field
The invention belongs to plant protection art, relate to bacillus cereus JC65 and volatile material thereof at plant growth-promoting
In application
Background technology
Along with the development of modern agriculture, the large-scale excess of chemical fertilizer is used and is caused environmental and human health impacts
Great damage, it has also become affect the key factor of environment, human health and social development.Excess uses chemical fertilizer,
The physicochemical character causing soil deteriorates, soil nutrient structural imbalance, fertility decline.Cause equally soil acidification with
And harden, finally cause arable soil to be degenerated.And the acidifying of soil can increase toxic heavy metal element such as hydrargyrum, lead
Deng the activity in soil, produce heavy metal pollution, affect plant growth.
Several chemical fertilizer the most partially executing single character are easily caused the nutritional disorder of crop, and chemical fertilizer contains mostly
Various different salts, wherein the chemical substance such as nitrogen, phosphorus, potassium is easily by soil consolidation, accumulates in soil,
Soil property salinization of soil, causes soil nutrient unbalance, affects and hinders crops to inhale the conversion of absorbed Nutrients
Receive, make fruit and vegerable growth traits inferior, crop mouthfeel and quality variation, Quality Down, as vegetable taste the most fragrant,
Melon and fruit taste sweetless, mouthfeel is poor, and perishable, should not deposit.Therefore, people have in the urgent need to safer
The replacement product of effect.In recent years, microorganism formulation because having the features such as environmental friendliness, growth-promoting volume increase, diseases prevention and
Receive much concern.Utilizing beneficial microbe in soil to develop into microbial manure is research and development focus in recent years.
CN102174431A discloses a kind of biocontrol bacterial strain JC65 of bacterial fruit blotch of watermelon that prevents and treats to west
The preventive effect of melon Acidovorax Avenae Subsp. Citrulli is 50-90%, and sick field effect of increasing production is 10-200%.Growth-promoting to anosis plant
Effect is up to 10-200%, and the effect of increasing production to anosis field is 5-20%.This microbial inoculum can be watered spraying and use, or
Make compost to use, it is possible to after dilution, when transplanting, pouring root uses.But and this antibacterial undisclosed possesses growth-promoting function.
Summary of the invention
It is an object of the invention to for the deficiencies in the prior art, it is provided that bacillus cereus JC65 is promoting plant growing
In application.
The volatile material that it is yet another object of the invention to provide bacillus cereus JC65 generation is promoting plant growing
In application.
The purpose of the present invention can be achieved through the following technical solutions:
Bacillus JC65 application in the growth promoting plant, described bacillus JC65 is in 2010
On December is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation for 15
Number it is CGMCC NO.4475.
Bacillus JC65 can promote the application in its microorganism formulation grown, described brood cell in preparation
Bacillus JC65 is preserved in the common micro-life of China Committee for Culture Collection of Microorganisms on the 15th in December in 2010
Thing center, culture presevation number is CGMCC NO.4475.
By described bacillus JC65 28 DEG C of 180rpm shaken cultivation 12-16h in LB culture fluid, then
6000rpm is centrifuged 10min and obtains thalline, with aquesterilisa, thalline is diluted making microbial inoculum, microbial inoculum finished product
Middle viable bacteria total concentration is 1 × 109-1×1010CFU/mL。
The volatile material that bacillus JC65 produces application in the growth promoting plant, described brood cell
Bacillus JC65 is preserved in the common micro-life of China Committee for Culture Collection of Microorganisms on the 15th in December in 2010
Thing center, culture presevation number is CGMCC NO.4475.
The volatile material that bacillus JC65 produces application in preparation promotes the preparation of plant growing, institute
The bacillus JC65 stated is preserved in China Committee for Culture Collection of Microorganisms on 15th in December in 2010
Common micro-organisms center, culture presevation number is CGMCC NO.4475.
The crops such as plant preferred vegetable of the present invention, grain and oil crop.
Beneficial effect
The present invention, by the exploitation again to existing bacterial strain, screens bacillus cereus JC65, its volatilization produced
Property material can wide spectrum, efficiently promote plant growing, enable in particular to promote that the crops such as vegetable, grain and oil crop are raw
Grow, improve its quality.Solve other method poor effect such as chemical fertilizer or have the problems such as residual, protection
Environment, promotes agricultural sustainable development, has preferable application prospect.
The plant height of plant and root length are had after processing and improve significantly by said preparation JC65, have plant necessarily
Growth-promoting functions, through JC65 process crop compared with matched group, growth-promoting effect surpasses 21.55%, reaches as high as
829.14%, and improve its quality.This bacillus microbial inoculum, can room temperature preservation 6 months-1 year.
Accompanying drawing explanation
Fig. 1, the microbial bacterial agent JC65 volatile material growth-promoting effect to crop
Fig. 2, the impact on tomato biological amount of the microbial bacterial agent JC65 volatile material
Biological deposits information
JC65, for bacillus (Bacillus sp), is preserved in China Microbiological bacterium on 15th in December in 2010
Plant preservation administration committee common micro-organisms center, address: section in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
The culture presevation number of institute of microbiology of institute is CGMCC NO.4475.
Detailed description of the invention
Embodiment 1 volatile material has the screening of growth-promoting effect bacterial strain
Method: use the method screening volatile material separating culture dish to have the bacterial strain of growth-promoting effect.South will be intended
Canola seed use after 3%NaClO solution disinfection 10min, ddH2O cleans 6 times.South is intended by off-the-shelf
Canola seed is layered in MS culture dish and is placed on after 4 DEG C of black out incubators carry out low temperature vernalization 2d, is placed on light
According to incubator is cultivated (12h illumination, 20 DEG C, 50% relative humidity, 40W fluorescent lamp) 2d.Then, will
The seedling of 2d size is layered on one side of the two compartment culture dish containing MS culture medium, instills 10 μ L's at another side
(concentration is 10 to bacterium solution7CFU/mL), seal culture dish with sealed membrane, continue to place in above-mentioned illumination box
Cultivate, each culture dish 3 strain seedling, each process 6 repetition.To be not added with bacterium solution as comparison.
Growth-promoting statistical effect: significant change be can be observed after about 21d, after taking out plant, adds up each strain plant
Biomass: the number of blade, leaf area, fresh weight, and with ultraviolet spectrophotometry detection arabidopsis Chlorophyll
Content.
As shown in table 1, microbial bacterial agent JC65 can produce the volatile material with growth-promoting effect.
Table 1 produces the growth-promoting effect of volatile material bacterial strain
Note: different lower case persons represent significant difference in 0.05 level.
Embodiment 2
The preparation method of microbial bacterial agent JC65 microbial inoculum is as follows:
Bacillus JC65 is 28 DEG C of 180rpm shaken cultivation 12-16h in LB culture fluid, and then 6000
Rpm is centrifuged 10min and obtains thalline, is diluted making microbial inoculum with aquesterilisa by thalline, lives in microbial inoculum finished product
Bacterium total concentration is 1 × 109-1×1010CFU/mL。
Embodiment 3 microbial bacterial agent JC65 produces volatile material and tests the growth-promoting of solanaceous vegetables
Method: by Fructus Lycopersici esculenti after 3%NaClO solution disinfection 5min, in MS culture medium after accelerating germination, will
The consistent seedling that sprouts is transferred in tissue culture bottle, places 1 for every bottle.It addition, again by sterilized containing MS
The 10mL small beaker of culture medium is placed in tissue culture bottle, and (concentration is to inoculate JC65 bacterium solution in small beaker
5.0×107CFU/mL)10μL.Tissue culture bottle is placed in illumination box cultivation (condition: light application time 12h,
Temperature 28 DEG C, relative humidity 50%).And it is right using escherichia coli (Escherichia coli) DH5 α as blank
According to group CK, each process 24 young plant, repeat for 3 times.
Add up after 28d that the plant height of Fructus Lycopersici esculenti, stem be thick, the number of blade, fresh weight.
It is fresh that Biomass increases (%)=(process group plant fresh weight blank group plant fresh weight)/adjoining tree
Weight × 100%
As shown in table 2, microbial bacterial agent JC65 produces volatile material and can remarkably promote solanaceous vegetables Fructus Lycopersici esculenti
Growth, shows the fresh weight of crop, and stem is long, and stem the most compared with the control, has significantly increase.Show, micro-
The volatile material that bacteria agent JC65 produces increases up to 829.14% to the Biomass of Fructus Lycopersici esculenti.
Table 2 microbial bacterial agent JC65 produces the volatile material impact on solanaceous vegetables tomato biological amount
Note: different lower case persons represent significant difference in 0.05 level.
Embodiment 4 microbial bacterial agent JC65 produces the volatile material impact on solanaceous vegetables quality
Method: according to the step in embodiment 3, by tomato seeds after 3%NaClO solution disinfection 5min,
In MS culture medium after accelerating germination, by sprouting, consistent seedling is transferred in tissue culture bottle, places 1 for every bottle.Separately
Outward, the more sterilized 10mL small beaker containing MS culture medium is placed in tissue culture bottle, and in small beaker
(concentration is 5.0 × 10 to inoculation JC65 bacterium solution7CFU/mL)10μL.Tissue culture bottle is placed in illumination box training
Support (condition: light application time 12h, temperature 28 DEG C, relative humidity 50%).After 14d, by tissue culture bottle
Tomato seedling is transplanted in basin alms bowl, is placed in greenhouse growth (condition: light application time 12h, temperature 28 DEG C, phase
To humidity 50%).And DH5 α is as blank group CK, often using escherichia coli (Escherichia coli)
Individual process 24 young plant, repeats for 3 times.
Finally add up the vitamin C of tamato fruit, soluble sugar, titratable acid.
As shown in table 3, microbial bacterial agent JC65 produces after volatile material processes, the vitamin C of Fructus Lycopersici esculenti, solvable
Property sugar and the content of titratable acid, and dramatically increase the sugar-acid ratio of Fructus Lycopersici esculenti.Show, microbial bacterial agent JC65
The volatile material produced has significant effect in terms of improving Tomato Quality.
Table 3 microbial bacterial agent JC65 produces the volatile material impact on Tomato Quality
Note: different lower case persons represent significant difference in 0.05 level.
Embodiment 5 microbial bacterial agent JC65 produces volatile material and tests the growth-promoting of leafy vegetable
Method: by water spinach seed after 3%NaClO solution disinfection 5min, soaks 5min in sterilized water,
Then accelerating germination in MS culture medium, by sprouting, consistent seedling is transferred in tissue culture bottle, places 1 for every bottle.
It addition, the more sterilized 10mL small beaker containing MS culture medium is placed in tissue culture bottle, and at small beaker
(concentration is 5.0 × 10 to middle inoculation JC65 bacterium solution7CFU/mL)10μL.Tissue culture bottle is placed in illumination box
Cultivate (condition: light application time 12h, temperature 28 DEG C, relative humidity 50%).And with escherichia coli (Escherichia
Coli) DH5 α is as blank group CK, each process 12 young plant, repeats for 3 times.
Add up after 14d that the plant height of water spinach, stem be thick, fresh weight.
As shown in table 4, microbial bacterial agent JC65 produces volatile material and can remarkably promote leafy vegetable water spinach
Growth, show that the stem length of crop, stem be thick, fresh weight compared with the control, have significantly increase.Show, micro-
The volatile material that bacteria agent JC65 produces reaches 42.09% to the growth-promoting effect of leafy vegetable water spinach.
Table 4 microbial bacterial agent JC65 produces the volatile material impact on leafy vegetable water spinach Biomass
Note: different lower case persons represent significant difference in 0.05 level.
Embodiment 6 microbial bacterial agent JC65 produces volatile material and tests the growth-promoting of grain and oil crop
Method: by corn seed after 3%NaClO solution disinfection 5min, soaks 5min in sterilized water,
Then accelerating germination in MS culture medium, by sprouting, consistent seedling is transferred in tissue culture bottle, places 1 for every bottle.
It addition, the more sterilized 10mL small beaker containing MS culture medium is placed in tissue culture bottle, and at small beaker
(concentration is 5.0 × 10 to middle inoculation JC65 bacterium solution7CFU/mL)10μL.Tissue culture bottle is placed in illumination box
Cultivate (condition: light application time 12h, temperature 28 DEG C, relative humidity 50%).And with escherichia coli (Escherichia
Coli) DH5 α is as blank group CK, each process 12 young plant, repeats for 3 times.
Add up after 14d that the plant height of Semen Maydis, stem be thick, fresh weight.
As shown in table 5, microbial bacterial agent JC65 produces volatile material and can remarkably promote the life of grain and oil crop Semen Maydis
Long, show that the stem length of crop, stem be thick, fresh weight compared with the control, have significantly increase.Show, micro-life
The volatile material that thing microbial inoculum JC65 produces reaches 21.55% to the growth-promoting effect of leafy vegetable water spinach.
Table 5 microbial bacterial agent JC65 produces the volatile material impact on grain and oil crop Corn Biomass
Note: different lower case persons represent significant difference in 0.05 level.
The collection of embodiment 7 volatile matter and qualification
Method: use solid phase micro-extraction method to collect the volatile material of bacterial strain JC65, will be for examination antibacterial JC65
Access in LB culture fluid and cultivate (condition of culture: 28 DEG C, 180r/min, 24h), then bacterium solution is diluted to
It is about 5.0 × 107CFU/mL is standby.Take 10 μ L bacterium solution and receive in the ml headspace bottle containing MS culture medium, 28 DEG C
Incubator is cultivated 5d.The syringe needle of solid-phase micro-extracting device is inserted above ml headspace bottle, fixing solid-phase microextraction
Handle, carefully releases fiber head (SPME fiber), then and start timing, under 50 DEG C of environment, and absorption
Take out after 40min；Insert the injection port of GC-MS instrument, thermal desorption 1min at 220 DEG C immediately
(Mohamed et al.,2006；Blom et al.,2011).
Chromatographic condition:
Chromatographic column: Supelcowax 10 (30m × 0.25mm inside diameter, 0.25um) (24079,
Supelco)。
Injector temperature: 250 DEG C；Heating schedule: 35 DEG C keep 3min, rise to 180 DEG C of guarantors with 10 DEG C/min
Hold 1min, finally rise to 240 DEG C with 4 DEG C/min and keep 4min；Carrier gas (He): flow velocity 1.0mL/min, no
Split sampling.
Mass Spectrometry Conditions: electron impact ion source (EI)；Electron energy 70eV；Transmission line temperature 220 DEG C；Activate
Voltage 1.5V；Mass scan range m/z:50-500.
Signals assigned uses NIST MS Search software V. to retrieve
As shown in table 6, the volatile material that bacterial strain JC65 produces mainly has ketone, alkanes, alcohols, fat
Class etc..
The volatile material that table 6 bacterial strain JC65 produces
The growth-promoting of plant is tested by embodiment 8 volatile material
Method: by arabidopsis seed use after 3%NaClO solution disinfection 10min, ddH2O cleans 6 times.
Off-the-shelf arabidopsis seed is layered in MS culture dish and is placed on 4 DEG C of black out incubators and carries out low temperature vernalization
After 2d, it is placed in illumination box cultivation (12h illumination, 20 DEG C, 50% relative humidity, 40W fluorescent lamp)
2d.Then, the Arabidopsis thaliana Seedlings of 2d size is layered on two compartment culture dish containing MS culture medium.
According to the analysis result of GC-MS, choose the compound methyl n-heptyl ketone of its specific component, 2-ethyl-1-hexanol,
The sterling of methyl n-undecyl ketone.Pure compounds solution: add 10 these materials of μ g and be dissolved in the DSM of 10 μ L,
Before use, by lanoline solution (lanoline solution: take 1.6g lanoline and be dissolved in the dichloromethane (DSM) of 10ml
In.) with pure compounds liquid is mixed in proportion, make final concentration be respectively 1ng, 10ng, 100ng, 1 μ g,
10μg、1mg/10mL.Take 20 μ L the most again to drop on the filter paper (diameter is 1cm) of sterilizing, dry
After 90s, be placed on the another side of two compartment culture dish, after sealing, as in incubator (12h illumination, 20 DEG C,
50% relative humidity, 40W fluorescent lamp) cultivate, statistics Biomass after 14d: fresh weight.
As shown in table 7, methyl n-heptyl ketone, 2-ethyl-1-hexanol, n-tridecane ketone are different dense under splitter plates system
Degree processes the growth-promoting functions to arabidopsis with varying strength, and methyl n-heptyl ketone is 10ng/10 μ L, 2-ethyl-1-hexanol
Being 10 μ g/10 μ L, n-tridecane is 100ng/10 μ L.Show, the volatility that microbial bacterial agent JC65 produces
Material has preferable growth-promoting effect.
The growth-promoting effect of table 7 volatile material
Note: " " represents that plant does not grows.
Claims (9)
1. bacillus JC65 promote plant growth in application, described bacillus JC65 in
Within 2010, December is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium on the 15th
Planting preserving number is CGMCC NO.4475.
Application the most according to claim 1, it is characterised in that described plant is vegetable, grain and oil crop.
3. bacillus JC65 can promote the application in its microorganism formulation grown in preparation, described
It is common that bacillus JC65 is preserved in China Committee for Culture Collection of Microorganisms on 15th in December in 2010
Microorganism center, culture presevation number is CGMCC NO.4475.
Application the most according to claim 3, it is characterised in that described plant is vegetable, grain and oil crop,
And improve its quality.
Application the most according to claim 3, it is characterised in that by described bacillus JC65 at LB
28 DEG C of 180rpm shaken cultivation 12-16h in culture fluid, then 6000rpm is centrifuged 10min and obtains thalline,
Being diluted making microbial inoculum with aquesterilisa by thalline, in microbial inoculum finished product, viable bacteria total concentration is 1 × 109-1×1010
CFU/mL。
6. the volatile material that bacillus JC65 produces application in the growth promoting plant, described
It is common that bacillus JC65 is preserved in China Committee for Culture Collection of Microorganisms on 15th in December in 2010
Microorganism center, culture presevation number is CGMCC NO.4475.
Application the most according to claim 6, it is characterised in that described plant is vegetable, grain and oil crop.
8. the volatile material that bacillus JC65 produces application in preparation promotes the preparation of plant growing,
Described bacillus JC65 is preserved in Chinese microorganism strain preservation conservator on the 15th in December in 2010
Meeting common micro-organisms center, culture presevation number is CGMCC NO.4475.
Application the most according to claim 8, it is characterised in that described plant is vegetable, grain and oil crop.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967632A (en) * | 2017-02-23 | 2017-07-21 | 南京农业大学 | It is a kind of efficiently to prevent and treat various crop gray mold and the microorganism mixture of capsicum resistance be lifted |
| CN113061559A (en) * | 2021-05-10 | 2021-07-02 | 南京农业大学 | Composite biological agent and application thereof in preventing and treating rice blast and promoting rice growth |
| CN113736712A (en) * | 2021-10-12 | 2021-12-03 | 绵阳市农业科学研究院 | Plant growth-promoting bacteria and microbial inoculum separated from kiwi fruits, separation and purification method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174431B (en) * | 2011-01-12 | 2012-12-12 | 南京农业大学 | Bio-control strain for preventing and controlling bacterial fruit blotch of watermelon |
| CN102584359B (en) * | 2011-12-14 | 2013-04-17 | 山东宝源生物有限公司 | Composite biological agent and preparation method thereof |
| CN103563996B (en) * | 2013-11-12 | 2016-08-17 | 无锡亚克生物科技有限公司 | Bacillus subtilis G1 application in promoting plant growing |
-
2016
- 2016-06-18 CN CN201610440616.XA patent/CN105961441B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967632A (en) * | 2017-02-23 | 2017-07-21 | 南京农业大学 | It is a kind of efficiently to prevent and treat various crop gray mold and the microorganism mixture of capsicum resistance be lifted |
| CN113061559A (en) * | 2021-05-10 | 2021-07-02 | 南京农业大学 | Composite biological agent and application thereof in preventing and treating rice blast and promoting rice growth |
| CN113736712A (en) * | 2021-10-12 | 2021-12-03 | 绵阳市农业科学研究院 | Plant growth-promoting bacteria and microbial inoculum separated from kiwi fruits, separation and purification method and application thereof |
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