CN108546655A - A kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria and its application - Google Patents
A kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria and its application Download PDFInfo
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- CN108546655A CN108546655A CN201810303948.2A CN201810303948A CN108546655A CN 108546655 A CN108546655 A CN 108546655A CN 201810303948 A CN201810303948 A CN 201810303948A CN 108546655 A CN108546655 A CN 108546655A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The present invention relates to a kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria and its application, degradation bacteria is the rice pseudomonad that dwells, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.15325, and the deposit date is on 01 29th, 2018.It can be used for preparing microbial bacterial agent, a concentration of 2.0 3.0 × the 10 of bacterium solution8cfu/ml.The microbial bacterial agent its rhizospere competition in Field information is good and safe and reliable, and there is good degradation to the suppression harmful bacteria in melon crop root exudates without the infection ability that causes a disease to interculture, the anti-adversity ability for promoting melon crop plant improves the yield and fruit quality of melon crop；Preparation method is simple, and formula is reasonable, and stable effect is with obvious effects to the Auto toxicity for alleviating melon crop.
Description
Technical field
The present invention relates to microorganisms and continuous cropping obstacle to repair field, and in particular to a kind of melon crop rhizosphere suppression harmful bacteria drop
Solve bacterium and its application.
Background technology
Melon crop crop is the Important Economic crop of China's industrialized agriculture, and the planting conditions of protecting field in cycles cause
Great changes will take place for soil physico-chemical property, and production performance declines, and the pathogenic microorganism content in soil accumulates year by year, produces tight
The continuous cropping obstacle of weight.Numerous studies find that the Auto toxicity of melon crop is the major reason for generating continuous cropping obstacle, continuous to plant
Ecological environment of soil is caused to change, plant invalid body and the metabolin of root exudates and pathogenic microorganism are to plant growth
With toxic action, seed sprouting, plant strain growth, cell division, pathogen activity and edaphon structure are influenced, to draw
From toxic action, lead to the generation of continuous cropping obstacle.
Numerous studies show can be with effectively preventing by Auto toxicity by adding the means such as beneficial microbe or Antagonistic Fungi
With the generation of continuous cropping obstacle caused by root system soil-borne disease etc..Endophyte of plant survives in inside each tissue of plant and organ,
But so that host plant is shown apparent infection symptoms, and there is the microbial population of degradation to plant suppression harmful bacteria,
By being detached out of plant body, beneficial microbe is screened and is inoculated with to decompose harmful substance present in continuous cropping soil, overcome certainly
Toxic action, while the anti-adversity ability of plant is improved, and nutrition and living space are competed with harmful bacteria, reduce the number of soil surface characters
It measures and its Soil-sickness Problem in actual production is solved to reduce disease incidence of soil-borne disease to the infection ability of root system.
Probiotics at present applied to melon crop continuous cropping obstacle and Auto toxicity improvement is fewer, mostly uses fungicide greatly
Soil remediation is carried out with chemical fertilizer, leads to soil hardening acidification, nutrient imbalance, secondary salinization, is lacked safely and effectively
Biological bacteria administers the improvement and sustainable development for seriously constraining melon crop continuous cropping obstacle, it has also become melon crop agricultural production
One of bottleneck problem of urgent need to resolve.
Be below applicant retrieval with the relevant bibliography of the application
1. Cheng Ying, Bai Shoufa, facilitating effects China of the .2011. such as China muskmelon stubble decomposition objects to fusarium droop is respected in the village
Agronomy is notified to, 27 (8):217-221.
2. Li Yong, Long Qiliang, the .2014. microbial degradations such as fourth Bandung are in the relieving effect China of ginseng Auto toxicity
Medicine magazine, 39 (15):2868-2872.
3. Zhao Ya is intelligent, Yuan Yunyun, the optimization peanuts of the .2015. peanut Allelochemical degradation bacterium fermentation parameters such as Du Haiyan
Report, 44 (4) 31-36.
4. Liu Zhao armies, the separation of the .2007. wild soybean rhizosphere microorganisms such as Wang Deguo, Li Tie and its alleviation Soybean Cropping
Research .26 (2) 176-180. of obstacle
Invention content
In order to make up above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of melon crop rhizosphere suppression harmful bacterias
Degradation bacteria and its application solve melon crop Soil-sickness Problem, play rush for the suppression harmful bacteria for melon crop rhizosphere of degrading
Into the purpose of plant strain growth and degradation Auto toxicity.
It is a further object of the invention to provide a kind of preparations of the microbial bacterial agent of degradation melon crop Auto toxicity
Method is dwelt rice pseudomonad (Pseudomonas oryzihabitans) and mineral nutrition by a kind of melon crop endogenetic bacteria
Element is mixed by proportion optimizing, and the purpose is to melon crop suppression harmful bacterias of degrading, and enhance the anti-adversity ability of plant, is reduced
The generation and harm of suppression harmful bacteria.
The purpose of the present invention is what is realized by following technical solution：A kind of melon crop rhizosphere suppression harmful bacteria degradation
Bacterium and its application, are characterized in that：
A kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria：The Classification And Nomenclature of the bacterial strain is the rice pseudomonad that dwells
(Pseudomonas oryzihabitans), depositary institution：In China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, deposit number CGMCC
No.15325, the deposit date is on 01 29th, 2018.
The bacterium colony and thalline of the bacterial strain are characterized as：The suppression harmful bacteria degradation bacteria is in NA culture mediums (yeast extract 1g；Beef extract
3g；Peptone 5g；Sucrose 10g；Agar 18g；Distilled water 1000ml；121 DEG C sterilizing 30min) on culture 48h after bacterium colony it is smooth
Or wrinkle, neat in edge or irregular, plano-convex is faint yellow, and colony diameter 2mm after 30 DEG C of culture 2d, thalline is rod-shaped, flagellum fortune
It is dynamic, do not produce gemma, Gram-negative.
The bacterial strain physiological and biochemical property is：Oxidase test is negative, catalase test is positive, methyl red test is negative, forms sediment
Powder hydrolysis experiment feminine gender, gelatin hydrolysis negative, the nitrate reduction test positive, H2S negatives, malonate utilize examination
Testing positive, citrate, using experiment, positive, glucose utilization tests the positive, lactose utilization negative, galactose utilization try
It is positive using experiment to test positive, maltose.
The optimum culture medium for rice pseudomonad strain of dwelling is LB culture mediums, and formula includes that tryptone 10g/L, yeast carry
It is the 2% of culture medium weight to take object 5g/L, sodium chloride (NaCl) 10g/L, inoculum concentration, and fermentation temperature is 28 DEG C, and mixing speed is
120rpm, fermentation time are 5 days.
The microbial inoculum can be used for preparing microbial bacterial agent；A concentration of 2.0-3.0 × 10 of bacterium solution8cfu/ml。
The application method of microbial inoculum：The method of administration spreaded manuer in holes with pouring root, every seedling microbial inoculum are used in the transplanting time of melon crop
Dosage is 250ml.
As the preferred embodiment of embodiment, mineral nutrient element, including potassium dihydrogen phosphate, sulphur is added in the microbial bacterial agent
Sour magnesium, calcium sulfate, molybdic acid, manganese sulfate, ferrous sulfate, zinc sulfate, active ingredient are：
Beneficial effects of the present invention：In Field information, its rhizospere competition is good and safely may be used for the microbial bacterial agent
Lean on, and to interculture (vegetables such as crowndaisy chrysanthemum, rape) without cause a disease infection ability in melon crop root exudates from
Noxious material has good degradation, promotes the anti-adversity ability of melon crop plant, improves yield and the fruit of melon crop
Real quality；Preparation method is simple, and formula is reasonable, and stable effect is with obvious effects to the Auto toxicity for alleviating melon crop.
Specific implementation mode
1. material and method
1.1 culture medium for detaching the rice pseudomonad that dwells is minimal medium, and formula includes MgSO4·7H2O
0.2g/L, CaCl2·2H2O 0.02g/L, FeCl30.05g/L, KH2PO41.0g/L, K2HPO41.0g/L, NH4NO3
1.0g/L, cholesterol 0.3g/L, agar 17g/L are respectively with concentration for the treatment of for 200 μ gmL-1P-hydroxybenzoic acid, benzoic acid
With cinnamic acid as sole carbon source, adjust pH value to neutrality.
1.2 method for detaching the rice pseudomonad that dwells：It is cut into segment after taking muskmelon rhizome intersection flowing water to rinse, 75%
Alcohol impregnates 1min, 0.1% mercuric chloride 30s, reuses rinsed with sterile water 4 times, grinds and be slurried in sterile mortar, lapping liquid is dilute
It is coated in minimal medium after releasing 3 times, cultivates 48h in 28 DEG C of incubators, obtain rice pseudomonad strain of dwelling.
Dwell rice pseudomonad (Pseudomonas oryzihabitans), depositary institution：Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Chinese Academy of Sciences microorganism is ground
Study carefully institute, strain number：20180101H16, deposit number are CGMCC No.15325, and the deposit date is on 01 29th, 2018.
1.3 prepare rice pseudomonad microbial inoculum of dwelling：Appropriate media for cultivating rice pseudomonad strain of dwelling and LB cultures
Base, formula include tryptone 10g/L, yeast extract 5g/L, sodium chloride (NaCl) 10g/L, and inoculum concentration is culture medium weight
2%, fermentation temperature be 28 DEG C, mixing speed 120rpm, fermentation time be 5 days, obtain bacteria suspension.
The preparation and application of the microbial bacterial agent of 1.4 degradation melon crop continuous cropping obstacles：Gained is dwelt rice pseudomonad bacterium
Suspension (a concentration of 2.0-3.0 × 108Cfu/ml mineral nutrient element, including potassium dihydrogen phosphate 100-150mg/L, sulfuric acid) is added
Magnesium 200-500mg/L, calcium sulfate 500-900mg/L, molybdic acid 0.5-1.0mg/L, manganese sulfate 15-30mg/L, ferrous sulfate 3-
5mg/L, zinc sulfate 5-10mg/L.100g muskmelon continuous cropping soil is mixed with microbial bacterial agent 50ml, and 30 are cultivated under the conditions of 25 DEG C of moisturizings
It；100g continuous croppings soil is mixed with 50ml sterile waters makees blank control；Each processing repeats three times.
Degradation detection method of 1.5 microbial bacterial agents to the phenol acid substance in soil：
(1) being placed in the centrifuge tube of 75mL for examination soil for 25g is taken, the 1molL- of 25mL is added into soil1's
NaOH solution, with being stood for 24 hours after whirlpool concussion instrument mixing, secondary Nikkei overturns well distributing rocker mixing 1h, ultrasonic oscillator concussion
30min detaches supernatant, using 12molL- after centrifugation1Hydrochloric acid adjust soil liquid pH value to 2.5, centrifuged after standing 2h
Humic acid is removed, 4 DEG C of separation supernatant is preserved to prepare to analyze and be measured.
(2) phenolic acid HPLC separation and identification
The preparation of standard solution：Precise 10mg P-hydroxybenzoic acid, vanillic aldehyde, syringic acid, vanillic aldehyde, coumaric acid,
Dibutyl phthalate, benzoic acid, cinnamic acid (being provided by the Shanghai bio tech ltd sigma) are respectively implanted 100mL
In volumetric flask, 100mL is dissolved and is settled to methanol, then the 100 μ gmL- of concentration of each phenolic acid of the standard solution1。
(3) chromatographic condition：The measurement of different types of phenolic acid is using HPLC methods (Agilent LC 1100, U.S. in soil
State).Impurity present in the filtering with microporous membrane removal solution with 0.22 μm is had to pass through before sample feeding, chromatography column is
SB-C18 (Agilent 4.6mm × 250mm), if flow velocity 1mLmin-1, 25 DEG C of column temperature, sample detection wavelength 280nm, every time
Sample size is 20 μ L, mobile phase A：Methanol (chromatographic grade) and Mobile phase B：The acetic acid aqueous solution (chromatographic grade) of pH=2.8, sample
Separation achievees the purpose that separation by the method for gradient elution and shortens phenolic acid retention time.Elution requirement B：70%
(0min) → 50% (15min) → 30% (16min) → 0% (30min) → end (30min).Determined using retention time
Property, it is quantified using peak area external standard method.
(4) data processing and statistical analysis
Often experiment is repeated 3 times, for statistical analysis to data using spss 17.0, and significant difference is analyzed between each processing
It is analyzed using Duncan methods.
Phenolic acid content R (mgkg in soil-1) calculation formula it is as follows：R(mg·kg-1)=
SSample×VSample×CMark/SSample× m (something phenolic contents in R- samples；SSampleSample peak area；VSampleSample constant volume body
Product, mL；CMarkConcentration of standard solution, mgL-1；SMarkStandard solution peak area；M- example weights, g.)
The calculation formula of degradation rate：Degradation rate=control group phenolic content-processing group phenolic content/control group phenolic content
×100
1.6 microbial bacterial agents prevent the field trial of Muskmelon Fusarium wilt
For trying melon variety：Especially big super early sugared king (Heilungkiang Jing Feng new and high technologies development corporation, Ltd.).
Test site：Agricultural University Of Shenyang proving ground.
The Muskmelon Planting previous moon carries out site preparation and fertilization, is colonized the 3-4 months, and field planting is spreaded manuer in holes 15 centimetres deeply, and melon seedling enters hole
After make on melon Tu Tuo 2-3 centimetres away from furrow face, around enclosed with fine earth, be colonized water in be added, microbial bacterial agent root irrigation, bacterium solution
A concentration of 2.0-3.0 × 108cfu/ml。
If not using microbial bacterial agent root irrigation for control, first batch, the phase investigation of yielding positive results of second batch of muskmelon it is withered
Sick incidence, picking time investigate yield, i.e., after field, muskmelon is beared fruit after maturation, to the melon fruit under each treatment conditions into
Row weighs and records data, calculates the melon yield under the conditions of different disposal.
Incidence (%)=morbidity total strain number/total strain number × 100
Disease index (%)=∑ (diseased plant numbers at different levels × typical value at different levels)/investigation total strain number × superlative degree typical value ×
100
2 results and analysis
Microbial bacterial agent to the degradation of the phenol acid substance in muskmelon continuous cropping soil the result shows that, the microbial inoculum is to muskmelon point
Main phenolic acid in secretion has excellent degradation, wherein the degradation to cinnamic acid is best (being shown in Table 1).
Degradation of 1 microbial bacterial agent of table to the phenol acid substance in muskmelon continuous cropping soil
Field experiment result of study shows (table 2) that external source, which applies microbial bacterial agent, can be conducive to the growth and production of enhancing plant
The raising of amount can be conducive to the raising of the growth and yield of enhancing plant, reduce the generation of Muskmelon Fusarium wilt.
Effect of 2 microbial bacterial agent of table to muskmelon plant height, yield and incidence
Claims (3)
1. a kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria and its application, it is characterized in that：
A kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria：The Classification And Nomenclature of the bacterial strain is the rice pseudomonad that dwells（Pseudomonas oryzihabitans）, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, address are Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No. 15325, are protected
It is on 01 29th, 2018 to hide the date；
The bacterium colony and thalline of the bacterial strain are characterized as：The suppression harmful bacteria degradation bacteria is in NA culture mediums（Yeast extract 1g；Beef extract 3g；
Peptone 5g；Sucrose 10g；Agar 18g；Distilled water 1000ml；121 DEG C of sterilizing 30min）After upper culture 48h bacterium colony it is smooth or
Wrinkle, neat in edge or irregular, plano-convex is faint yellow, and colony diameter 2mm after 30 DEG C of culture 2d, thalline is rod-shaped, flagellum fortune
It is dynamic, do not produce gemma, Gram-negative；
The bacterial strain physiological and biochemical property is：Oxidase test is negative, catalase test is positive, methyl red test is negative, starch water
Solve negative, gelatin hydrolysis negative, the nitrate reduction test positive, H2S negatives, malonate utilization test sun
Property, citrate is positive using experiment, glucose utilization experiment positive, the experiment of lactose utilization negative, galactose utilization are positive
Property, maltose it is positive using experiment；
The optimum culture medium for rice pseudomonad strain of dwelling is LB culture mediums, and formula includes tryptone 10g/L, yeast extract
5g/L, sodium chloride（NaCl）10g/L, inoculum concentration are the 2% of culture medium weight, and fermentation temperature is 28 DEG C, and mixing speed is
120rpm, fermentation time are 5 days；
The rice pseudomonad that dwells can be used for preparing microbial bacterial agent, a concentration of 2.0-3.0 × 10 of bacterium solution8cfu/ml。
2. a kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria and its application, it is characterized in that：The rice pseudomonad microbial inoculum of dwelling
Application method：It is 250 ml to use the method for administration spreaded manuer in holes with pouring root, every seedling microbial inoculum dosage in the transplanting time of melon crop.
3. a kind of melon crop rhizosphere suppression harmful bacteria degradation bacteria according to claim 1 and its application, it is characterized in that：It is described
Microbial bacterial agent be added mineral nutrient element, including potassium dihydrogen phosphate, magnesium sulfate, calcium sulfate, molybdic acid, manganese sulfate, ferrous sulfate,
Zinc sulfate, active ingredient are：
Potassium dihydrogen sulfate 100-150 mg/L；
Magnesium sulfate 200-500 mg/L；
Calcium sulfate 500-900 mg/L；
Molybdic acid 0.5-1.0 mg/L；
Manganese sulfate 15-30 mg/L；
Ferrous sulfate 3-5 mg/L；
Zinc sulfate 5-10 mg/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109997875A (en) * | 2019-04-23 | 2019-07-12 | 山东中创亿丰肥料集团有限公司 | A kind of microbial bacterial agent and preparation method thereof with pest-resistant sterilizing function |
| CN110041139A (en) * | 2019-05-30 | 2019-07-23 | 沈阳农业大学 | A kind of agent of biological bacteria sandwich tape and application method for continuous cropping soil reparation |
| CN116326412A (en) * | 2022-05-18 | 2023-06-27 | 北京农学院 | Method for regulating and controlling rhizosphere activating effect of apples |
-
2018
- 2018-04-03 CN CN201810303948.2A patent/CN108546655A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109997875A (en) * | 2019-04-23 | 2019-07-12 | 山东中创亿丰肥料集团有限公司 | A kind of microbial bacterial agent and preparation method thereof with pest-resistant sterilizing function |
| CN110041139A (en) * | 2019-05-30 | 2019-07-23 | 沈阳农业大学 | A kind of agent of biological bacteria sandwich tape and application method for continuous cropping soil reparation |
| CN110041139B (en) * | 2019-05-30 | 2021-11-30 | 沈阳农业大学 | Biological bacterium multilayer tape agent for continuous cropping soil remediation and use method |
| CN116326412A (en) * | 2022-05-18 | 2023-06-27 | 北京农学院 | Method for regulating and controlling rhizosphere activating effect of apples |
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Application publication date: 20180918 |