CN105713841A - Fusarium oxysporum and application of fusarium oxysporum in germination pretreatment of fagopyrum tataricum seeds - Google Patents
Fusarium oxysporum and application of fusarium oxysporum in germination pretreatment of fagopyrum tataricum seeds Download PDFInfo
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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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
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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
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- 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 fusarium oxysporum which is preserved in the CGMCC (China General Microbiological Culture Collection Center) of the microbiological culture preservation management committee on November 21, 2014 with the preservation number CGMCC No.10102 and also discloses an application of the fusarium oxysporum in germination pretreatment of fagopyrum tataricum seeds. The method for pretreating the fagopyrum tataricum seeds by the fusarium oxysporum is simple and easy to implement and operate and has no overhigh technical requirements, the cost is lower, and the effect on increasing the germination rate of the fagopyrum tataricum seeds and shortening the germination time is very remarkable.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of fungus and the application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment thereof, particularly relate to a kind of fusarium oxysporum and the application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment thereof.
Background technology
Radix Et Rhizoma Fagopyri Tatarici, also known as hull buckwheat (Fagopyrumtataricum), is a kind of famous food and medicament dual-purpose rain fed crops, has significantly high nutritive value and health care.The trophic function compositions such as Radix Et Rhizoma Fagopyri Tatarici rich in proteins, starch, fat, vitamin, mineral and trace element, the biological flavone active components such as especially contained in Radix Et Rhizoma Fagopyri Tatarici rutin, Quercetin, have the effects such as reduction blood glucose, cholesterol, blood fat and enhancing body immunity.China is the first big Radix Et Rhizoma Fagopyri Tatarici plant development state in the world, and its long-term cultivated area is more than 6,000,000 mu, and annual production about 400,000 tons, main producing region is concentrated mainly on the provinces and regions such as the Sichuan on the south the Changjiang river, Yunnan, Guizhou and Tibet.
The fresh Radix Et Rhizoma Fagopyri Tatarici Semen Tritici aestivi germination percentage of current this season results is generally more than 95%.But, Radix Et Rhizoma Fagopyri Tatarici seed is along with the increase of the time of storage in natural environment storing process, and degeneration occurs its pheron gradually, and the nutrient in endosperm reduces to some extent, thus causing germination Quality Down, causing germination percentage to reduce, wherein after 1 year, its germination percentage reduces to 80-85%, is reduced to 50-60% after 2 years, after 3 years, germination percentage is only 20-25%, even not germinateing, quality and the vigor of its seedling are also greatly lowered, and have a strong impact on the plant development of Radix Et Rhizoma Fagopyri Tatarici and develop further.
Summary of the invention
The technical problem to be solved in the present invention is in that, for the defect of prior art, it is provided that a kind of fusarium oxysporum and the application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment thereof.Fusarium oxysporum is simple to the preprocess method of Radix Et Rhizoma Fagopyri Tatarici seed, operation with low cost, easy, without too high technology requirement, and is improving Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination and to shorten sprout time effect fairly obvious.
The technical solution adopted for the present invention to solve the technical problems is:
The present invention provides a kind of novel fusarium oxysporum, called after fusarium oxysporum (Fusariumoxysporum) Fataf9, described fusarium oxysporum is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.10102 on November 21st, 2014.Fusarium oxysporum (Fusariumoxysporum) Fataf9 is separating obtained in healthy Radix Et Rhizoma Fagopyri Tatarici plant, is a strain Radix Et Rhizoma Fagopyri Tatarici endogenetic fungus.
Fusarium oxysporum (Fusariumoxysporum) Fataf9 has the feature that
Bacterium colony on PDA plate is purple, edge white, back wheel stricture of vagina shape, white and purple are alternate, and colony edge is neat, and aerial hyphae is longer, the nearly sickleshaped of megaspore or oblong, 2～3 every, sporidiole is oval or ellipse, and two kinds of spore sizes are (4.3 μm～17.5 μm) × (1.8 μm～4.7 μm).
This fusarium oxysporum (Fusariumoxysporum) 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.Carrying out it addition, fusarium oxysporum (Fusariumoxysporum) Fataf9 cultivates at aerobic condition, the advantageous pH range of culture medium is pH6.0～pH7.5.
The ITS sequence of described fusarium oxysporum is:
The phylogenetic tree of the genetic homology of fusarium oxysporum Fataf9 is:
Fusarium oxysporum can have concrete application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment.
The application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment of the described fusarium oxysporum, including following processing method:
A, fusarium oxysporum mycelia water propose the preparation of polysaccharide: cultivated by fusarium oxysporum mycelium inoculation, gather in the crops mycelium；Mycelium first removes monosaccharide, disaccharide and lipid material after drying, then water carries, refining after obtain mycelia water and carry polysaccharide；
B, purity testing: measure fusarium oxysporum Fataf9 mycelia water and carry sugared content and corresponding purity, the with glucose as a standard product Criterion curve of polysaccharide；
C, treatment fluid preparation: taking fusarium oxysporum mycelia water and carry polysaccharide and be dissolved in sterilized water, adjusting its concentration is 15-25mg/mL, standby after filtration；
D, Radix Et Rhizoma Fagopyri Tatarici seed disinfection: choose mature and plump, in the same size, without the Radix Et Rhizoma Fagopyri Tatarici seed disinfectant soaking disinfection that goes mouldy, then clean with sterilized water, dry；
E, Radix Et Rhizoma Fagopyri Tatarici seed treatment: the Radix Et Rhizoma Fagopyri Tatarici seed after sterilization is placed in treatment fluid that is obtained by step C and that dilute, 25 DEG C, soak 10-16h under dark condition, the volume ratio of Radix Et Rhizoma Fagopyri Tatarici seed weight and treatment fluid is 1:10 (m/v)；
Described fusarium oxysporum application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment, it is preferable that in described step A, when fusarium oxysporum mycelium inoculation is cultivated, adopts by the mycelium inoculation of activation in PD culture medium, and described PD culture medium contains Radix Et Rhizoma Fagopyri Tatarici powder Aqueous extracts.
In the application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment of the described fusarium oxysporum, it is preferable that in described step E, the concentration of the treatment fluid of dilution is 150-200 μ g/mL.
The present invention useful endogenetic fungus to be derived from Radix Et Rhizoma Fagopyri Tatarici self--Radix Et Rhizoma Fagopyri Tatarici seed is carried out pretreatment for inducer by active metabolite produced by fusarium oxysporum Fataf9 (mycelia water carries polysaccharide WPS), improve percentage of seedgermination, and to Radix Et Rhizoma Fagopyri Tatarici seed nonhazardous effect, safety is high, promotes to sprout effect notable.Fusarium oxysporum Fataf9 prepares convenient, is conducive to scale in production to use.The processing method that employing fusarium oxysporum Fataf9 carries out pretreatment is simple, with low cost, it is easy to operation, and without too high technology requirement, and it is fairly obvious with shortening sprout time effect to improve Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination.Meanwhile, the method has good economic society value and application prospect.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is the Radix Et Rhizoma Fagopyri Tatarici seed that difference stores the time limit, the embodiment of the present invention and untreated percentage of seedgermination comparison diagram；
Fig. 2 is the Radix Et Rhizoma Fagopyri Tatarici seed that difference stores the time limit, the embodiment of the present invention and untreated percentage of seedgermination comparison diagram.
Fig. 1 diagram often organizes the percentage of seedgermination after 6 cylinders in bar diagram represent western buckwheat 1 (comparison), western buckwheat 1 (WPS process), river buckwheat 1 (comparison), river buckwheat 1 (WPS process), rice buckwheat No. 1 (comparison) and rice buckwheat 1 (WPS process) respectively successively.
Fig. 2 diagram often organizes the percentage of seedgermination after 6 cylinders in bar diagram represent western buckwheat 1 (comparison), western buckwheat 1 (WPS process), river buckwheat 1 (comparison), river buckwheat 1 (WPS process), rice buckwheat No. 1 (comparison) and rice buckwheat 1 (WPS process) respectively successively.
Detailed description of the invention
In order to the technical characteristic of the present invention, purpose and effect are more clearly understood from, now comparison accompanying drawing describes the specific embodiment of the present invention in detail.
One, the separation and Culture of fusarium oxysporum Fataf9 and qualification
Separate purification
1, Radix Et Rhizoma Fagopyri Tatarici processes: choose Radix Et Rhizoma Fagopyri Tatarici root and the stem of health, to its rhizome surface first with 70% Ethanol Treatment 30s, then process 20min with 0.2% mercuric chloride and carry out surface sterilization, remove the rhizome epidermis of Radix Et Rhizoma Fagopyri Tatarici, root, stem are divided into the block section of about 0.5cm × 0.5cm × 0.5cm size, are placed in potato dextrose agar (PDA) culture medium.
2, cultivate: first suppress antibacterial to disturb by PDA culture medium adds 200 μ g/mL streptomycin sulfate (Streptomycinsulfate), be then placed at 25 DEG C and carry out light culture.
3, purification is separated: within the 3rd day, start to observe from cultivating, observe the fungus colony grown in Radix Et Rhizoma Fagopyri Tatarici root, stem piece of tissue in PDA plate, be easily separated cultivation with its edge mycelia of Inoculating needle picking under aseptic technique；It is easily separated purification to being separated the fungus colony obtained further, continuous purification 4-5 time, consistent to colonial morphology, namely obtain Radix Et Rhizoma Fagopyri Tatarici fusarium oxysporum Fataf9.
Then Radix Et Rhizoma Fagopyri Tatarici fusarium oxysporum Fataf9 is saved on PDA test tube slant and flat board, and it is carried out morphological characteristic observation and molecular biology identification.
Cultural colony is observed
Bacterium cake will be bought by well-grown fusarium oxysporum Fataf9 on PDA plate, bacterium cake diameter is Ф=5mm, each PDA plate is placed the cellophane of one piece of diameter 6cm, by on pure culture biscuits involvng inoculation to cellophane, dark culturing at 25 DEG C, the shape of every 24h observed and recorded Fataf9 bacterium colony, size, color and luster change, quality, speed of growth, whether chromogenesis etc..
This fusarium oxysporum (Fusariumoxysporum) 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.Carrying out it addition, fusarium oxysporum (Fusariumoxysporum) Fataf9 cultivates at aerobic condition, the advantageous pH range of culture medium is pH6.0～pH7.5.
Microexamination
Cellophane is taken off from PDA plate, is cut into 1cm2Size, it is placed on microscope slide, after making specimen with sterilized water for floating supporting agent, examine under a microscope the details such as mycelial thickness, the inserted part of conidiome, shape, type, size, product spore mode, and conidial mode of sprouting, whether have every, the morphological characteristic such as size, shape, surface features, color and luster.
Bacterium colony on PDA plate is purple, edge white, back wheel stricture of vagina shape, white and purple are alternate, and colony edge is neat, and aerial hyphae is longer, the nearly sickleshaped of megaspore or oblong, 2～3 every, sporidiole is oval or ellipse, and two kinds of spore sizes are (4.3 μm～17.5 μm) × (1.8 μm～4.7 μm).
Molecular biology identification
Activation Radix Et Rhizoma Fagopyri Tatarici fusarium oxysporum Fataf9 buys bacterium cake, and bacterium cake diameter is Ф=5mm, is inoculated on PD fluid medium, and 25 DEG C, results after light culture 6 days under 150rpm, vacuum filtration, to dry, gathers in the crops fresh mycelia.Then CTAB freeze-thaw method is adopted to extract the genomic DNA of fusarium oxysporum Fataf9.
Then select fungus universal primer: sequence 1:ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') and sequence 2:ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ') that the ITS district of fusarium oxysporum Fataf9 is carried out pcr amplification.The result that sequence after amplification obtains is compared with the fungus ITS complete sequence of other kind in GenBank, finds out the fungus that the highest sibship of similarity is nearest.
The ITS sequence (sequence 3) of described fusarium oxysporum is:
Two, described fusarium oxysporum Fataf9 application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment:
1, concrete application is to include following processing method:
A, fusarium oxysporum Fataf9 mycelia water propose the preparation of polysaccharide: cultivated by fusarium oxysporum mycelium inoculation, gather in the crops mycelium；Mycelium first removes monosaccharide, disaccharide and lipid material after drying, then water carries, refining after obtain mycelia water and carry polysaccharide.
Particularly as follows: from a little fusarium oxysporum mycelia of picking inclined-plane to activation culture on PDA plate, then by the fusarium oxysporum Fataf9 mycelium inoculation activated in the triangular flask filling PD culture medium, preferred Radix Et Rhizoma Fagopyri Tatarici powder Aqueous extracts containing 2.5g/L in PD culture medium, by it at 25 DEG C, results mycelium after light culture 7d under 150rpm；Gained fusarium oxysporum Fataf9 mycelium is carried out vacuum lyophilization, pulverizes, adopt 95% ethanol: it is carried out circumfluence distillation by petroleum ether (1:1, v/v), to remove monosaccharide, disaccharide and lipid material.Then weigh the fusarium oxysporum Fataf9 mycelia powder of quantitative pretreatment, adopt 90 DEG C of hot water returns extractions to obtain fusarium oxysporum Fataf9 mycelia water in conjunction with the method such as precipitate with ethanol, dialysis and carry polysaccharide (WPS).
B, purity testing: measure fusarium oxysporum Fataf9 mycelia water and carry sugared content and corresponding purity, the with glucose as a standard product Criterion curve of polysaccharide；Purity testing is to adopt sulfuric acid anthrone colorimetric method to measure fusarium oxysporum Fataf9 mycelia water to put forward the sugared content of polysaccharide and corresponding purity.
C, treatment fluid preparation: taking mycelia water and carry polysaccharide and be dissolved in sterilized water, adjusting its concentration is 15-25mg/mL, after filtration treatment fluid is standby；Filter type can adopt multiple, and the present embodiment adopts and filters through microporous filter membrane (0.4 μm).
D, Radix Et Rhizoma Fagopyri Tatarici seed disinfection: choose mature and plump, in the same size, without the Radix Et Rhizoma Fagopyri Tatarici seed disinfectant soaking disinfection that goes mouldy, then clean with sterilized water, dry；Disinfectant adopts 0.5% liquor natrii hypochloritis, it would however also be possible to employ other are suitable for the disinfectant of the present invention.Sterilization soak time is 10-15min.
E, Radix Et Rhizoma Fagopyri Tatarici seed treatment: the Radix Et Rhizoma Fagopyri Tatarici seed after sterilization is placed in the treatment fluid obtained by step C, 25 DEG C, soak 10-16h under dark condition, the volume ratio of Radix Et Rhizoma Fagopyri Tatarici seed weight and treatment fluid is 1:10 (m/v), and namely the seed after process can be used for planting.
The germination percentage of Radix Et Rhizoma Fagopyri Tatarici seed is verified by germination culture experiment.
The condition cultivated of germinateing is: cultivation temperature 25 DEG C, humidity 70-75%, every day, light application time was 10-12 hour.
The application of the present invention is described with specific embodiment below:
Embodiment 1
1, fusarium oxysporum Fataf9 application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment includes following processing method:
(1) fusarium oxysporum Fataf9 mycelia water proposes the preparation of polysaccharide:
A. the fermentation culture of fusarium oxysporum Fataf9: from a little fusarium oxysporum mycelia of picking test tube slant to activation culture on PDA plate 5 days, then by the fusarium oxysporum mycelium inoculation of activation in PD culture medium, wherein need to add the Radix Et Rhizoma Fagopyri Tatarici powder Aqueous extracts 20mL meter of 20.0-25.0g/L according to every 200mLPD solution, PD culture medium adds Radix Et Rhizoma Fagopyri Tatarici powder Aqueous extracts.After inoculation in 25 DEG C, 150rpm low suspension cultivate results after 7 days, centrifugal (4000rpm) obtains fusarium oxysporum Fataf9 fresh mycelia.
B. fusarium oxysporum Fataf9 mycelia water proposes the preparation of polysaccharide:
The gained fresh mycelium of fusarium oxysporum Fataf9 is carried out vacuum lyophilization, pulverize, then 95% ethanol is adopted: petroleum ether (1:1, v/v) it is carried out circumfluence distillation 2h at 50-55 DEG C, to remove monosaccharide in mycelium, disaccharide and lipid material, centrifugal, collect residue, dry at 50-55 DEG C.
Water carries: weigh quantitative pretreatment Fataf9 mycelia powder, it is placed in circumfluence distillation device, add a certain proportion of distilled water and carry out circumfluence distillation, extraction conditions is: ratio of water to material is 25:1-30:1 (v/w), Extracting temperature 90 DEG C, extraction time 2-3h, extract 2 times altogether, united extraction liquid.
Separation and purification: by centrifugal mode extraction fluid and mycelium residue, centrifuged supernatant is concentrated into certain volume, adds 3 times of volume 95% ethanol, 48～72h is precipitated at 4 DEG C, then adopt centrifugation precipitation and supernatant to be separated, collect precipitation, successively with dehydrated alcohol, washing with acetone.
Further its solution is placed in bag filter and dialyses, an extracellular fluid dialysis (the present embodiment adopts deionized water as extracellular fluid dialysis) is changed every 12h, after 48h, solution in bag filter is carried out vacuum lyophilization, obtain Radix Et Rhizoma Fagopyri Tatarici fusarium oxysporum Fat9 mycelia water and carry polysaccharide (WPS).
C. the preparation for the treatment of fluid: take a certain amount of Radix Et Rhizoma Fagopyri Tatarici fusarium oxysporum Fat9 mycelia water and carry polysaccharide (through sulfuric acid anthrone colorimetric method mensuration, its purity reaches 85.6%), being dissolved in sterilized water, adjusting its concentration is 20.0mg/mL, standby after microporous filter membrane (0.4 μm) filters.
(2) the choosing of Radix Et Rhizoma Fagopyri Tatarici seed: select mature and plump, be of moderate size, uniformity, without the Radix Et Rhizoma Fagopyri Tatarici seed of health of going mouldy as material.
(3) sterilization of Radix Et Rhizoma Fagopyri Tatarici seed: the Radix Et Rhizoma Fagopyri Tatarici seed 0.5% liquor natrii hypochloritis immersion treatment 10min that will choose, then cleans with sterilized water, is placed on aseptic filter paper and dries.
(4) Fataf9 mycelia water puies forward polysaccharide induced and processes Radix Et Rhizoma Fagopyri Tatarici seed: be placed in the treatment fluid WPS solution obtained by (1) (concentration is adjusted to 150 μ g/mL) by the Radix Et Rhizoma Fagopyri Tatarici seed after sterilization, 25 DEG C, soak 12-16h under dark condition, the volume ratio of seed weight and mycelia polysaccharide solution is 1:10 (m/v).
(5) Radix Et Rhizoma Fagopyri Tatarici germination is cultivated: be uniformly placed in the germination box (120 × 120 × 50mm) spreading the moistening filter paper that haves three layers by the Radix Et Rhizoma Fagopyri Tatarici seed processed through step (4), 100 seeds of every box, then (temperature 25 DEG C in growth cabinet it is placed on, humidity 70-75%, light application time 12h/ days) carry out germinateing and cultivate, the germination cycle is 2-4d.After germination terminates, add up its germination percentage.
Embodiment 2
(1) fusarium oxysporum Fataf9 mycelia water puies forward the preparation method of polysaccharide and treatment fluid with embodiment 1.
(2) the choosing of Radix Et Rhizoma Fagopyri Tatarici seed: select mature and plump, be of moderate size, uniformity, without the Radix Et Rhizoma Fagopyri Tatarici seed of health of going mouldy as material.
(3) sterilization of Radix Et Rhizoma Fagopyri Tatarici seed: the Radix Et Rhizoma Fagopyri Tatarici seed 0.5% liquor natrii hypochloritis immersion treatment 10min that will choose, then cleans with sterilized water, is placed on filter paper and dries.
(4) Fataf9 mycelia water puies forward polysaccharide induced and processes Radix Et Rhizoma Fagopyri Tatarici seed: be placed in the treatment fluid WPS solution obtained by (1) (concentration is adjusted to 200 μ g/mL) by the Radix Et Rhizoma Fagopyri Tatarici seed after sterilization, 25 DEG C, soak 10-12h under dark condition, the volume ratio of seed weight and mycelia polysaccharide solution is 1:10 (m/v).
(5) Radix Et Rhizoma Fagopyri Tatarici germination is cultivated: be uniformly placed in the germination box (120 × 120 × 50mm) spreading the moistening filter paper that haves three layers by the Radix Et Rhizoma Fagopyri Tatarici seed processed through step (4), 100 seeds of every box, then (temperature 25 DEG C in growth cabinet it is placed on, humidity 70-75%, light application time 10h/ days) carry out germinateing and cultivate, the germination cycle is 2-4d.After germination terminates, add up its germination percentage.
Adopt following experimental verification effect of the present invention:
Contrast experiment 1:
The preprocess method improving Radix Et Rhizoma Fagopyri Tatarici seed germination carries out according to following steps:
The method adopting embodiment 1, the Radix Et Rhizoma Fagopyri Tatarici fusarium oxysporum Fataf9 mycelia water that preparation concentration is 150 μ g/mL carries polysaccharide (WPS) solution, standby.
Fresh Radix Et Rhizoma Fagopyri Tatarici seed (0 year) and the room temperature of choosing this season results store 0.5 year, 1 year, 1.5 years, 2.0 years, 2.5 years naturally, and the Radix Et Rhizoma Fagopyri Tatarici seed of 3.0 years (western buckwheat 1) is experiment material.
Difference is stored the Radix Et Rhizoma Fagopyri Tatarici seed 0.5% liquor natrii hypochloritis immersion treatment 10min of phase, then cleans with sterilized water, be placed on filter paper and dry.
Radix Et Rhizoma Fagopyri Tatarici seed after sterilization is respectively placed in WPS solution (concentration is adjusted to 150 μ g/mL), 25 DEG C, soak 16h under dark condition, the volume ratio of seed weight and mycelia polysaccharide solution is 1:10 (m/v).
Five, each Radix Et Rhizoma Fagopyri Tatarici seed after process is uniformly placed in the germination box spreading the moistening filter paper that haves three layers, 100 seeds of every box, be then placed in growth cabinet (temperature 25 DEG C, humidity 70-75%, light application time 12h/ days) carry out germinateing and cultivate.After germination terminates, add up its germination percentage.
Blank: by the Radix Et Rhizoma Fagopyri Tatarici seed of each storage time limit (1.0,1.5,2.0,2.5,3.0 years) without WPS solution-treated, and being only placed in sterilized water, soaking temperature, time and solid-liquid ratio are all consistent with above-mentioned processing method.
As shown in Figure 1, this contrast experiment 1 respectively stores the time limit (1.0 after Fataf9 mycelia polysaccharide solution-treated, 1.5, 2.0, 2.5, 3.0 years) Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination respectively reach 91%, 86.3%, 83.0%, 72.0%, 61.0%, it is followed successively by bar diagram the 3rd group in FIG respectively, 4th group, 5th group, 6th group neutralizes the 2nd cylinder in the 7th group, the germination percentage of corresponding blank is 82.3%, 74.7%, 56%, 35%, 21.3%, it is followed successively by bar diagram the 3rd group in FIG respectively, 4th group, 5th group, 6th group neutralizes the 1st cylinder in the 7th group, the germination percentage of the present invention is 1.1-2.9 times of blank, average sprout time effectively shortens to 60 hours.
Contrast experiment 2:
Naturally storing the Radix Et Rhizoma Fagopyri Tatarici seed " river buckwheat 1 " of 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years as experiment material using the fresh Radix Et Rhizoma Fagopyri Tatarici seed (0 year) of this season results and room temperature, all the other preprocess methods improving Radix Et Rhizoma Fagopyri Tatarici seed germination are identical with contrast experiment 1.
As shown in Figure 1, this experiment respectively stores the time limit (1.0 after Fataf9 mycelia polysaccharide solution-treated, 1.5, 2.0, 2.5, 3.0 years) Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination respectively reach 90.3%, 85.3%, 82.0%, 73.0%, 62.3%, it is followed successively by bar diagram the 3rd group in FIG respectively, 4th group, 5th group, 6th group neutralizes the 4th cylinder in the 7th group, the germination percentage of corresponding blank is 82.0%, 73.7%, 55.7%, 34.0%, 22.7%, it is followed successively by bar diagram the 3rd group in FIG respectively, 4th group, 5th group, 6th group neutralizes the 3rd cylinder in the 7th group, the germination percentage of the present invention is 1.1-2.7 times of blank, average sprout time effectively shortens to 60 hours.
Contrast experiment 3:
Naturally storing the Radix Et Rhizoma Fagopyri Tatarici seed " rice buckwheat 1 " of 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years as experiment material using the fresh Radix Et Rhizoma Fagopyri Tatarici seed (0 year) of this season results and room temperature, all the other preprocess methods improving Radix Et Rhizoma Fagopyri Tatarici seed germination are identical with contrast experiment 1.
As shown in Figure 1, this experiment respectively stores the time limit (1.0 after Fataf9 mycelia polysaccharide solution-treated, 1.5, 2.0, 2.5, 3.0 years) Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination respectively reach 91.3%, 86.0%, 81.0%, 69.0%, 60.3%, it is followed successively by bar diagram the 3rd group in FIG respectively, 4th group, 5th group, 6th group neutralizes the 6th cylinder in the 7th group, the germination percentage of corresponding blank is 83.0%, 76.0%, 58.0%, 38.0%, 22.3%, it is followed successively by bar diagram the 3rd group in FIG respectively, 4th group, 5th group, 6th group neutralizes the 5th cylinder in the 7th group), the germination percentage of the present invention is 1.1-2.7 times of blank germination percentage, average sprout time effectively shortens to 48 hours.
Contrast experiment 4:
The preprocess method improving Radix Et Rhizoma Fagopyri Tatarici seed germination carries out according to following steps:
According to embodiment 2, the Radix Et Rhizoma Fagopyri Tatarici fusarium oxysporum Fataf9 mycelia water that preparation concentration is 200 μ g/mL carries polysaccharide (WPS) solution as treatment fluid, standby.
Fresh Radix Et Rhizoma Fagopyri Tatarici seed (0 year) and the room temperature of choosing this season results store 0.5 year, 1 year, 1.5 years, 2.0 years, 2.5 years naturally, and the Radix Et Rhizoma Fagopyri Tatarici seed of 3.0 years (western buckwheat 1) is experiment material.
Difference is stored the Radix Et Rhizoma Fagopyri Tatarici seed 0.5% liquor natrii hypochloritis immersion treatment 10min of phase, then cleans with sterilized water, be placed on filter paper and dry.
Radix Et Rhizoma Fagopyri Tatarici seed after sterilization is respectively placed in WPS solution (concentration is adjusted to 200 μ g/mL), 25 DEG C, soak 12h under dark condition, the volume ratio of seed weight and mycelia polysaccharide solution is 1:10 (m/v), blank: each Radix Et Rhizoma Fagopyri Tatarici seed is placed in sterilized water, soaking temperature, time and solid-liquid ratio are all consistent with above-mentioned processing method.
Each Radix Et Rhizoma Fagopyri Tatarici seed after processing uniformly is placed in the germination box spreading the moistening filter paper that haves three layers, 100 seeds of every box, be then placed in growth cabinet (temperature 25 DEG C, humidity 70-75%, light application time 10h/ days) carry out germinateing and cultivate.
As shown in Figure 2, this experiment respectively stores the time limit (1.0 after Fataf9 mycelia polysaccharide solution-treated, 1.5, 2.0, 2.5, 3.0 years) Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination respectively reach 91.7%, 87.7%, 81.7%, 71.0%, 58.7%, it is followed successively by bar diagram the 3rd group in fig. 2 respectively, 4th group, 5th group, 6th group neutralizes the 2nd cylinder in the 7th group, the germination percentage of corresponding blank is 82.3%, 74.7%, 56%, 35%, 21.3%, it is followed successively by bar diagram the 3rd group in fig. 2 respectively, 4th group, 5th group, 6th group neutralizes the 1st cylinder in the 7th group, the germination percentage of the present invention is 1.1-2.8 times of blank, average sprout time effectively shortens to 48 hours.
Contrast experiment 5:
Naturally storing the Radix Et Rhizoma Fagopyri Tatarici seed " river buckwheat 1 " of 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years as experiment material using the fresh Radix Et Rhizoma Fagopyri Tatarici seed (0 year) of this season results and room temperature, all the other preprocess methods improving Radix Et Rhizoma Fagopyri Tatarici seed germination are identical with experiment four.
As shown in Figure 2, this experiment respectively stores the time limit (1.0 after Fataf9 mycelia polysaccharide solution-treated, 1.5, 2.0, 2.5, 3.0 years) Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination respectively reach 90.0%, 86.3%, 81.3%, 74.0%, 60.7%, it is followed successively by bar diagram the 3rd group in fig. 2 respectively, 4th group, 5th group, 6th group neutralizes the 4th cylinder in the 7th group, the germination percentage of corresponding blank is 82.0%, 73.7%, 55.7%, 34.0%, 22.7%, it is followed successively by bar diagram the 3rd group in fig. 2 respectively, 4th group, 5th group, 6th group neutralizes the 3rd cylinder in the 7th group, the germination percentage of the present invention is 1.1-2.7 times of blank, average sprout time effectively shortens to 54 hours.
Contrast experiment 6:
Naturally storing the Radix Et Rhizoma Fagopyri Tatarici seed " rice buckwheat 1 " of 0.5 year, 1.0 years, 1.5 years, 2.0 years, 2.5 years and 3.0 years as experiment material using the fresh Radix Et Rhizoma Fagopyri Tatarici seed (0 year) of this season results and room temperature, all the other preprocess methods improving Radix Et Rhizoma Fagopyri Tatarici seed germination are identical with experiment four, five.This experiment respectively stores the time limit (1.0 after Fataf9 mycelia polysaccharide solution-treated, 1.5, 2.0, 2.5, 3.0 years) Radix Et Rhizoma Fagopyri Tatarici percentage of seedgermination respectively reach 90.3%, 86.3%, 80.0%, 70.0%, 58.7%, it is followed successively by bar diagram the 3rd group in fig. 2 respectively, 4th group, 5th group, 6th group neutralizes the 6th cylinder in the 7th group, the germination percentage of corresponding blank is 83.0%, 76.0%, 58.0%, 38.0%, 22.3%, it is followed successively by bar diagram the 3rd group in fig. 2 respectively, 4th group, 5th group, 6th group neutralizes the 5th cylinder in the 7th group, the germination percentage of the present invention is 1.1-2.6 times of blank, average sprout time effectively shortens to 36 hours.
Sequence table
Claims (7)
1. a fusarium oxysporum, it is characterised in that fusarium oxysporum Fataf9 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.10102 on November 21st, 2014.
2. fusarium oxysporum according to claim 1, it is characterised in that have the feature that
Bacterium colony on PDA plate is purple, edge white, back wheel stricture of vagina shape, white and purple are alternate, and colony edge is neat, and aerial hyphae is longer, the nearly sickleshaped of megaspore or oblong, 2～3 every, sporidiole is oval or ellipse, and two kinds of spore sizes are (4.3 μm～17.5 μm) × (1.8 μm～4.7 μm).
3. fusarium oxysporum according to claim 1, it is characterised in that the ITS sequence of described fusarium oxysporum is:
4. the fusarium oxysporum application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment.
5. the fusarium oxysporum according to claim 4 application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment, it is characterised in that include following processing method:
A, fusarium oxysporum mycelia water propose the preparation of polysaccharide: cultivated by fusarium oxysporum mycelium inoculation, gather in the crops mycelium；Mycelium first removes monosaccharide, disaccharide and lipid material after drying, then water carries, refining after obtain fusarium oxysporum mycelia water and carry polysaccharide；
B, purity testing: measure fusarium oxysporum Fataf9 mycelia water and carry sugared content and corresponding purity, the with glucose as a standard product Criterion curve of polysaccharide；
C, treatment fluid preparation: taking fusarium oxysporum mycelia water and carry polysaccharide and be dissolved in sterilized water, adjusting its concentration is 15-25mg/mL, standby after filtration obtains treatment fluid；
D, Radix Et Rhizoma Fagopyri Tatarici seed disinfection: choose mature and plump, in the same size, without the Radix Et Rhizoma Fagopyri Tatarici seed disinfectant soaking disinfection that goes mouldy, then clean with sterilized water, dry；
E, Radix Et Rhizoma Fagopyri Tatarici seed treatment: the Radix Et Rhizoma Fagopyri Tatarici seed after sterilization is placed in obtained by step C and in the treatment fluid that dilutes, 25 DEG C, soak 10-16h under dark condition, the volume ratio of Radix Et Rhizoma Fagopyri Tatarici seed weight and treatment fluid is 1:10 (m/v).
6. the fusarium oxysporum according to claim 5 application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment, it is characterized in that, in described step A, when fusarium oxysporum mycelium inoculation is cultivated, adopting by the mycelium inoculation of activation in PD culture medium, described PD culture medium contains Radix Et Rhizoma Fagopyri Tatarici powder Aqueous extracts.
7. the fusarium oxysporum according to claim 5 application in Radix Et Rhizoma Fagopyri Tatarici seed germination pretreatment, it is characterised in that in described step E, the concentration of the treatment fluid of dilution is 150-200 μ g/mL.
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| CN107494541A (en) * | 2017-08-28 | 2017-12-22 | 成都大学 | The method of conditioning agent resistant to lodging and raising crops seedling stage lodging tolerance |
| CN109182144A (en) * | 2018-09-29 | 2019-01-11 | 江苏农林职业技术学院 | The fungal elicitor and the preparation method and application thereof for promoting purple dendrobium seed to sprout |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107494541A (en) * | 2017-08-28 | 2017-12-22 | 成都大学 | The method of conditioning agent resistant to lodging and raising crops seedling stage lodging tolerance |
| CN107494541B (en) * | 2017-08-28 | 2020-09-11 | 成都大学 | Lodging-resistant regulator and method for improving lodging resistance of crops in seedling stage |
| CN107432135A (en) * | 2017-09-18 | 2017-12-05 | 山西大学 | Promote the method for cynomorium songaricum seed sprouting using fungi |
| CN109182144A (en) * | 2018-09-29 | 2019-01-11 | 江苏农林职业技术学院 | The fungal elicitor and the preparation method and application thereof for promoting purple dendrobium seed to sprout |
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