CN105961441B - The application of bacillus JC65 and its volatile materials in plant growth-promoting - Google Patents

The application of bacillus JC65 and its volatile materials in plant growth-promoting Download PDF

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CN105961441B
CN105961441B CN201610440616.XA CN201610440616A CN105961441B CN 105961441 B CN105961441 B CN 105961441B CN 201610440616 A CN201610440616 A CN 201610440616A CN 105961441 B CN105961441 B CN 105961441B
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bacillus
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CN105961441A (en
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郭坚华
谢越盛
蒋春号
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Nanjing Agricultural University
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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
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • 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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  • Engineering & Computer Science (AREA)
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Abstract

The invention discloses the application of bacillus JC65 and its volatile materials in plant growth-promoting.Invention screens bacillus JC65 by the exploitation again to existing bacterial strain, the volatile materials generated can wide spectrum, efficiently promote plant growth, more particularly to promote the crop growths such as vegetables, grain and oil crop, growth-promoting effect surpasses 21.55%, reaches as high as 829.14%, and improves its quality.It is ineffective or have the problems such as residual to solve other methods such as chemical fertilizer, protects environment, promotes agricultural sustainable development, with good application prospect.

Description

The application of bacillus JC65 and its volatile materials in plant growth-promoting
Technical field
The invention belongs to plant protection arts, are related to bacillus JC65 and its volatile materials answering in plant growth-promoting With
Background technique
With the development of modern agriculture, the large-scale excessive application of chemical fertilizer causes pole to environmental and human health impacts Major injury, it has also become influence environment, human health and an important factor for social development.Excessive application chemical fertilizer, causes the reason of soil Change character deterioration, the decline of soil nutrient structural imbalance, fertility.Soil acidification and hardened is equally caused, arable land soil is finally caused Earth is degenerated.And the acidification of soil will increase the activity in the soil such as toxic heavy metal element such as mercury, lead, generate heavy metal-polluted Dye, influences plant growth.
Several chemical fertilizer for applying single mass partially too much are easy to cause the trophic disturbance of crop, and chemical fertilizer is containing various mostly Different salts, wherein the chemical substances such as nitrogen, phosphorus, potassium accumulate in the soil easily by soil consolidation, and soil property salinization of soil causes Soil nutrient is unbalance, influences and hinders crops to absorb to the conversion of absorbed Nutrients, keep fruits and vegetables growth traits inferior, makees Object mouthfeel and quality variation, quality decline, as vegetables not fragrant, melon and fruit of tasting is tasted sweetless, poor taste, and it is perishable, no Preferably store.Therefore, there is an urgent need to safer and more effective to replace product by people.In recent years, microorganism formulation because have it is environmental-friendly, Growth-promoting volume increase, diseases prevention the features such as and be concerned.Being developed using beneficial microbe in soil into microbial manure is in recent years Research and development focus.
A kind of biocontrol bacterial strain JC65 of prevention and treatment bacterial fruit blotch of watermelon is disclosed in CN102174431A to watermelon bacterium Property fruit blotch preventive effect be 50-90%, sick field effect of increasing production be 10-200%.To the growth-promoting effect of disease-free plant up to 10- 200%, the effect of increasing production to disease-free field is 5-20%.The microbial inoculum can be watered spraying use, or be made into compost use, can also be dilute In transplanting, pouring root is used after releasing.But it does not disclose the bacterium and has growth-promoting function.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide bacillus JC65 to promote answering in plant growth With.
It is yet another object of the invention to provide the volatile materials of bacillus JC65 generation in promoting plant growth Using.
The purpose of the present invention can be achieved through the following technical solutions:
Application of the bacillus JC65 in the growth for promoting plant, the bacillus JC65 was December 15 in 2010 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center day, culture presevation number is CGMCC NO.4475.
Application of the bacillus JC65 in the microorganism formulation that preparation can promote it to grow, the bacillus JC65 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation on December 15th, 2010 Number be CGMCC NO.4475.
By the bacillus JC65 in LB culture solution 28 DEG C of 180rpm shaken cultivation 12-16h, then 6000rpm from Heart 10min obtains thallus, is diluted thallus with aqua sterilisa and microbial inoculum is made, and viable bacteria total concentration is 1 × 10 in microbial inoculum finished product9-1 ×1010CFU/mL。
Application of the volatile materials that bacillus JC65 is generated in the growth for promoting plant, the bacillus JC65 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation on December 15th, 2010 Number be CGMCC NO.4475.
Application of the volatile materials that bacillus JC65 is generated in the preparation that preparation promotes plant growth, the bud Born of the same parents bacillus JC65 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium on December 15th, 2010 Kind deposit 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 screens bacillus JC65 by the exploitation again to existing bacterial strain, the volatile materials generated Can wide spectrum, efficiently promote plant growth, more particularly to promote the crop growths such as vegetables, grain and oil crop, improve its quality.Solution Other methods such as chemical fertilizer of having determined are ineffective or have the problems such as residual, protect environment, promote agricultural sustainable development, tool There is preferable application prospect.
Have after said preparation JC65 processing to the plant height and root long of plant and significantly improve, there is certain growth-promoting to plant Effect, compared with the control group, growth-promoting effect surpasses 21.55% to the crop handled through JC65, reaches as high as 829.14%, and improve it Quality.The bacillus microbial inoculum, can be room temperature preservation -1 year 6 months.
Detailed description of the invention
Fig. 1, microbial bacterial agent JC65 volatile materials are to the growth-promoting effect of crop
The influence of Fig. 2, microbial bacterial agent JC65 volatile materials to tomato biological amount
Biological deposits information
JC65 is bacillus (Bacillus sp), is preserved in Chinese microorganism strain preservation on December 15th, 2010 Administration committee's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology, Chinese Academy of Sciences strain Deposit number is CGMCC NO.4475.
Specific embodiment
1 volatile materials of embodiment has the screening of growth-promoting effect bacterial strain
Method: there is the bacterial strain of growth-promoting effect using the method screening volatile materials for separating culture dish.By arabidopsis kind Son after 3%NaClO solution disinfection 10min with using, ddH2O is cleaned 6 times.The arabidopsis seed being ready for is layered on MS culture dish In be placed on after 4 DEG C of black out incubators carry out low temperature vernalization 2d, be placed on culture in illumination box (12h illumination, 20 DEG C, 50% relative humidity, 40W fluorescent lamp) 2d.Then, the seedling of 2d size is layered on to the one of the two compartment culture dish of the culture medium containing MS Side instills the bacterium solution (concentration 10 of 10 μ L in another side7CFU/mL), culture dish is sealed with sealed membrane, continues to place above-mentioned light According to being cultivated in incubator, 3 plants of seedling of each culture dish, 6 repetitions of each processing.Bacterium solution is not added as control.
Growth-promoting effect statistics: can be observed significant change after about 21d, after taking out plant, count the biology of each plant of plant Amount: 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 generate the volatile materials with growth-promoting effect.
The growth-promoting effect of the production volatile materials bacterial strain of table 1
Note: different lowercase persons indicate in 0.05 horizontal upper significant difference.
Embodiment 2
Microbial bacterial agent JC65 microbial inoculum the preparation method is as follows:
Bacillus JC65 28 DEG C of 180rpm shaken cultivation 12-16h in LB culture solution, then 6000rpm is centrifuged 10min Thallus is obtained, thallus is diluted with aqua sterilisa, microbial inoculum is made, viable bacteria total concentration is 1 × 10 in microbial inoculum finished product9-1× 1010CFU/mL。
3 microbial bacterial agent JC65 of embodiment produces volatile materials and tests the growth-promoting of solanaceous vegetables
Method: by tomato after 3%NaClO solution disinfection 5min, on MS culture medium after vernalization, will sprout consistent children Seedling is transferred in tissue culture bottle, and every bottle is placed 1.In addition, the 10mL small beaker by sterilized containing MS culture medium is placed on group again It trains in bottle, and (concentration is 5.0 × 10 to inoculation JC65 bacterium solution in small beaker7CFU/mL)10μL.Tissue culture bottle is placed in illumination training Cultivated in feeding case (condition: light application time 12h, 28 DEG C of temperature, relative humidity 50%).And with Escherichia coli (Escherichia Coli) DH5 α is as blank control group CK, 24 young plants of each processing, 3 repetitions.
Plant height, stem thickness, the number of blade, fresh weight of tomato are counted after 28d.
Biomass increase (%)=(processing group plant fresh weight-blank control group plant fresh weight)/adjoining tree fresh weight × 100%
As shown in table 2, microbial bacterial agent JC65, which produces volatile materials, can remarkably promote the growth of solanaceous vegetables tomato, Show the fresh weight of crop, stem length, stem thickness compared with the control, there is significant increase.Show what microbial bacterial agent JC65 was generated Volatile materials increases up to 829.14% to the biomass of tomato.
2 microbial bacterial agent JC65 of table produces influence of the volatile materials to solanaceous vegetables tomato biological amount
Note: different lowercase persons indicate in 0.05 horizontal upper significant difference.
4 microbial bacterial agent JC65 of embodiment produces influence of the volatile materials to solanaceous vegetables quality
Method: it according to the step in embodiment 3, by tomato seeds after 3%NaClO solution disinfection 5min, is cultivated in MS On base after vernalization, the consistent seedling that will sprout is transferred in tissue culture bottle, and every bottle is placed 1.In addition, containing MS for sterilized again The 10mL small beaker of culture medium is placed in tissue culture bottle, and (concentration is 5.0 × 10 to inoculation JC65 bacterium solution in small beaker7CFU/mL) 10μL.Tissue culture bottle is set into culture (condition: light application time 12h, 28 DEG C of temperature, relative humidity 50%) in the light incubator.14d Afterwards, the tomato seedling in tissue culture bottle is transplanted in basin alms bowl, place in the greenhouse growth (condition: light application time 12h, 28 DEG C of temperature, Relative humidity 50%).And using Escherichia coli (Escherichia coli) DH5 α as blank control group CK, each 24 plants of processing Seedling, 3 repetitions.
Finally count vitamin C, soluble sugar, the titratable acid of tamato fruit.
As shown in table 3, after microbial bacterial agent JC65 produces volatile materials processing, the vitamin C of tomato, soluble sugar and The content of titratable acid, and dramatically increase the sugar-acid ratio of tomato.The volatile materials for showing that microbial bacterial agent JC65 is generated is changing There is significant effect in terms of kind Tomato Quality.
3 microbial bacterial agent JC65 of table produces influence of the volatile materials to Tomato Quality
Note: different lowercase persons indicate in 0.05 horizontal upper significant difference.
5 microbial bacterial agent JC65 of embodiment produces volatile materials and tests the growth-promoting of leafy vegetable
Method: by water spinach seed after 3%NaClO solution disinfection 5min, 5min is impregnated in sterile water, then in MS Vernalization on culture medium, the consistent seedling that will sprout are transferred in tissue culture bottle, and every bottle is placed 1.In addition, again by it is sterilized containing The 10mL small beaker of MS culture medium is placed in tissue culture bottle, and (concentration is 5.0 × 10 to inoculation JC65 bacterium solution in small beaker7CFU/ mL)10μL.Tissue culture bottle is set into culture (condition: light application time 12h, 28 DEG C of temperature, relative humidity 50%) in the light incubator. And using Escherichia coli (Escherichia coli) DH5 α as blank control group CK, 12 young plants of each processing, 3 repetitions.
Plant height, the stem thickness, fresh weight of water spinach are counted after 14d.
As shown in table 4, microbial bacterial agent JC65, which produces volatile materials, can remarkably promote the growth of leafy vegetable water spinach, The stem length, stem thickness, fresh weight for showing crop compared with the control, there is significant increase.Show what microbial bacterial agent JC65 was generated Volatile materials reaches 42.09% to the growth-promoting effect of leafy vegetable water spinach.
4 microbial bacterial agent JC65 of table produces influence of the volatile materials to leafy vegetable water spinach biomass
Note: different lowercase persons indicate in 0.05 horizontal upper significant difference.
6 microbial bacterial agent JC65 of embodiment produces volatile materials and tests the growth-promoting of grain and oil crop
Method: by corn seed after 3%NaClO solution disinfection 5min, impregnating 5min in sterile water, then trains in MS Vernalization on base is supported, the consistent seedling that will sprout is transferred in tissue culture bottle, and every bottle is placed 1.In addition, containing MS for sterilized again The 10mL small beaker of culture medium is placed in tissue culture bottle, and (concentration is 5.0 × 10 to inoculation JC65 bacterium solution in small beaker7CFU/mL) 10μL.Tissue culture bottle is set into culture (condition: light application time 12h, 28 DEG C of temperature, relative humidity 50%) in the light incubator.And Using Escherichia coli (Escherichia coli) DH5 α as blank control group CK, 12 young plants of each processing, 3 repetitions.
Plant height, the stem thickness, fresh weight of corn are counted after 14d.
As shown in table 5, microbial bacterial agent JC65, which produces volatile materials, can remarkably promote the growth of grain and oil crop corn, table The stem length, stem thickness of crop, fresh weight compared with the control, there is significant increase now.Show waving for microbial bacterial agent JC65 generation Volatile material reaches 21.55% to the growth-promoting effect of leafy vegetable water spinach.
5 microbial bacterial agent JC65 of table produces influence of the volatile materials to grain and oil crop Corn Biomass
Note: different lowercase persons indicate in 0.05 horizontal upper significant difference.
The collection and identification of 7 volatile matter of embodiment
Method: collecting the volatile materials of bacterial strain JC65 using solid phase micro-extraction method, will access LB for examination bacterium JC65 Cultivated in culture solution (condition of culture: 28 DEG C, 180r/min, for 24 hours), bacterium solution is then diluted to about 5.0 × 107CFU/mL is standby With.It takes 10 μ L bacterium solutions to be connected in the ml headspace bottle containing MS culture medium, cultivates 5d in 28 DEG C of incubators.By solid-phase micro-extracting device Syringe needle is inserted into above ml headspace bottle, and fixed solid phase microextraction handle carefully releases fiber head (SPME fiber), then and starts to count When, under 50 DEG C of environment, taken out after adsorbing 40min；It is inserted into the injection port of GC-MS instrument, the 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；Temperature program: 35 DEG C of holding 3min rise to 180 DEG C of holding 1min with 10 DEG C/min, most 240 DEG C of holding 4min are risen to 4 DEG C/min afterwards；Carrier gas (He): flow velocity 1.0mL/min, Splitless injecting samples.
Mass Spectrometry Conditions: electron impact ion source (EI)；Electron energy 70eV；220 DEG C of transmission line temperature；Activate voltage 1.5V；Mass scan range m/z:50-500.
Signals assigned is retrieved using NIST MS Search software V.
As shown in table 6, the volatile materials that bacterial strain JC65 is generated mainly has ketone, alkanes, alcohols, lipid etc..
The volatile materials that 6 bacterial strain JC65 of table is generated
8 volatile materials of embodiment tests the growth-promoting of plant
Method: by arabidopsis seed with being used after 3%NaClO solution disinfection 10min, ddH2O is cleaned 6 times.It will be ready for Arabidopsis seed be layered in MS culture dish and be placed on after 4 DEG C of black out incubators carry out low temperature vernalization 2d, be placed on illumination cultivation (12h illumination, 20 DEG C, 50% relative humidity, 40W fluorescent lamp) 2d is cultivated in case.Then, the Arabidopsis thaliana Seedlings of 2d size are layered on One side of the two compartment culture dish of the culture medium containing MS.
According to the analysis of GC-MS as a result, choosing the compound methyl n-heptyl ketone of its specific component, 2- ethyl -1- hexanol, 2- 13 The sterling of alkanone.Pure compounds solution: the 10 μ g substances are added to be dissolved into the DSM of 10 μ L, before use, by wool lipoprotein solution (wool lipoprotein solution: 1.6g lanolin is taken to be dissolved in the methylene chloride (DSM) of 10ml.) mixed in proportion with pure compounds liquid, It is respectively 1ng, 10ng, 100ng, 1 μ g, 10 μ g, 1mg/10mL that final concentration, which is made,.Then take 20 μ L drops in the filter paper of sterilizing again On (diameter is 1cm), after drying 90s, it is placed on the another side of two compartment culture dish, after sealing, as (12h light in incubator According to 20 DEG C, 50% relative humidity, 40W fluorescent lamp) it cultivates, biomass is counted after 14d: fresh weight.
As shown in table 7, methyl n-heptyl ketone, 2- ethyl -1- hexanol, n-tridecane the ketone various concentration under splitter plates system are handled There are the growth-promoting functions of varying strength to arabidopsis, methyl n-heptyl ketone is that 10ng/10 μ L, 2- ethyl -1- hexanol is 10 μ g/10 μ L, just Tridecane is 100ng/10 μ L.Show that the volatile materials that microbial bacterial agent JC65 is generated has preferable growth-promoting effect.
The growth-promoting effect of 7 volatile materials of table
Note: " --- " indicates that plant does not grow.

Claims (2)

1. application of the volatile materials that bacillus JC65 is generated in the growth for promoting vegetables, grain and oil crop, the bud Born of the same parents bacillus JC65 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium on December 15th, 2010 Kind deposit number is CGMCC NO.4475.
2. application of the volatile materials that bacillus JC65 is generated in the preparation that preparation promotes vegetables, grain and oil crop to grow, The bacillus JC65 is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on December 15th, 2010 Object center, culture presevation number are CGMCC NO.4475.
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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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Publication number Priority date Publication date Assignee Title
CN102174431A (en) * 2011-01-12 2011-09-07 南京农业大学 Bio-control strain for preventing and controlling bacterial fruit blotch of watermelon
CN102584359A (en) * 2011-12-14 2012-07-18 山东宝源生物有限公司 Composite biological agent and preparation method thereof
CN103563996A (en) * 2013-11-12 2014-02-12 无锡亚克生物科技有限公司 Application of bacillus subtilis G1 in plant growth promoting

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174431A (en) * 2011-01-12 2011-09-07 南京农业大学 Bio-control strain for preventing and controlling bacterial fruit blotch of watermelon
CN102584359A (en) * 2011-12-14 2012-07-18 山东宝源生物有限公司 Composite biological agent and preparation method thereof
CN103563996A (en) * 2013-11-12 2014-02-12 无锡亚克生物科技有限公司 Application of bacillus subtilis G1 in plant growth promoting

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