CN105462885B - One plant of phenolic acid degradation bacteria and its biological organic fertilizer and application - Google Patents
One plant of phenolic acid degradation bacteria and its biological organic fertilizer and application Download PDFInfo
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- CN105462885B CN105462885B CN201510966625.8A CN201510966625A CN105462885B CN 105462885 B CN105462885 B CN 105462885B CN 201510966625 A CN201510966625 A CN 201510966625A CN 105462885 B CN105462885 B CN 105462885B
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Abstract
The present invention relates to one plant of phenolic acid degradation bacteria and its biological organic fertilizer and applications.One plant of Novosphingobium (Novosphingobium Subarcticum) AMCC100069, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address are preserved on July 31st, 2015：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, culture presevation number is CGMCC No.11163.Novosphingobium (Novosphingobium Subarcticum) AMCC100069 of the present invention has notable degradation effect to phenolic acids such as the phloridzins that causes continuous cropping obstacle of apple, there is extremely significant facilitation to fruit tree plant strain growth, and continuous cropping obstacle of apple problem in production can be effectively relieved.
Description
Technical field
The present invention relates to one plant of phenolic acid degradation bacteria and its biological organic fertilizer and applications, belong to microbial technique neck
Domain.
Background technology
China's apple production has leapt to the first in the world at present, but as replacement is newly set in existing orchard into veteran successively
Phase, fruit tree are also faced with serious Soil-sickness Problem.It is mainly shown as that planting shoot (seedling) growth is weak, plant downgrades internode
The features such as shortening, blade is small, and root system development is bad, easily contaminates rot disease, and tree body resistance is low.
The allelopathic that plant is generated by approach such as the decompositions of the volatilization of cauline leaf, leaching, the secretion of root system and plant stubble
Substance be influence rhizospheric environment key problem, research find the phenolic acid in root exudates be report at present it is main
Allelochemical, including benzoic acid, chlorogenic acid, caffeic acid, P-hydroxybenzoic acid, phloroglucin, syringic acid, cinnamic acid, phloridzin
Deng.Through investigation, the phenolic acid in the continuous cropping soil of Chinese loess plateau apple producing region be mainly phloridzin, chlorogenic acid and
Benzoic acid (Song Weihong etc., 2012)；Main phenolic acid in the continuous cropping soil of Chinese Bohai Sea Gulf apple producing region may be root skin
Glycosides, pyrogallic acid and chlorogenic acid (Sun Haibing etc., 2011).The phloridzin of one of an important factor for causing orchard continuous cropping obstacle
Mainly it is discharged into soil by the decomposition of the secretion of root system and residuum.Cui Xiaojing (2010) researchs find soil in orchard
The content of the phloridzin of earth is related with the age of tree of fruit tree in orchard, and the content for increasing phloridzin with the age of tree also increases.It is existing to grind
Study carefully and shows that the continuous cropping obstacle of fruit tree and the accumulated concentrations of fruit tree rhizosphere phloridzin have prodigious correlation, the phloridzin of high concentration
Accumulation can cause slow-growing lower stubble apple tree, yield decline, quality to become of inferior quality.
It is increasingly closed with the research for alleviating plant continuous cropping obstacle using phenolic acid in function stem degradation environment
Note, some indigenous beneficial microbes can be grown by sole carbon source and the energy of phenolic acid, to reach biodegradable mesh
's.Therefore it is gained great popularity with microbial degradation phenolic acid to alleviate continuous cropping obstacle scheme.
Chinese patent literature CN104099272A (application number 201410325083.1) discloses one plant of phenolic acid class allelopathic object
The preparation and application of matter degradation bacteria and its microbial inoculum；The BJS-1-3 bacterial strains that the invention is related to derive from the perennial willow rhizosphere in Tai'an
Soil is obtained through being manually enriched with, screening and purifying；Bacterial strain para Toluic Acid has stronger degradation capability；It can be utilized respectively
The phenolic acid class such as ferulic acid, P-hydroxybenzoic acid, vanillic aldehyde, cinnamic acid, phloridzin allelochemical is as sole carbon source growth and breeding；
It can be colonized in willow, apple, cucumber rhizosphere.The microbial inoculum prepared using the bacterial strain can be prevented effectively because of phenolic acid class allelopathic object
The continuous cropping obstacle of the plants such as willow, apple, cucumber caused by matter accumulates.
Invention content
The Novosphingobium that can drop phenolic acid in view of the deficiencies of the prior art, the present invention provides one plant and its micro-
Bio-organic fertilizer can be applied to alleviate apple tree continuous cropping obstacle.
One plant of Novosphingobium (Novosphingobium Subarcticum) AMCC100069, July 31 in 2015
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, culture presevation number is CGMCC No.11163.
In Novosphingobium (Novosphingobium Subarcticum) AMCC100069 is seen from Qixia City of Shandong Province
The town villages Shan Houbo apple is replanted isolated in Orchard Soil, and Main Biological is：Bacterium colony smaller round, yellow is glossy,
The smooth moistening in surface, bacterium colony center semi-cylindrical hill, not chromogenic element, Gram-negative bacteria G-, indole test, V-P experiments, nitric acid
Salt restores and gelatin liquefaction is feminine gender, methyl red test, catalase test, citrate utilization, 3- ketone groups lactose, phenylalanine
Deaminase is the positive.
The bacterium there is stronger degradation capability, liquid submerged culture 12h to be reached to the degradation rate of phloridzin phenolic acid
81.78%, it cultivates for 24 hours to phloridzin degradation rate up to 96.88%；The focusability gallic acid acid of liquid submerged culture 12h, benzene first
Acid, chlorogenic acid, caffeic acid, P-hydroxybenzoic acid, phloroglucin, syringic acid degradation rate be 85.0%, 33.0%, 27.0%,
24.0%, 20.0%, 18.0%, 15.0%.
Above-mentioned Novosphingobium (Novosphingobium Subarcticum) AMCC100069 is preparing alleviation fruit tree
Application in the biological organic fertilizer of continuous cropping obstacle.
Above application includes the following steps：
(1) after being activated to Novosphingobium (Novosphingobium Subarcticum) AMCC100069, through expanding
After culture, it is 0.5 × 10 that viable bacteria concentration, which is made,10~1.0 × 1010The bacterium solution of cfu/ml；
(2) by bacterium solution made from step (1) and organic fertilizer according to volume mass ratio (40~50):1 ratio mixing is equal
Even, volume mass compares unit：Living bacteria count 2~4 × 10 is made in L/t8The biological organic fertilizer of cfu/g.
According to currently preferred, the organic fertilizer in the step (2) is to meet NY 525 (organic fertilizer) standard to want
The organic fertilizer asked.
According to currently preferred, the step of activation in the step (1), is as follows：
By Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains inoculation to work
Change in culture medium and carry out activation culture for 24 hours, temperature is 28~30 DEG C, speed of agitator is 180~200r/min；
Every liter of component of activation medium is as follows：
Phloridzin 0.96g, MgSO4·7H2O 0.2g、NH4H2PO41g, KCl 0.2g, water are settled to 1000mL, pH7.0,
121 DEG C of sterilizing 20min.
According to currently preferred, the step of expansion culture in the step (1), is as follows：
Novosphingobium (Novosphingobium Subarcticum) AMCC100069 after activation is pressed into volume hundred
The inoculum concentration than 2% is divided to be inoculated into fermentation medium, in 28~30 DEG C of temperature, the item that speed of agitator is 180~220r/min
Under part, fermented and cultured is carried out；
Every liter of component of fermentation medium is as follows：
Phloridzin 0.96g, MgSO4·7H2O 0.2g、NH4H2PO41g, KCl 0.2g, water are settled to 1000mL, pH7.0,
121 DEG C of sterilizing 20min.
Advantageous effect
1, Novosphingobium (Novosphingobium Subarcticum) AMCC100069 of the present invention is to causing
The phenolic acids such as the phloridzin of continuous cropping obstacle of apple have notable degradation effect, drops of the liquid submerged culture 12h to phloridzin
Solution rate is cultivated up to 81.78% for 24 hours to phloridzin degradation rate up to 96.88%；The focusability gallic acids of liquid submerged culture 12h
Acid, benzoic acid, chlorogenic acid, caffeic acid, P-hydroxybenzoic acid, phloroglucin, syringic acid degradation rate be 85.0%, 33.0%,
27.0%, 24.0%, 20.0%, 18.0%, 15.0%；
2, the present invention is raw made from Novosphingobium (Novosphingobium Subarcticum) AMCC100069
Organic fertilizer, the function stems of the phenolic acids such as the efficient degradation phloridzin content in bio-organic fertilizer reaches 2~4 ×
108Cfu/g effectively can be colonized and play in the soil effect；Organic fertilizer can also provide abundant for fruit tree growth simultaneously
Nutrition, and promotion functions bacterium plays effect, improves the effect of the soil texture.
3, the experimental results showed that, it is right using after biological organic fertilizer of the present invention in the serious soil of continuous cropping obstacle
Plant strain growth has extremely significant facilitation, and biomass increases by 158.40% (P compared with the control<0.01), apple connects
It is effectively relieved as obstacle phenomenon.
Description of the drawings
The colonial morphology of Fig. 1 Novosphingobiums (Novosphingobium Subarcticum) AMCC100069 bacterial strains
And microscopic morphology；
Fig. 2 Novosphingobiums (Novosphingobium Subarcticum) AMCC100069 bacterial strains are to phloridzin
Degradation capability and thalli growth relationship；
Specific implementation mode
Technical scheme of the present invention is described further with reference to embodiment, but institute's protection domain of the present invention is not limited to
This.
Experimental method in embodiment is unless otherwise specified this field conventional method.It is tested used in embodiment
Material is unless otherwise specified ordinary commercial products.Quantitative test in following embodiment, is respectively provided with three repeated experiments,
Results are averaged.
Organic fertilizer described in embodiment is purchased from green source organic fertilizer Co., Ltd of Yantai City；
Phloridzin is purchased from Nanjing Zelang Pharmaceutical Technology Inc..
Separation, identification and the preservation of 1 bacterial strain of embodiment
One, the separation of Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains
Sampling time：In September, 2011
Sampling position：The Qixia City of Shandong Province towns the Guan Li villages Shan Houbo
It is acquired from Circum-Bohai Sea one with apple continuous cropping garden rhizosphere and has separated 74 plants in soil sample with degradation apple root skin
The bacterial strain of the phenolic acids ability such as glycosides, wherein one plant of name are AMCC100069 bacterial strains.
Two, the identification of Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains
The colonial morphology and microscopic morphology of AMCC100069 bacterial strains are shown in Fig. 1.
Bacterium colony circle is smaller, and yellow is glossy, the smooth moistening in surface, bacterium colony center semi-cylindrical hill, not chromogenic element, and leather is blue
Family name's negative bacterium G-, indole test, V-P experiments, nitrate reduction and gelatin liquefaction are feminine gender, methyl red test, catalase test,
Citrate utilization, 3- ketone groups lactose, phenylalanine deaminase are the positive.
The 16SrDNA sequences of AMCC100069 bacterial strains are as shown in the sequence 1 of sequence table.
Three, the preservation of Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains
It is identified, AMCC100069 bacterial strain categories Novosphingobium (Novosphingobium Subarcticum), in
On July 31st, 2015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address：North
The institute 3 of the Chaoyang Districts Jing Shi North Star West Road 1, deposit number are CGMCC No.11163.
The degradation of embodiment 2, Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains
Ability indoor measurement
One, the degradation capability of Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains
Indoor measurement
By Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains inoculation to work
Change in culture medium and carry out activation culture for 24 hours, temperature is 28~30 DEG C, speed of agitator is 180~200r/min；
Every liter of component of activation medium is as follows：
Phloridzin 0.96g, MgSO47H2O 0.2g、NH4H2PO41g, KCl 0.2g, water are settled to 1000mL,
PH7.0,121 DEG C of sterilizing 20min；
Novosphingobium (Novosphingobium Subarcticum) AMCC100069 after activation is pressed into 2% (matter
Amount percentage) inoculum concentration be inoculated into 2mmol/L phloridzin fluid nutrient medium (culture medium form and quality：Phloridzin
0.96g、MgSO4.7H2O 0.2g、NH4H2PO41g, KCl 0.2g, distilled water are settled to 1000mL, 7.0,121 DEG C of sterilizings of pH
20min), 30 DEG C, 160r/min shaking table cultures and measure phloridzin degradation rate.
The result shows that：Novosphingobium (Novosphingobium Subarcticum) AMCC100069 after culture 12h
Bacterial strain, up to 81.78%, cultivates rear Novosphingobium (Novosphingobium for 24 hours to the degradation rate of phloridzin
Subarcticum) AMCC100069 bacterial strains to phloridzin degradation rate up to 96.88%.There is significant degradation to imitate phloridzin
Fruit.
Novosphingobium (Novosphingobium Subarcticum) AMCC100069 after activation is pressed into 2% (matter
Amount percentage) inoculum concentration be inoculated into chlorogenic acid containing 2mmol/L, phloroglucin, pyrogallic acid, P-hydroxybenzoic acid,
(culture medium forms and quality for the different phenolic acid fluid nutrient medium of syringic acid, caffeic acid, seven kinds of benzoic acid：Phenolic acid
0.96g、MgSO4.7H2O 0.2g、NH4H2PO41g, KCl 0.2g, distilled water are settled to 1000mL, 7.0,121 DEG C of sterilizings of pH
20min), 30 DEG C, measure to different phenolic acid degradation rates after 160r/min shaking table cultures 12h.The result shows that the bacterium pair seven
Kind phenolic acid all has certain degradation capability.
Table 1AMCC100069 bacterial strains compose (12h) to the degradation of different phenolic acid
Two, Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains are to phloridzin
Degradation curve
Novosphingobium (Novosphingobium Subarcticum) AMCC100069 after activation is pressed into 2% (matter
Amount percentage) inoculum concentration be inoculated into 2mmol/L phloridzin fluid nutrient medium (culture medium form and quality：Phloridzin
0.96g、MgSO4.7H2O 0.2g、NH4H2PO41g, KCl 0.2g, distilled water are settled to 1000mL, 7.0,121 DEG C of sterilizings of pH
20min), 30 DEG C, 160r/min shaking table culture 20h take a sample 8000r/min centrifugations 10min to collect zymotic fluid every 4h.With
Ethyl acetate is with zymotic fluid (v:v)1:Phloridzin in 1 ratio extractive fermentation liquid takes organic phase to carry out rotary evaporation finally molten
In the methanol of 2mL, 13000r/min centrifuges 10min deproteinizeds, with 0.45 μm of membrane filtration before loading, then with efficient liquid
Chromatography (C18 columns) measures the residual volume of phloridzin, calculates the degradation rate of phloridzin and measures thalli growth situation.
Testing conditions are：Detection wavelength is 270nm, and 30 DEG C of column temperature, sample size is 10 μ L, the acetic acid of volume fraction 0.6%
Aqueous solution (95%) elutes 40min with acetonitrile (5%) with flow velocity 1mL/min.
The result shows that：With the increase of processing time, the degradation rate of the phloridzin of processing group constantly increases, and in new sheath ammonia
It is between alcohol bacillus (Novosphingobium Subarcticum) AMCC100069 bacterial strain thalli growth amounts and phloridzin degradation rate
Positive correlation (Fig. 2).
Embodiment 3, the preparation of microbial organic fertilizer and pot experiment
One, the preparation of microbial organic fertilizer
1. Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strain inoculations are arrived
Activation culture is carried out in activation medium for 24 hours, and temperature is 28~30 DEG C, speed of agitator is 180~200r/min；
Every liter of component of activation medium is as follows：
Phloridzin 0.96g, MgSO47H2O 0.2g、NH4H2PO41g, KCl 0.2g, water are settled to 1000mL, pH
7.0,121 DEG C of sterilizing 20min；
Novosphingobium (Novosphingobium Subarcticum) AMCC100069 after activation is pressed into 2% (matter
Amount percentage) inoculum concentration be inoculated into fermentation medium, fermentation temperature is 28~30 DEG C, speed of agitator is 180~220r/
Min makes thalline content in zymotic fluid be no less than 1.0 × 1010cfu/g；
Every liter of component of fermentation medium is as follows：
Phloridzin 0.96g, MgSO4.7H2O 0.2g、NH4H2PO41g, KCl 0.2g, distilled water 1000mL, pH 7.0,
121 DEG C of sterilizing 20min.
2. by Novosphingobium described in 40L (Novosphingobium Subarcticum) AMCC100069 bacterial strains
Zymotic fluid is uniformly mixed with 1 ton of organic fertilizer, and living bacteria count 2~4 × 10 is made8The biological organic fertilizer of cfu/g.
The organic fertilizer can be the organic fertilizer for meeting the requirement of NY 525 (organic fertilizer) standard.The organic fertilizer specifically may be used
For the green rosy clouds organic fertilizer of the green source organic fertilizer Co., Ltd production in Yantai City.Obtain living bacteria count in the bio-organic fertilizer
It is 2~4 × 108cfu/g；
Two, pot experiment
Pot experiment is carried out in country of Shandong Agricultural University apple engineering science and technology research center base.By Novosphingobium
Phloridzin fluid nutrient medium (training of (Novosphingobium Subarcticum) the AMCC100069 inoculations to 2mmol/L
Support base composition and quality：Phloridzin 0.96g, MgSO4.7H2O 0.2g、NH4H2PO41g, KCl 0.2g, distilled water are settled to
7.0,121 DEG C of 1000mL, pH sterilizing 20min) in 30 DEG C, 160r/min cultivate 12 hours, thalline were collected by centrifugation, then uses nothing
The resuspension of bacterium water washing makes a concentration of the 1 × 10 of its bacteria suspension8CFU/mL, it is spare.
The Malus hupenensis Seedlings for having grown 4~6 true leaves are taken to carry out potting, routinely management is irrigated and removed and is miscellaneous
Grass, cultivation detect each index after 5 months.Specific experiment design is as follows：
2 potted plant experiment of table designs
Note：- indicate to be not added with.
The result shows that：Compared with CK, T1 processing ground, underground part fresh weight reduce by about 31.24%, 43.41% (P respectively<
0.01), show that the phloridzin of high concentration has extremely significant inhibiting effect to plant strain growth；T2 processing ground, underground part fresh weight
Increase separately about 18.67%, 18.81% (P<0.01)；T3 processing on the ground, underground part fresh weight increase separately about 135.68,
93.87% (P<0.01).Show that Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterium are added
The processing and addition of liquid include the life of Novosphingobium (Novosphingobium Subarcticum) AMCC100069 bacterial strains
The processing of organic fertilizer can be effectively relieved all to having extremely significant facilitation to plant strain growth because high concentration phloridzin accumulates
It is tired and caused by continuous cropping obstacle of apple problem.(as shown in table 3)
The biomass of 3 different disposal plant of table
Note：Average data in the same row uses Multiple range test, the numerical value that all tail portions indicate different letters to indicate them
Between there were significant differences (lowercase P<0.05, difference reaches the level of signifiance；Capital P<0.01, difference reaches extremely notable water
It is flat).
Claims (6)
1. one plant of Novosphingobium（Novosphingobium Subarcticum）AMCC100069, on July 31st, 2015
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center（CGMCC）, address：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, culture presevation number is CGMCC No.11163.
2. Novosphingobium described in claim 1（Novosphingobium Subarcticum）It is prepared by AMCC100069
Alleviate the application in the biological organic fertilizer of apple tree continuous cropping obstacle.
3. application as claimed in claim 2, which is characterized in that include the following steps：
（1）To Novosphingobium（Novosphingobium Subarcticum）After AMCC100069 activation, cultivated through expanding
Afterwards, it is 0.5 × 10 that viable bacteria concentration, which is made,10~1.0 × 1010The bacterium solution of cfu/ml；
（2）By step（1）Bacterium solution obtained is with organic fertilizer according to volume mass ratio（40~50）:1 ratio is uniformly mixed, body
Product quality compares unit：Living bacteria count 2~4 × 10 is made in L/t8The biological organic fertilizer of cfu/g.
4. application as claimed in claim 3, which is characterized in that the step（2）In organic fertilizer be meet NY 525 mark
The organic fertilizer of alignment request.
5. application as claimed in claim 3, which is characterized in that the step（1）In activation the step of it is as follows：
By Novosphingobium（Novosphingobium Subarcticum）AMCC100069 inoculations are to activation medium
For 24 hours, temperature is 28~30 DEG C to middle progress activation culture, speed of agitator is 180~200r/min；
Every liter of component of activation medium is as follows：
Phloridzin 0.96 g, MgSO4·7H2O 0.2 g、NH4H2PO41 0.2 g of g, KCl, water are settled to 1000mL, pH
7.0。
6. application as claimed in claim 3, which is characterized in that the step（1）In expansion culture the step of it is as follows：
By the Novosphingobium after activation（Novosphingobium Subarcticum）AMCC100069 presses percent by volume
2% inoculum concentration is inoculated into fermentation medium, under conditions of 28~30 DEG C of temperature, speed of agitator are 180~220r/min,
Carry out fermented and cultured；
Every liter of component of fermentation medium is as follows：
Phloridzin 0.96 g, MgSO4·7H2O 0.2 g、NH4H2PO41 0.2 g of g, KCl, water are settled to 1000mL, pH
7.0。
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| CN111455015A (en) * | 2020-04-02 | 2020-07-28 | 山东省科学院生态研究所 | Rapid screening method of continuous cropping obstacle factor strains for degrading phenolic acid substances |
| CN112028708A (en) * | 2020-06-17 | 2020-12-04 | 内蒙古自治区农牧业科学院 | Compound fertilizer for removing continuous cropping obstacles of cucumber soil, preparation method thereof, diversity fertilization method and method for removing continuous cropping obstacles |
| CN115261255B (en) * | 2022-05-16 | 2023-06-16 | 西北农林科技大学 | Sphingobacterium and application thereof |
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| CN104099272A (en) * | 2014-07-09 | 2014-10-15 | 山东农业大学 | Phenolic acid type allelochemical degrading bacterium and preparation and application of microbial inoculum thereof |
| CN105062937A (en) * | 2015-09-24 | 2015-11-18 | 山东佐田氏生物科技有限公司 | Bacillus amyloliquefaciens strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099272A (en) * | 2014-07-09 | 2014-10-15 | 山东农业大学 | Phenolic acid type allelochemical degrading bacterium and preparation and application of microbial inoculum thereof |
| CN105062937A (en) * | 2015-09-24 | 2015-11-18 | 山东佐田氏生物科技有限公司 | Bacillus amyloliquefaciens strain and application thereof |
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| Title |
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| VLASTA ČATSKÁ等.Rhizosphere micro-organisms in relation to the apple replant problem.《Plant and Soil》.1982,第69卷187-197页. * |
| 果树连作障碍机理及控制途径;谢洪刚 等;《辽宁农业职业技术学院学报》;20080731;第10卷(第4期);6-8页 * |
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