CN105713841B - A kind of fusarium oxysporum and its application in the sprouting pretreatment of bitter buckwheat Wheat Seeds - Google Patents

A kind of fusarium oxysporum and its application in the sprouting pretreatment of bitter buckwheat Wheat Seeds Download PDF

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CN105713841B
CN105713841B CN201610184554.0A CN201610184554A CN105713841B CN 105713841 B CN105713841 B CN 105713841B CN 201610184554 A CN201610184554 A CN 201610184554A CN 105713841 B CN105713841 B CN 105713841B
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fusarium oxysporum
bitter buckwheat
fataf9
mycelia
polysaccharide
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CN105713841A (en
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赵江林
赵钢
钟灵允
彭镰心
邹亮
向达兵
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Huantai Biotechnology Co ltd
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Chengdu University
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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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/77—Fusarium
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/02—Germinating apparatus; Determining germination capacity of seeds or the like

Abstract

The invention discloses a kind of fusarium oxysporum, the fusarium oxysporum is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.10102 on November 21st, 2014.It also discloses fusarium oxysporum and sprouts the application in pretreatment in bitter buckwheat Wheat Seeds.Fusarium oxysporum of the present invention is simple and easy to the preprocess method of bitter buckwheat Wheat Seeds, low in cost, easy to operate, without excessively high technical requirements, and improves bitter buckwheat percentage of seedgermination and to shorten sprout time effect fairly obvious.

Description

A kind of fusarium oxysporum and its application in the sprouting pretreatment of bitter buckwheat Wheat Seeds
Technical field
The invention belongs to microorganisms technical fields, are related to a kind of fungi and its answering on bitter buckwheat Wheat Seeds are sprouted and pre-processed With more particularly to a kind of fusarium oxysporum and its bitter buckwheat Wheat Seeds sprout pretreatment on application.
Background technique
Bitter buckwheat is also known as hull buckwheat (Fagopyrum tataricum), is a kind of famous food and medicament dual-purpose rain fed crops, tool There are very high nutritive value and healthcare function.Bitter buckwheat is rich in protein, starch, fat, vitamin, minerals and microelement etc. The biological flavones active component such as rutin contained in trophic function ingredient, especially bitter buckwheat, Quercetin, having reduces blood It sugar, cholesterol, blood lipid and enhances human immunity and other effects.China is the first big bitter buckwheat plant development state in the world, throughout the year Cultivated area is at 6,000,000 mu or more, and about 400,000 tons of annual output, main producing region is concentrated mainly on Sichuan, Yunnan, Guizhou on the south the Changjiang river With the provinces and regions such as Tibet.
The fresh bitter buckwheat wheat germination percentage of this season harvest is usually 95% or more at present.However, bitter buckwheat Wheat Seeds are in nature With the increase of storage time in environment storing process, zymoprotein is gradually denaturalized, and the nutrient in endosperm is reduced, from And lead to quality decline of germinateing, it causes germination percentage to reduce, wherein a Nian Houqi germination percentage is reduced to 80-85%, is reduced to after 2 years 50-60%, germination percentage is only 20-25% after 3 years, or even is not germinateed, and the quality and vigor of seedling are also greatly lowered, seriously The plant development for influencing tartary buckwheat and further development and utilization.
Summary of the invention
The technical problem to be solved in the present invention is that in view of the drawbacks of the prior art, provide a kind of fusarium oxysporum and its Bitter buckwheat Wheat Seeds sprout the application in pretreatment.Fusarium oxysporum is simple and easy to the preprocess method of bitter buckwheat Wheat Seeds, at low cost It is honest and clean, easy to operate, without excessively high technical requirements, and improving bitter buckwheat percentage of seedgermination and to shorten sprout time effect ten clearly demarcated It is aobvious.
The technical solution adopted by the present invention to solve the technical problems is:
The present invention provides a kind of novel fusarium oxysporum, is named as fusarium oxysporum (Fusarium oxysporum) Fataf9, the fusarium oxysporum are preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on November 21st, 2014 Object center, deposit number CGMCC No.10102.Fusarium oxysporum (Fusarium oxysporum) Fataf9 is planted from healthy bitter buckwheat It is separating obtained in strain, it is one plant of bitter buckwheat endogenetic fungus.
Fusarium oxysporum (Fusarium oxysporum) Fataf9 has the feature that
Bacterium colony on PDA plate is purple, and edge white, back wheel line shape, white is alternate with purple, and colony edge is whole Together, aerial hyphae is longer, the nearly sickle-shaped or oblong of megaspore, and 2~3 are oval or ellipse every, microspore, and two kinds Spore size is (4.3 μm~17.5 μm) × (1.8 μm~4.7 μm).
The fusarium oxysporum (Fusarium oxysporum) Fataf9 well-grown in PDA culture medium, suitable growth Temperature is 20 DEG C~32 DEG C, and optimum growth temp is 25 DEG C~28 DEG C.In addition, fusarium oxysporum (Fusarium oxysporum) Fataf9 culture is carried out in aerobic condition, and the advantageous pH range of culture medium is pH6.0~pH7.5.
The ITS sequence of the fusarium oxysporum are as follows:
The phylogenetic tree of the genetic homology of fusarium oxysporum Fataf9 are as follows:
Fusarium oxysporum can be sprouted in pretreatment in bitter buckwheat Wheat Seeds concrete application.
The fusarium oxysporum sprouts the application in pretreatment, including following processing method in bitter buckwheat Wheat Seeds:
A, fusarium oxysporum mycelia water proposes the preparation of polysaccharide: by fusarium oxysporum mycelia inoculated and cultured, harvesting mycelium；Mycelium First remove monosaccharide, disaccharides and lipid material after drying, then water mention, refine after obtain mycelia water and mention polysaccharide；
B, purity testing: measurement fusarium oxysporum Fataf9 mycelia water proposes the sugared content and corresponding purity of polysaccharide, with glucose Standard curve is established for standard items；
C, the preparation for the treatment of fluid: taking fusarium oxysporum mycelia water to mention polysaccharide and be dissolved in sterile water, and adjusting its concentration is 15- 25mg/mL, it is spare after filtering；
D, tartary buckwheat seed disinfection: mature and plump is chosen, bitter buckwheat Wheat Seeds in the same size, without mildew are impregnated with disinfectant Then disinfection is cleaned with sterile water, is dried；
E, tartary buckwheat seed treatment: the bitter buckwheat Wheat Seeds after disinfection are placed in obtained by step C and diluted treatment fluid In, impregnate 10-16h under 25 DEG C, dark condition, the volume ratio of tartary buckwheat seed weight and treatment fluid is 1:10 (m/v)；
The fusarium oxysporum sprouts the application in pretreatment, in the preferably described step A, fusarium oxysporum in bitter buckwheat Wheat Seeds When mycelium inoculation culture, using the mycelium inoculation that will be activated on PD culture medium, the PD culture medium contains buckwheat powder Aqueous extracts.
The fusarium oxysporum is sprouted in the application in pretreatment in bitter buckwheat Wheat Seeds, diluted in the preferably described step E The concentration for the treatment of fluid is 150-200 μ g/mL.
The present invention is with beneficial endogenetic fungus -- the active metabolism caused by fusarium oxysporum Fataf9 from tartary buckwheat itself Object (mycelia water mentions polysaccharide WPS) be inducer bitter buckwheat seed is pre-processed, improve percentage of seedgermination, and to bitter buckwheat seed without Toxic action, it is highly-safe, promote to sprout significant effect.Fusarium oxysporum Fataf9 preparation is convenient, is conducive to scale in production It uses.It is simple and easy using the pretreated processing method of fusarium oxysporum Fataf9 progress, it is low in cost, it is easy to operate, without excessively high Technical requirements, and improve bitter buckwheat percentage of seedgermination and shorten sprout time effect it is fairly obvious.Meanwhile this method has very Good economic society value and application prospect.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples, in attached drawing:
Fig. 1 is the bitter buckwheat Wheat Seeds to the different storage time limits, and the embodiment of the present invention and untreated percentage of seedgermination compare Figure；
Fig. 2 is the bitter buckwheat Wheat Seeds to the different storage time limits, and the embodiment of the present invention and untreated percentage of seedgermination compare Figure.
6 cylinders successively represent western buckwheat 1 (control), western buckwheat 1 (at WPS respectively in every group of column diagram in Fig. 1 diagram Reason), river buckwheat 1 (control), river buckwheat 1 (WPS processing), the seed hair after meter Qiao 1 (control) and rice buckwheat 1 (WPS processing) Bud rate.
6 cylinders successively represent western buckwheat 1 (control), western buckwheat 1 (at WPS respectively in every group of column diagram in Fig. 2 diagram Reason), river buckwheat 1 (control), river buckwheat 1 (WPS processing), the seed hair after meter Qiao 1 (control) and rice buckwheat 1 (WPS processing) Bud rate.
Specific embodiment
For a clearer understanding of the technical characteristics, objects and effects of the present invention, now control attached drawing is described in detail A specific embodiment of the invention.
One, fusarium oxysporum Fataf9 being separately cultured and identifies
It isolates and purifies
1, tartary buckwheat is handled: the tartary buckwheat root and stem of health are chosen, 70% alcohol treatment 30s is first used to its rhizome surface, Then surface sterilization is carried out with 0.2% mercury chloride processing 20min, removes the rhizome epidermis of tartary buckwheat, root, stem is divided into about 0.5cm × 0.5cm × 0.5cm size block is placed on potato dextrose agar (PDA) culture medium.
2, it cultivates: the suppression of 200 μ g/mL streptomycin sulphates (Streptomycin sulfate) will first be added in PDA culture medium Bacterium interference processed, then places it in and carries out dark culture at 25 DEG C.
3, isolate and purify: from since culture the 3rd day, observe bitter buckwheat root in PDA plate, grow on stem tissue block it is true Bacterium bacterium colony, with transfer needle picking, its edge mycelia is separately cultured under aseptic technique；Further obtained to separated Fungus colony isolated and purified, continuous purification 4-5 times, until colonial morphology unanimously to get arrive tartary buckwheat fusarium oxysporum Fataf9。
Then bitter buckwheat fusarium oxysporum Fataf9 is stored on the test tube slant PDA and plate, and morphological feature is carried out to it Observation and molecular biology identification.
Cultural colony observation
Bacteria cake will be beaten by well-grown fusarium oxysporum Fataf9 on PDA plate, bacteria cake diameter is Ф=5mm, each PDA The glassine paper that one piece of diameter 6cm is placed on plate, by pure culture biscuits involvng inoculation to glassine paper, dark culturing at 25 DEG C is remembered per observation for 24 hours Record the shape of Fataf9 bacterium colony, size, color variation, quality, the speed of growth, whether chromogenesis etc..
The fusarium oxysporum (Fusarium oxysporum) Fataf9 well-grown in PDA culture medium, suitable growth Temperature is 20 DEG C~32 DEG C, and optimum growth temp is 25 DEG C~28 DEG C.In addition, fusarium oxysporum (Fusarium oxysporum) Fataf9 culture is carried out in aerobic condition, and the advantageous pH range of culture medium is pH6.0~pH7.5.
Microexamination
Glassine paper is removed from PDA plate, is cut into 1cm2Size places it on glass slide, is floating carry with sterile water After sample is made in agent, observe under the microscope mycelial thickness, the inserted part of conidiome, shape, type, size, Produce details and conidial budding mode such as spore mode, whether have every, size, shape, surface features, color etc. Morphological feature.
Bacterium colony on PDA plate is purple, and edge white, back wheel line shape, white is alternate with purple, and colony edge is whole Together, aerial hyphae is longer, the nearly sickle-shaped or oblong of megaspore, and 2~3 are oval or ellipse every, microspore, and two kinds Spore size is (4.3 μm~17.5 μm) × (1.8 μm~4.7 μm).
Molecular biology identification
Activation bitter buckwheat fusarium oxysporum Fataf9 beats bacteria cake, and bacteria cake diameter is Ф=5mm, is inoculated into PD fluid nutrient medium On, dark culture harvests after 6 days at 25 DEG C, 150rpm, and vacuum filtration harvests fresh mycelia to doing.Then CTAB freeze thawing is used The genomic DNA of method extraction fusarium oxysporum Fataf9.
Then fungi universal primer: sequence 1:ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') and sequence 2 is selected: ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ') carries out PCR amplification to the area ITS of fusarium oxysporum Fataf9.Sequence after amplification The result that column sequencing obtains is compared with other kinds in GenBank of fungi ITS complete sequence, finds out the highest relationship of similitude The nearest fungi of relationship.
The ITS sequence (sequence 3) of the fusarium oxysporum are as follows:
Two, the fusarium oxysporum Fataf9 sprouts the application in pretreatment in bitter buckwheat Wheat Seeds:
1, concrete application be include following processing method:
A, fusarium oxysporum Fataf9 mycelia water proposes the preparation of polysaccharide: by fusarium oxysporum mycelia inoculated and cultured, harvesting mycelium； First remove monosaccharide, disaccharides and lipid material after mycelium is dry, then water mention, refine after obtain mycelia water and mention polysaccharide.
Specifically: the activation culture on a little fusarium oxysporum mycelia to PDA plate of picking from inclined-plane, then by fusarium oxysporum The mycelium inoculation of Fataf9 activation is in the triangular flask for filling PD culture medium, the preferred buckwheat powder water containing 2.5g/L in PD culture medium It is harvested mycelium after dark culture 7d at 25 DEG C, 150rpm by extract；Gained fusarium oxysporum Fataf9 mycelium is carried out true Vacuum freecing-dry crushes, and using 95% ethyl alcohol: petroleum ether (1:1, v/v) carries out circumfluence distillation to it, to remove monosaccharide, two Sugar and lipid material.Then quantitatively pretreated fusarium oxysporum Fataf9 mycelia powder is weighed, is extracted using 90 DEG C of hot water returns Fusarium oxysporum Fataf9 mycelia water, which is obtained, in conjunction with the methods of alcohol precipitation, dialysis mentions polysaccharide (WPS).
B, purity testing: measurement fusarium oxysporum Fataf9 mycelia water proposes the sugared content and corresponding purity of polysaccharide, with glucose Standard curve is established for standard items；Purity testing be using sulfuric acid anthrone colorimetric method measurement fusarium oxysporum Fataf9 mycelia water mention it is more The sugared content and corresponding purity of sugar.
C, the preparation for the treatment of fluid: taking mycelia water to mention polysaccharide and be dissolved in sterile water, and adjusting its concentration is 15-25mg/mL, filtering It is spare that treatment fluid is obtained afterwards；Filter type can be using a variety of, and the present embodiment is used to be filtered through (0.4 μm) of miillpore filter.
D, tartary buckwheat seed disinfection: mature and plump is chosen, bitter buckwheat Wheat Seeds in the same size, without mildew are impregnated with disinfectant Then disinfection is cleaned with sterile water, is dried；Disinfectant uses 0.5% liquor natrii hypochloritis, can also be applicable in this hair using other Bright disinfectant.Disinfection soaking time is 10-15min.
E, tartary buckwheat seed treatment: the bitter buckwheat Wheat Seeds after disinfection are placed in treatment fluid obtained by step C, 25 DEG C, impregnate 10-16h under dark condition, the volume ratio of tartary buckwheat seed weight and treatment fluid is 1:10 (m/v), kind that treated Son can be used to plant.
The germination percentage of bitter buckwheat Wheat Seeds is verified by culture experiment of germinateing.
The condition of germination culture are as follows: 25 DEG C of cultivation temperature, humidity 70-75%, daily light application time are 10-12 hours.
Application of the invention is illustrated with specific embodiment below:
Embodiment 1
1, it includes following processing method that fusarium oxysporum Fataf9, which sprouts the application in pretreatment in bitter buckwheat Wheat Seeds:
(1) fusarium oxysporum Fataf9 mycelia water proposes the preparation of polysaccharide:
A. the fermented and cultured of fusarium oxysporum Fataf9: a little fusarium oxysporum mycelia of picking is to PDA plate from test tube slant Then the fusarium oxysporum mycelia of activation is inoculated in PD culture medium by upper activation culture 5 days, wherein needing according to every 200mL PD solution The buckwheat powder Aqueous extracts 20mL meter of 20.0-25.0g/L is added, buckwheat powder Aqueous extracts are added in PD culture medium.In 25 after inoculation DEG C, suspend culture 7 days under 150rpm after harvest, centrifugation (4000rpm) obtains fusarium oxysporum Fataf9 fresh mycelia.
B. fusarium oxysporum Fataf9 mycelia water proposes the preparation of polysaccharide:
The fresh mycelium of gained fusarium oxysporum Fataf9 is subjected to vacuum freeze drying, crushes, then uses 95% ethyl alcohol: stone Oily ether (1:1, v/v) carries out circumfluence distillation 2h to it at 50-55 DEG C, to remove the monosaccharide in mycelium, disaccharides and lipid Substance, centrifugation are collected residue, are dried at 50-55 DEG C.
Water mentions: weighing quantitative pretreatment Fataf9 mycelia powder, places it in circumfluence distillation device, adds certain proportion Distilled water carry out circumfluence distillation, extraction conditions are as follows: ratio of water to material is 25:1-30:1 (v/w), 90 DEG C of Extracting temperature, when extraction Between 2-3h, altogether extract 2 times, combined extract.
Separation and purification: extraction fluid and mycelium residue, centrifuged supernatant are concentrated into certain body by way of centrifugation Product, adds 3 times of 95% ethyl alcohol of volume, and 48~72h is precipitated at 4 DEG C, then will be precipitated using centrifugation and supernatant separates, Precipitating is collected, dehydrated alcohol, acetone washing are successively used.
Further its solution is placed in bag filter and is dialysed, replacing an extracellular fluid dialysis every 12h, (the present embodiment is used and gone Ionized water is as extracellular fluid dialysis), after 48h, solution in bag filter is subjected to vacuum freeze drying to get bitter buckwheat fusarium oxysporum Fat9 Mycelia water mentions polysaccharide (WPS).
C. the preparation for the treatment of fluid: a certain amount of bitter buckwheat fusarium oxysporum Fat9 mycelia water is taken to mention polysaccharide (through sulfuric acid anthrone colorimetric 85.6%) method measurement, purity reach, are dissolved in sterile water, adjusting its concentration is 20.0mg/mL, through miillpore filter It is spare after (0.4 μm) filtering.
(2) selection of bitter buckwheat seed: selection mature and plump is of moderate size, uniformity, the bitter buckwheat seed without mildew health As material.
(3) disinfection of bitter buckwheat seed: by the bitter buckwheat seed of selection with 0.5% liquor natrii hypochloritis immersion treatment 10min, so It is cleaned afterwards with sterile water, places it on aseptic filter paper and dry.
(4) Fataf9 mycelia water mentions polysaccharide induced processing bitter buckwheat seed: it is obtained that the bitter buckwheat seed after disinfection is placed in (1) Treatment fluid WPS solution in (concentration is adjusted to 150 μ g/mL), 12-16h, seed weight and bacterium are impregnated under 25 DEG C, dark condition The volume ratio of silk polysaccharide solution is 1:10 (m/v).
(5) bitter buckwheat germination is cultivated: will uniformly be placed on paving by the bitter buckwheat seed of step (4) processing and have three layers wetting In the germination box (120 × 120 × 50mm) of filter paper, then 100 seeds of every box place it in growth cabinet (temperature 25 DEG C, humidity 70-75%, light application time 12h/ days) germination culture is carried out, the germination period is 2-4d.After germination, its hair is counted Bud rate.
Embodiment 2
(1) fusarium oxysporum Fataf9 mycelia water mentions polysaccharide and treatment fluid the preparation method is the same as that of Example 1.
(2) selection of bitter buckwheat seed: selection mature and plump is of moderate size, uniformity, the bitter buckwheat seed without mildew health As material.
(3) disinfection of bitter buckwheat seed: by the bitter buckwheat seed of selection with 0.5% liquor natrii hypochloritis immersion treatment 10min, so It is cleaned afterwards with sterile water, places it on filter paper and dry.
(4) Fataf9 mycelia water mentions polysaccharide induced processing bitter buckwheat seed: it is obtained that the bitter buckwheat seed after disinfection is placed in (1) Treatment fluid WPS solution in (concentration is adjusted to 200 μ g/mL), 10-12h, seed weight and bacterium are impregnated under 25 DEG C, dark condition The volume ratio of silk polysaccharide solution is 1:10 (m/v).
(5) bitter buckwheat germination is cultivated: will uniformly be placed on paving by the bitter buckwheat seed of step (4) processing and have three layers wetting In the germination box (120 × 120 × 50mm) of filter paper, then 100 seeds of every box place it in growth cabinet (temperature 25 DEG C, humidity 70-75%, light application time 10h/ days) germination culture is carried out, the germination period is 2-4d.After germination, its hair is counted Bud rate.
Using following experimental verifications effect of the present invention:
Comparative experiments 1:
The preprocess method that bitter buckwheat Wheat Seeds are sprouted is improved to follow the steps below:
Using the method for embodiment 1, prepares the bitter buckwheat fusarium oxysporum Fataf9 mycelia water that concentration is 150 μ g/mL and mention polysaccharide (WPS) solution, it is spare.
Choose this season harvest fresh bitter buckwheat seed (0 year) and room temperature store naturally 0.5 year, 1 year, 1.5 years, 2.0 years, 2.5 years and 3.0 years bitter buckwheat seeds (western buckwheat 1) are experimental material.
By the bitter buckwheat Wheat Seeds of different Storage periods with 0.5% liquor natrii hypochloritis immersion treatment 10min, sterile water is then used It cleans, places it on filter paper and dry.
Bitter buckwheat seed after disinfection is respectively placed in WPS solution (concentration is adjusted to 150 μ g/mL), in 25 DEG C, dark condition The volume ratio of lower immersion 16h, seed weight and mycelia polysaccharide solution is 1:10 (m/v).
Five, treated each bitter buckwheat seed is uniformly placed on to paving to have three layers in the germination box for soaking filter paper, 100, every box Then seed places it in growth cabinet (25 DEG C of temperature, humidity 70-75%, light application time 12h/ days) and carries out germination training It supports.After germination, its germination percentage is counted.
Blank control: by the bitter buckwheat seed of each storage time limit (1.0,1.5,2.0,2.5,3.0 years) without WPS solution at Reason, and be only placed in sterile water, soaking temperature, time and solid-liquid ratio are consistent with above-mentioned processing method.
As shown in Figure 1, in this comparative experiments 1 through Fataf9 mycelia polysaccharide solution processing after respectively store the time limit (1.0,1.5, 2.0,2.5,3.0 years) bitter buckwheat percentage of seedgermination respectively reach 91%, 86.3%, 83.0%, 72.0%, 61.0%, in Fig. 1 It is middle to be followed successively by the 3rd group of column diagram, the 4th group, the 5th group, the 2nd cylinder in the 6th group the 7th group of neutralization, corresponding blank control respectively Germination percentage be 82.3%, 74.7%, 56%, 35%, 21.3%, be followed successively by respectively in Fig. 1 the 3rd group of column diagram, the 4th group, The 1st cylinder in 5th group, the 6th group the 7th group of neutralization, germination percentage of the invention are 1.1-2.9 times of blank control, are averagely sprouted It is 60 hours that the hair time, which effectively shortens,.
Comparative experiments 2:
With this season harvest fresh bitter buckwheat seed (0 year) and room temperature store naturally 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years bitter buckwheat seeds " river buckwheat 1 " are used as experimental material, remaining improves the pretreatment side that bitter buckwheat Wheat Seeds are sprouted Method is identical as comparative experiments 1.
As shown in Figure 1, in this experiment through Fataf9 mycelia polysaccharide solution processing after respectively store the time limit (1.0,1.5,2.0, 2.5,3.0 years) bitter buckwheat percentage of seedgermination respectively reach 90.3%, 85.3%, 82.0%, 73.0%, 62.3%, in Fig. 1 It is followed successively by the 3rd group of column diagram, the 4th group, the 5th group, the 4th cylinder in the 6th group the 7th group of neutralization respectively, corresponding blank control Germination percentage is 82.0%, 73.7%, 55.7%, 34.0%, 22.7%, is followed successively by the 3rd group of column diagram, the 4th respectively in Fig. 1 Group, the 5th group, the 3rd cylinder in the 6th group the 7th group of neutralization, germination percentage of the invention are 1.1-2.7 times of blank control, are averaged It is 60 hours that sprout time, which effectively shortens,.
Comparative experiments 3:
With this season harvest fresh bitter buckwheat seed (0 year) and room temperature store naturally 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years bitter buckwheat seeds " rice buckwheat 1 " are used as experimental material, remaining improves the pretreatment side that bitter buckwheat Wheat Seeds are sprouted Method is identical as comparative experiments 1.
As shown in Figure 1, in this experiment through Fataf9 mycelia polysaccharide solution processing after respectively store the time limit (1.0,1.5,2.0, 2.5,3.0 years) bitter buckwheat percentage of seedgermination respectively reach 91.3%, 86.0%, 81.0%, 69.0%, 60.3%, in Fig. 1 It is followed successively by the 3rd group of column diagram, the 4th group, the 5th group, the 6th cylinder in the 6th group the 7th group of neutralization respectively, corresponding blank control Germination percentage is 83.0%, 76.0%, 58.0%, 38.0%, 22.3%, is followed successively by the 3rd group of column diagram, the 4th respectively in Fig. 1 Group, the 5th group, the 5th cylinder in the 6th group the 7th group of neutralization), germination percentage of the invention is the 1.1-2.7 of blank control germination percentage Times, it is 48 hours that average sprout time, which effectively shortens,.
Comparative experiments 4:
The preprocess method that bitter buckwheat Wheat Seeds are sprouted is improved to follow the steps below:
According to embodiment 2, it is molten that the bitter buckwheat fusarium oxysporum Fataf9 mycelia water that preparation concentration is 200 μ g/mL mentions polysaccharide (WPS) Liquid is spare as treatment fluid.
Choose this season harvest fresh bitter buckwheat seed (0 year) and room temperature store naturally 0.5 year, 1 year, 1.5 years, 2.0 years, 2.5 years and 3.0 years bitter buckwheat seeds (western buckwheat 1) are experimental material.
By the bitter buckwheat Wheat Seeds of different Storage periods with 0.5% liquor natrii hypochloritis immersion treatment 10min, sterile water is then used It cleans, places it on filter paper and dry.
Bitter buckwheat seed after disinfection is respectively placed in WPS solution (concentration is adjusted to 200 μ g/mL), in 25 DEG C, dark condition The volume ratio of lower immersion 12h, seed weight and mycelia polysaccharide solution is 1:10 (m/v), and blank control: each bitter buckwheat seed is placed in In sterile water, soaking temperature, time and solid-liquid ratio are consistent with above-mentioned processing method.
Treated each bitter buckwheat seed is uniformly placed on to paving to have three layers in the germination box for soaking filter paper, 100 kinds of every box Then son places it in growth cabinet (25 DEG C of temperature, humidity 70-75%, light application time 10h/ days) and carries out germination culture.
As shown in Fig. 2, in this experiment through Fataf9 mycelia polysaccharide solution processing after respectively store the time limit (1.0,1.5,2.0, 2.5,3.0 years) bitter buckwheat percentage of seedgermination respectively reach 91.7%, 87.7%, 81.7%, 71.0%, 58.7%, in Fig. 2 It is followed successively by the 3rd group of column diagram, the 4th group, the 5th group, the 2nd cylinder in the 6th group the 7th group of neutralization respectively, corresponding blank control Germination percentage is 82.3%, 74.7%, 56%, 35%, 21.3%, is followed successively by the 3rd group of column diagram, the 4th group, the 5th respectively in Fig. 2 Group, the 1st cylinder in the 6th group the 7th group of neutralization, when germination percentage of the invention is 1.1-2.8 times of blank control, averagely sprouting Between to effectively shorten be 48 hours.
Comparative experiments 5:
With this season harvest fresh bitter buckwheat seed (0 year) and room temperature store naturally 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years bitter buckwheat seeds " river buckwheat 1 " are used as experimental material, remaining improves the pretreatment side that bitter buckwheat Wheat Seeds are sprouted Method is identical as experiment four.
As shown in Fig. 2, in this experiment through Fataf9 mycelia polysaccharide solution processing after respectively store the time limit (1.0,1.5,2.0, 2.5,3.0 years) bitter buckwheat percentage of seedgermination respectively reach 90.0%, 86.3%, 81.3%, 74.0%, 60.7%, in Fig. 2 It is followed successively by the 3rd group of column diagram, the 4th group, the 5th group, the 4th cylinder in the 6th group the 7th group of neutralization respectively, corresponding blank control Germination percentage is 82.0%, 73.7%, 55.7%, 34.0%, 22.7%, is followed successively by the 3rd group of column diagram, the 4th respectively in Fig. 2 Group, the 5th group, the 3rd cylinder in the 6th group the 7th group of neutralization, germination percentage of the invention are 1.1-2.7 times of blank control, are averaged It is 54 hours that sprout time, which effectively shortens,.
Comparative experiments 6:
With this season harvest fresh bitter buckwheat seed (0 year) and room temperature store naturally 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years bitter buckwheat seeds " rice buckwheat 1 " are used as experimental material, remaining improves the pretreatment side that bitter buckwheat Wheat Seeds are sprouted Method is identical as experiment four, five.In this experiment through Fataf9 mycelia polysaccharide solution processing after respectively store the time limit (1.0,1.5,2.0, 2.5,3.0 years) bitter buckwheat percentage of seedgermination respectively reach 90.3%, 86.3%, 80.0%, 70.0%, 58.7%, in Fig. 2 It is followed successively by the 3rd group of column diagram, the 4th group, the 5th group, the 6th cylinder in the 6th group the 7th group of neutralization respectively, corresponding blank control Germination percentage is 83.0%, 76.0%, 58.0%, 38.0%, 22.3%, is followed successively by the 3rd group of column diagram, the 4th respectively in Fig. 2 Group, the 5th group, the 5th cylinder in the 6th group the 7th group of neutralization, germination percentage of the invention are 1.1-2.6 times of blank control, are averaged It is 36 hours that sprout time, which effectively shortens,.

Claims (2)

1. a kind of fusarium oxysporum mycelia water mention polysaccharide bitter buckwheat Wheat Seeds sprout pretreatment on application, fusarium oxysporum Fataf9 in On November 21st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.10102。
2. fusarium oxysporum mycelia water according to claim 1, which mentions polysaccharide, sprouts the application in pretreatment in bitter buckwheat Wheat Seeds, It is characterised in that it includes following processing method:
A, fusarium oxysporum Fataf9 mycelia water proposes the preparation of polysaccharide: by fusarium oxysporum Fataf9 mycelium inoculation culture, harvesting mycelia Body；First remove monosaccharide, disaccharides and lipid material after mycelium is dry, then water mention, refine after obtain fusarium oxysporum Fataf9 mycelia Water mentions polysaccharide；
B, purity testing: measurement fusarium oxysporum Fataf9 mycelia water proposes the sugared content and corresponding purity of polysaccharide, is mark with glucose Quasi- product establish standard curve；
C, the preparation for the treatment of fluid: taking fusarium oxysporum Fataf9 mycelia water to mention polysaccharide and be dissolved in sterile water, and adjusting its concentration is 15- 25mg/mL, it is spare after filtering to obtain treatment fluid；
D, tartary buckwheat seed disinfection: mature and plump is chosen, bitter buckwheat Wheat Seeds disinfectant immersion in the same size, without mildew disappears Then poison is cleaned with sterile water, is dried；
E, tartary buckwheat seed treatment: the bitter buckwheat Wheat Seeds after disinfection are placed in obtained by step C and in diluted treatment fluid, 25 DEG C, impregnate 10-16h under dark condition, the volume ratio of tartary buckwheat seed weight and treatment fluid is 1:10 (m/v)；
In the step A, when fusarium oxysporum Fataf9 mycelium inoculation culture, using the mycelium inoculation that will be activated in PD culture medium On, the PD culture medium contains buckwheat powder Aqueous extracts；
In the step E, the concentration of diluted treatment fluid is 150-200 μ g/mL.
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