CN104911108B - The preparation method and purposes of sea-buckthorn endogenetic fungus and its extract - Google Patents
The preparation method and purposes of sea-buckthorn endogenetic fungus and its extract Download PDFInfo
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Abstract
The present invention relates to an a kind of fungal strain in the wild seabuckthorn root in Changbai Mountain and the application on turfgrass drought tolerance is improved, the sea-buckthorn endogenetic fungus is Alternaria（Alternaria Sp）, deposit number is CGMCC NO.10117, preservation date 2014.12.11, depositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center.The concentration of mycelia extract is that nanogram level is other in the present invention, illustrates that the endophyte extract activity is very high, 0.5ng/ml concentration sprays turfgrass, it is possible to induces turfgrass to obtain preferable drought tolerance.
Description
Technical field
The invention belongs to microbial technology field, more particularly to a kind of one plant derived from the wild seabuckthorn root in Changbai Mountain is very
Bacterium and the application on turfgrass drought tolerance is improved.
Background technology
With the quickening of urban ecological construction process, requirement of the people to urban green space construction also increasingly improves.Lawn
Careless plant is used widely as a kind of excellent greening species in the construction of urban green space.But due to China's territory
Vast, geographic climate is complicated, and same turfgrass often has completely different growth performance in different regions.Cold
Under cold, high temperature, arid, waterlogging and the poor environment such as saline and alkaline, turfgrass plant shows the suffertibility of certain limit.But
When environment stress has exceeded the tolerance range of turfgrass, the enzyme in turfgrass body is destroyed, and normal metabolism is obstructed,
Cause termination even cell death of growing.By various artificial cultivation managements (such as rational fertilising, irrigating) or first
Enter the utilization of technological means (such as heat-resisting, cold-resistant, Resistant Gene imports), can to greatest extent slow down or reduce adverse circumstance
Injury to turfgrass.
At present, to improve Turfgrass Growth and strengthen resistance compared in the research of lawn using Phytochemistry regulation measure
An active field, it is careless slightly in application using upper also variant because growth regulator species is various
Easily cause unnecessary harm.
The content of the invention
The present invention is directed to above-mentioned problems of the prior art, there is provided one kind is in the wild seabuckthorn root in Changbai Mountain
One plant of endogenetic fungus, extract the activity that can improve turfgrass drought tolerance and yield in the mycelia manually cultivated from the fungi
Composition.
Technical scheme is as follows：
Sea-buckthorn endogenetic fungus is Alternaria（Alternaria Sp）, deposit number is CGMCC NO.10117, preservation day
Phase is 2014.12.11, depositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Preservation list
Bit address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The production technology of sea-buckthorn Endophytic Fungal Hyphae extract of the present invention comprises the following steps：
Step 1, the separation of sea-buckthorn endogenetic fungus：1. immediately treat, clean after the wild sea-buckthorn sample collection of health, then
Seabuckthorn root is cut into segment, with 75% alcohol rinse, 18 minutes are washed with 10% pasteurization liquid, finally with aseptic water washing 4 times；
2. the segment that the flushed seabuckthorn root of above-mentioned sterilization is aseptically respectively cut into 0.5cm length is inoculated in
On rose bengal medium flat board, after being placed in 25-30 DEG C of degree incubator culture 3-7 days, you can see the edge length that sample is cut through
Go out mycelia, separate, purify repeatedly through flat board；
Step 2, sea-buckthorn endogenetic fungus liquid fermentation and culture：
The fungi of above-mentioned steps 2. middle separation is connected in flat board PDA culture medium, 20-28 DEG C is cultivated 4-8 days, with punching
Device agar digs block and is inoculated in the 250mL conical flasks equipped with 50mLPDA, under 25-30 DEG C, 100-150r/min on rotary shaker
Culture is used as seed in 2-4 days, is inoculated into 10% volume in the 500mL triangular flasks equipped with 150mLPDA, in 25-30 DEG C, 100-
Cultivated 5 days on rotary shaker under 150r/min, terminate fermentation, be put into 4 DEG C of refrigerators and save backup；
Step 3, the preparation of sea-buckthorn endogenetic fungus extract：
It will be dried at 50-70 DEG C after above-mentioned mycelium washing, weigh, then crushed through high speed disintegrator, after crushing
Bacterium powder and 20wt% ethanol by volume 1:1 mixing extraction 20-28h, extracts 2-4 times, is mixed with magnetic stirring apparatus, ultrasound
Ripple shakes 0.8-1.5h, vacuum filtration, it is standby to collect filtrate.
The fungi separated in described step 2 is connected in flat board PDA culture medium, and 25 DEG C are cultivated 6 days；In 28 DEG C, 120r/
Cultivated 3 days on rotary shaker under min and be used as seed；In 28 DEG C, cultivated 5 days on rotary shaker under 120r/min, terminate fermentation.
In described step 3, dried after mycelium is washed at 60 DEG C, extract 24h, extracted 3 times, ultrasonic oscillation 1h.
The extract of obtained sea-buckthorn endogenetic fungus is applied to promote turfgrass to improve resistance and improve yield.
Compared with the mode of prior art, advantages of the present invention effect is as follows：
1st, the endophyte that can improve turfgrass drought resistance function that the present invention separates from wild sea-buckthorn, the bacterium is rod method
Bacterium, analyzed from its activity of the mycelia extract of the bacterium：Because its activity is very high, thus it is speculated that be not that existing known five major classes swash
Element.And before this also not on from Endophytes from Hippophae rod method（Alternaria Sp）Produce induction turfgrass and improve drought resistance
Material report, especially these active components can influence root activity, chlorophyll content, soluble sugar content, dried meat ammonia
The physiological and biochemical index such as acid content and mda content induces the drought-enduring function of turfgrass.The sea-buckthorn endogenetic fungus of the present invention
The drought resistance of plant can be improved with turfgrass Symbiotic evolution, endophyte of plant is formed during being evolved with plant symbiosis
Reciprocal mechanism, these mechanism are progressively clear by the analysis meeting to active component caused by endophyte.By manually producing
A large amount of endophyte of plant are come to obtain the active material of useful crop plants growth be the environment-friendly industry of an economical and effective.
2nd, absolute dosage is few.The concentration of mycelia extract is that nanogram level is other in the present invention, illustrates the endophyte
Extract activity is very high, and 0.5ng/ml concentration sprays turfgrass, it is possible to induces turfgrass to obtain preferable drought tolerance.
Brief description of the drawings
Influence schematic diagram of Fig. 1 drought stresses to turfgrass root activity.
Influence schematic diagram of Fig. 2 drought stresses to turfgrass chlorophyll content.
Fig. 3 drought stresses influence schematic diagram to turfgrass soluble sugar content.
Influence schematic diagram of Fig. 4 drought stresses to turfgrass proline content.
Influence schematic diagram of Fig. 5 drought stresses to turfgrass mda content.
Fig. 6 a fungies CBS1 colonial morphology schematic diagram.
Fig. 6 b fungies CBS1 spore shape schematic diagram.
Fig. 7 a are CBS1 fungi DNA amplification sequence charts.
Fig. 7 b are that chadogram schematic diagram is developed in the systematization that CBS1 is established.
Fig. 8 CBS1 strain mycelia extract production process flow charts.
Embodiment
Embodiment 1
One plant of inducible turfgrass raising drought resistance bacterium CBS1 is separated to from picking up from wild sea-buckthorn endogenetic fungus sample.Should
Strain growth is fast, and turfgrass can be made to improve its drought resistance under conditions of simulating drought.Reflected according to its morphology and genetic analysis
It is Alternaria to determine CBS1 bacterial strains（Alternaria Sp）, deposit number is CGMCC NO.10117, and preservation date is
2014.12.11, depositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Depositary institution
Location：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The production technology of sea-buckthorn Endophytic Fungal Hyphae extract of the present invention comprises the following steps：
Step 1, the separation of sea-buckthorn endogenetic fungus：1. immediately treat, clean after the wild sea-buckthorn sample collection of health, then
Seabuckthorn root is cut into segment, with 75% alcohol rinse, 18 minutes are washed with 10% pasteurization liquid, finally with aseptic water washing 4 times；
2. the segment that the flushed seabuckthorn root of above-mentioned sterilization is aseptically respectively cut into 0.5cm length is inoculated in
On rose bengal medium flat board, after being placed in 28 DEG C of degree incubator cultures 5 days, you can see that the edge that sample is cut through grows bacterium
Silk, separates, purifies repeatedly through flat board；
Step 2, sea-buckthorn endogenetic fungus liquid fermentation and culture：
The fungi of above-mentioned steps 2. middle separation is connected in flat board PDA culture medium, 25 DEG C are cultivated 6 days, with card punch agar
Dig block and be inoculated in the 250mL conical flasks equipped with 50mLPDA, in 28 DEG C, 3 days are cultivated as kind on rotary shaker under 120r/min
Son, it is inoculated with the 500mL triangular flasks equipped with 150mLPDA with 10% volume, in 28 DEG C, is trained under 120r/min on rotary shaker
Support 5 days, terminate fermentation, be put into 4 DEG C of refrigerators and save backup；
Step 3, the preparation of sea-buckthorn endogenetic fungus extract：
It will be dried at 60 DEG C after above-mentioned mycelium washing, weigh, then crushed through high speed disintegrator, the bacterium powder after crushing
With 20wt% ethanol by volume 1:1 mixing extraction 24h, extracts 3 times, is mixed with magnetic stirring apparatus, ultrasonic oscillation 1h,
Vacuum filtration, it is standby to collect filtrate.
The extract of obtained sea-buckthorn endogenetic fungus is applied to promote turfgrass to improve resistance and improve yield.
Embodiment 2
The fungi separated in described step 2 is connected in flat board PDA culture medium, and 20 DEG C are cultivated 8 days；In 25 DEG C, 150r/
Cultivated 4 days on rotary shaker under min and be used as seed；In 25 DEG C, cultivated 5 days on rotary shaker under 150r/min, terminate fermentation.
In described step 3, dried after mycelium is washed at 50 DEG C, extract 28h, extracted 4 times, ultrasonic oscillation
0.8h.Other steps are the same as embodiment 1.
Embodiment 3
The fungi separated in described step 2 is connected in flat board PDA culture medium, and 28 DEG C are cultivated 4 days；In 30 DEG C, 100r/
Cultivated 2 days on rotary shaker under min and be used as seed；In 30 DEG C, cultivated 2 days on rotary shaker under 100r/min, terminate fermentation.
In described step 3, dried after mycelium is washed at 70 DEG C, extract 20h, extracted 2 times, ultrasonic oscillation
1.5h.Other steps are the same as embodiment 1.
Using perennial English grass as test material, the PEG-6000 Drought stress simulation conditions of 4 kinds of concentration are have studied
Under, the change of the turfgrass morphological index and physiological and biochemical index of sprinkling plant endogenesis epiphyte CBS1 alcohol extracting things.As a result show,
Under various concentrations PEG-6000 stress, CBS1 can mitigate the influence of drought stress to a certain degree, wherein spraying 20% PEG-
6000（Medium drought）When, mitigation effect is the most notable, and the turfgrass plant height, grass cuttings amount, Root dry weight for spraying S compare respectively
Spray clear water processing（Control）25.1%, 77.4%, 24.6% is improved, root activity is 1.75 times of control, and chlorophyll content is more right
According to being higher by 46.0%, Proline Accumulation amount is 1.4 times of control, and control mda content is spray S 1.15 times.Show in
Spend under drought stress, plant endogenesis epiphyte CBS1 alcohol extracting things can effectively slow down the destructiveness of effect of drought stress on turfgrass.
The drought-enduring experiment case study in lawn is as follows：
This experiment is matched as sand by volume using Culture basin (cm of diameter 28, high 30 cm) turf plant grass, matrix: grass
Charcoal: organic fertilizer=4: 1: 2, and every basin loads the matrix of equal quality.After turfgrass Cheng Ping, equably in foliage-spray plant
Raw mykol extract C BS1（0.5 ng/ml ）, spray clear water is as control.After 2 d, with the polyethylene glycol of various concentrations（PEG-
6000）Solution（0%、10%、20%、30%）To turfgrass simulating drought (nothing, slight, moderate, severe) Stress treatment, respectively at place
8d carries out the measure of morphological index and physical signs after reason, each handles 3 repetitions.
1st, morphological index determines
8th d being handled since drought stress, determined using five point sampling per basin biomass.
（1）Plant height determines：Plant height has a net increase of height=plant plant height-turf-mown height.
（2）Grass cuttings measure fixed：Finish 1 h per the grass cuttings collected by basin mid diameter 20cm parts at 105 DEG C, then
Permanent quality is dried at 80 DEG C, claims quality afterwards.
（3）Root system amount of dry matter determines：Plant root is taken out, root fine sand is gently washed away, made root system in order in water,
Be put into the root disk for filling with water, blotting paper be stained with it is dry, take under ground portion carry out root system analysis.
An important factor for quality of turfgrass root system is influence lawn quality, and dry-matter accumulation is reflection Turfgrass Growth
Another important indicator of situation.As known from Table 1, under various concentrations PEG-6000 stress, CBS1 turfgrass plant height, grass cuttings
Amount, Root dry weight are above spray clear water（Control）Processing；With spray clear water（Control）Compare, without drought stress（0% PEG-
6000）When, CBS1 turfgrass plant height, grass cuttings amount, Root dry weight increase by 3.4%, 3.3%, 3.8% respectively；Mild stress（10%
PEG-6000）When, CBS1 turfgrass plant height, grass cuttings amount, Root dry weight are respectively increased 6.5%, 3.1%, 11.6%；Moderate is coerced
（20% PEG-6000）When, CBS1 turfgrass plant height, grass cuttings amount, Root dry weight are respectively increased 25.1%, 77.4%, 24.6%；Severe
Stress（30% PEG-6000）When, control turfgrass does not almost grow, and the Turfgrass Growth of CBS1 processing is slow.Show to use
Endophyte of plant alcohol extracting thing CBS1 turfgrass, the drought-resistant ability of turfgrass is significantly improved under drought stress, and completely
Low, medium drought can be resisted.
The measure of physical signs：
This explanation has carried out the PEG-6000 Drought stress simulations processing of 4 kinds of concentration to turfgrass, is carried by spraying mykol
Thing CBS1 is taken, by raw to root activity, chlorophyll content, soluble sugar content, proline content and mda content etc.
The measure of biochemical indicator is managed, to illustrate that it can improve turfgrass drought tolerance.
As a result show, when PEG-6000 concentration is coerced up to 20% moderate, CBS1 processing significantly improves the root system of turfgrass
Vigor, remarkable effect is played to turfgrass drought-resistant ability；It can be seen that under moderate and Severe drought stress, grass can be improved by spraying CBS1
Level ground blade of grass chlorophyll contents, strengthen the photosynthetic capacity of blade and green degree, remarkable effect is risen to turfgrass drought resisting；Show in 20% and 30%
Drought stress simulation environment in, the accumulation of sugar in turfgrass body can be promoted by spraying CBS1, so as to improve the drought resisting of turfgrass
Property；Turfgrass Proline Accumulation amount is more big more is advantageous to drought resisting, thus infers that the turfgrass for spraying CBS1 is advantageous to maintain blade
The flow of water, when PEG-6000 concentration is up to 20%, turfgrass is most strong to the adaptability of drought stress, and drought resistance is relatively preferable；Show
CBS1 significantly reduces the speed of lipid peroxidation under 20% drought stress, reduces MDA to cyto-architectural destruction, such as schemes
Shown in 1-5.
Wild sea-buckthorn endogenetic fungus CBS1 strains form and classification foundation：A fungal strain is sifted out in wild seabuckthorn root portion
CBS1, as shown in Figure 6 a.Isodiametric growth, be just white, after fade to blue or green brown to crineous, neat in edge, in wavy or split
Sheet, in media surface it can be seen that the sorus for having black is formed.Ripe conidium has 4-8 diaphragm, 1-5
Individual mediastinum film, the shape of falling club is avette, and ellipse, spore is clear, colorless when young, is in dark brown after ripe, as shown in Figure 6 b.
This explanation carries out the extraction of genomic DNA using CTAB methods to the fungi of screening, and with ITS4 and a pair of ITS5
Universal primer is expanded to the fungal bacterial strain separated from wild sea-buckthorn root, and amplified production is sequenced, base
Searched in blaSt, choose and carried out with the sequence of the research immediate kind of strain sequence or hithermost evolutionary branching representative strain
Compare, establish chadogram using DNAStar softwares, as shown in Figure 7b.As a result show that CBS1 fungies and Alternaria Sp's is same
Source similitude reaches 99%.
Influence of the drought stress of table 1 to each processing turfgrass biomass
Claims (2)
1. the production technology of sea-buckthorn Endophytic Fungal Hyphae extract, described sea-buckthorn endogenetic fungus is Alternaria
（Alternaria Sp）, deposit number is CGMCC NO.10117, preservation date 2014.12.11, depositary institution's title：In
State's Microbiological Culture Collection administration committee common micro-organisms center；Depositary institution address：BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3；
It is characterized in that comprise the following steps：
Step 1, sea-buckthorn endogenetic fungus liquid fermentation and culture：
Gained sea-buckthorn endogenetic fungus is connected in flat board PDA culture medium, 25 DEG C are cultivated 6 days, and digging block with card punch agar is inoculated in
250mL conical flasks equipped with 50mLPDA, cultivated 3 days on rotary shaker in 28 DEG C, under 120r/min and be used as seed, with volume
Amount 10% is inoculated with the 500mL triangular flasks equipped with 150mLPDA, in 28 DEG C, is cultivated 5 days on rotary shaker under 120r/min, eventually
Only ferment, be put into 4 DEG C of refrigerators and save backup；
Step 2, the preparation of sea-buckthorn endogenetic fungus extract：
To be dried at 60 DEG C after above-mentioned mycelium washing, and weigh, then crushed through high speed disintegrator, the bacterium powder after crushing with
20wt% ethanol by volume 1:1 mixing extraction 24h, extracts 3 times, is mixed with magnetic stirring apparatus, ultrasonic oscillation 1h, very
Sky filters, and it is standby to collect filtrate.
2. the extract of sea-buckthorn endogenetic fungus according to claim 1 is in promoting turfgrass to improve resistance and yield
Using.
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| CN107699531A (en) * | 2017-12-04 | 2018-02-16 | 西北师范大学 | A kind of hippophae plant Frankia isolated culture method |
| CN116218929B (en) * | 2023-02-01 | 2024-03-15 | 山东蓬勃生物科技有限公司 | Fermentation crude extract, preparation method and application |
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| CN102086439A (en) * | 2010-11-18 | 2011-06-08 | 辽宁省农业科学院 | Method for preparing ginseng fungus and ginseng fungus extract and application of ginseng fungus extract |
| CN103828828A (en) * | 2012-11-27 | 2014-06-04 | 陕西汤普森生物科技有限公司 | Plant growth regulation composition containing plant activator protein |
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| CN102086439A (en) * | 2010-11-18 | 2011-06-08 | 辽宁省农业科学院 | Method for preparing ginseng fungus and ginseng fungus extract and application of ginseng fungus extract |
| CN103828828A (en) * | 2012-11-27 | 2014-06-04 | 陕西汤普森生物科技有限公司 | Plant growth regulation composition containing plant activator protein |
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| 细极链格孢菌蛋白激发子对棉株光合特性的影响;张志刚等;《湖南农业大学学报(自然科学版)》;20080228;第34卷(第1期);第1-5页 * |
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