CN106497800A - Starch method and the mould application that Chinese sorghum Phoma sp is separated on grass from safflower - Google Patents
Starch method and the mould application that Chinese sorghum Phoma sp is separated on grass from safflower Download PDFInfo
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- CN106497800A CN106497800A CN201611117036.3A CN201611117036A CN106497800A CN 106497800 A CN106497800 A CN 106497800A CN 201611117036 A CN201611117036 A CN 201611117036A CN 106497800 A CN106497800 A CN 106497800A
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- safflower
- phoma
- grass
- chinese sorghum
- bacterium
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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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
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a kind of starch method and the mould application for separating Chinese sorghum Phoma sp on grass from safflower, separation method is：The safflower slurry grass that collection is fallen ill, water cut the tissue that size is 5 × 5mm in the strong intersection of disease after rinsing；Tissue is immersed in alcohol and liquor natrii hypochloritis that mass fraction is 5% successively, is finally rinsed 3 times with aqua sterilisa, and be placed in PDA culture medium and cultivate；The bacterium colony of different shape is switched in new PDA culture medium and is cultivated and is repeated to purify；The present invention is by proposing the method for separating Chinese sorghum Phoma sp from the safflower slurry grass of morbidity, and by designing primer, the combining form characterization bacterium is Chinese sorghum Phoma sp；The preventive effect experiment carried out starching safflower grass using the bacterium, detection draw the bacterium in concentration 1 × 106‑1×109Careless preventive effect is starched during conidia/ml to safflower more than 80%, it was demonstrated that extract the prevention and control field that the Chinese sorghum Phoma sp for obtaining can be efficiently applied to starch safflower grass from safflower slurry grass of falling ill.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of slurries from safflower separate on grass Chinese sorghum Phoma sp (Phoma
Sorghina method) and application.
Background technology
Safflower slurry grass is commonly called as corymb woodsorrel herb or root, pintongs dish, Xi Jiangcao sections perennial root broad leaved weed, originates in America heat
Band area, is a kind of malignant weed of whole world distribution, and China north and south various places are distributed, and Kweiyang and solely is mainly originated in Guizhou
Mountain, initially introduces as ornamental plant in China, also becomes now farmland malignant weed；Safflower slurry grass itself has extremely strong
Fertility and energy for growth, are mainly bred with underground bulb, and through statistics, the serious plot of safflower slurry grass harm can make capsicum
The short crop failure 50%-75% such as underproduction 50%-60%, corn underproduction 30-50%, peanut, soybean are aggrieved the most serious
It is open country watermelon, loss reaches 50-85%, or even some plot reaches 100%, preventing and treating safflower slurry grass has become upland weedses is prevented
The important process for removing, in view of specializing in terms of the careless pests occurrence rule of relevant safflower slurry and preventing and treating report very few, so for
The careless decreasing and increasing law of safflower slurry, Development and reproduction characteristic etc. carry out systematic research, are that preventing and treating safflower slurry grass is carried
It is problem in the urgent need to address at present for theoretical foundation.
So be directed to the problems referred to above, the invention provides a kind of from safflower starch grass on separate Chinese sorghum Phoma sp method and
The mould application.
Content of the invention
It is an object of the invention to provide a kind of starch from safflower the method that separates Chinese sorghum Phoma sp on grass and this mould should
It is used for preventing and treating dry crop from safflower slurry grass infringement, to solve the problems, such as to propose in above-mentioned background technology.
For achieving the above object, the present invention provides following technical scheme：
A kind of method for starching separately Chinese sorghum Phoma sp (Phoma sorghina) on grass from safflower, comprises the following steps：
1) separate：
1.1) the safflower slurry grass that collection is fallen ill, first rinses surface with running water；5 × 5mm of size is cut in the strong intersection of disease
Some pieces of tissue；
1.2) by step 1.1) in the tissue of shearing immerse 30s in alcohol successively, immerse the hypochlorous acid that mass fraction is 5%
45s in sodium solution, is finally rinsed 3 times with aqua sterilisa, and is placed in PDA culture medium dark culturing 5 days in 28 DEG C of incubators；
1.3) by step 1.2) in the bacterium colony of different shape that obtains of culture be switched in new PDA culture medium and carry out
Culture purified, and repeat purifying 2-3 time；
2) Morphological Identification：By step 1.3) in the card punch of bacterium colony diameter 0.5cm that purified lay bacteria cake and be placed on
In PDA culture medium, colony characteristicses are observed, daily including form, color, quality, bacterium in dark culturing 7 days in 28 DEG C of incubators
Silk and conidium form；
3) molecular biology identification：The DNA of bacterium colony during kit extraction step sample 2), first by ITS universal primers,
Enter performing PCR, after PCR, carry out glue reclaim sequencing, sequencing result submits NCBI to, and No. Genbank is respectively：KX758542；According to ITS
Sequencing result is Phoma (Phoma sp.), designs primer LSU and TUB according to the conserved sequence of the category and designs 2 pairs of primers,
Enter performing PCR, after PCR, carry out glue reclaim sequencing, sequencing result submits NCBI to, and No. Genbank is respectively：、KY000557、
KY000558.
As further scheme of the invention：The step 2) in, colonial morphology is white, radial, densification；Mycelia is saturating
Bright nothing every；Conidium is monospore, ellipse；According to step 2) in Morphological Identification and step 3) in molecular biology mirror
Fixed, the bacterial classification is drawn for deuteromycetes (Fungi Imperficti), the mould mesh of shell (Sphaeropsicaceae), Phoma
(Phoma sp.) Chinese sorghum Phoma sp (Phoma sorghina).
With the safflower slurry grass that the conidial suspension of detached pure culture bacterium is seeded to Seedling Stage, after inoculation, 2d occurs
Obvious water soaking mode spot, rear blade move back green turn white, cut incidence tissue and be separately cultured again, the separator of gained is connect with original
Plant pathogen consistent, complete the checking of Ke's Huo Shi rules, it was demonstrated that separator is the pathogenic bacteria of safflower slurry grass.
Study its transformant and find that the bacterium, to sugarcane, paddy rice, wheat, seeding corn and other crops safety, does not have to eleusine indica and lady's-grass
Have pathogenic, safflower slurry grass is pathogenic strong, to show the bacterium have larger potential development into weeding biocontrol microorganisms.
Cultural method：The bacterium can be cultivated with following media, and induces generation conidium,
1st, PDA culture medium：Potato 200g, glucose 20g, agar 15g, water 1L；
2nd, safflower slurry grass leaching juice culture medium：Safflower slurry blade of grass piece (dry, crush, crossing 200 mesh sieves) 200g, glucose
20g, agar 15g, water 1L；
3rd, maize powder medium is beautiful：Ground rice 30g, glucose 20g, agar 15g, water 1L
4th, oat medium：Oatmeal 30g, glucose 20g, agar 15g, water 1L.
As further scheme of the invention：For the Chinese sorghum Phoma sp obtained using the method, its application is utilization
Chinese sorghum Phoma sp (Phoma sorghina) carries out the careless preventive effect experiment of safflower slurry, and experimental procedure is as follows：1) by Chinese sorghum Phoma sp
28 DEG C of condition bottom fermentations of PDB culture mediums 7 or 14 days, zymotic fluid spraying treatment safflower slurry grass is taken；
2) safflower slurry blade of grass piece, concentration 1 × 10 can be processed with conidium or the mycelium aqueous solution6-
109Conidia/ml, or conidium is mixedly configured into suspension with all kinds of adjuvants；
After testing, Chinese sorghum Phoma sp starches careless preventive effect more than 80% to safflower, shows Chinese sorghum Phoma sp effective for red
The preventing and treating of flower slurry grass.
Compared with prior art, the invention has the beneficial effects as follows：The invention provides a kind of safflower slurry grass from morbidity
On isolate the method for Chinese sorghum Phoma sp (Phoma sorghina), and the form by the Morphological Identification bacterium on PDA is special
Levy：Pycnidia size 150-220 μm, conidium size 3.5-8.5 × 2.2-3.0 μm, by molecular biology identification,
DNA is extracted and ITS, LSU and TUB is sequenced, and the combining form characterization bacterium is Chinese sorghum Phoma sp (Phoma sorghina)；
The condition of culture wide adaptability of the bacterium, can grow between pH4-9 and temperature 15-35 DEG C, and culture medium cost is inexpensively easily obtained,
Spore can be sprouted between 15-35 DEG C, to most of crop safety, further, by starching careless preventive effect experiment to safflower,
Detection draws the bacterium in concentration 1 × 106-1×109Careless preventive effect is starched during conidia/ml to safflower more than 80%, it was demonstrated that from suffering from
The prevention and control field that the Chinese sorghum Phoma sp for obtaining can be efficiently applied to starch safflower grass is extracted in sick safflower slurry grass.
Description of the drawings
Fig. 1 is Chinese sorghum Phoma sp of the present invention (Phoma sorghina) spore structural representation under an optical microscope.
Specific embodiment
Below in conjunction with the embodiment of the present invention, to the embodiment of the present invention in technical scheme be clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiment.In based on the present invention
Embodiment, the every other embodiment obtained under the premise of creative work is not made by those of ordinary skill in the art, all
Belong to the scope of protection of the invention.
Embodiment 1
Fig. 1 is referred to, and one kind starches the method that Chinese sorghum Phoma sp (Phoma sorghina) is separated on grass from safflower, including
Following steps：
1) sample：The safflower slurry grass that in March, 2015 falls ill in Guangxi Shanglin County farmland sample collection, is preserved with polybag
Take back laboratory standby；
2) separate：
2.1) sample pre-treatments：First sample surfaces are rinsed with running water, be then used by the strong intersection of scissor cut disease that sterilizes
Tissue, size 5mm*5mm are transferred to the sterilizing of 75% ethanol in point cup with tweezers；
2.2) sample treatment：30s is soaked in the separate cup equipped with absolute ethyl alcohol, it is 5% to be transferred to equipped with mass fraction
Liquor natrii hypochloritis sterilizing separate cup in, soak 45s, then with aqua sterilisa rinse 3 times, be transferred in PDA culture medium；
2.3) tissue cultures：PDA culture medium is placed in the water-impermeable incubator under the conditions of 28 DEG C, dark culturing 5 days；
2.4) purify：The bacterium colony of picking different shape is forwarded to the water isolation type in new PDA culture medium, under the conditions of 28 DEG C
In incubator, dark culturing is separated and is purified for 5 days, and repeats purifying 2-3 time；
3) Morphological Identification：The card punch of the bacterium colony diameter 0.5cm for having purified is laid bacteria cake and is placed on PDA culture medium
On, under the conditions of 28 DEG C in water-impermeable incubator dark culturing 7 days, daily observe colony characteristicses, including form, color, matter
Ground, mycelia and conidium form；Record colony colour, bacterium colony quality, and the speed of growth is measured with ruler；Cultivate the 7th day
When, mycelia, ascus and spore feature is detected under Nikon Ni-E light microscopes with transfer needle picking mycelia and spore：Bacterium colony shape
State is white, radial, fine and close；The transparent nothing of mycelia every；Conidium is monospore, ellipse；Chinese sorghum Phoma sp (Phoma of the present invention
Sorghina) architectural feature is as shown in Figure 1 under an optical microscope；
4) molecular biology identification：Sampling kit extracts the DNA of bacterium colony, enters performing PCR first by ITS universal primers,
Glue reclaim sequencing is carried out after PCR；According to ITS sequencing results, then 2 pairs of primers of design LSU and TUB designs, enter performing PCR, after PCR
Carry out glue reclaim sequencing；Sequencing result submits NCBI to, and No. Genbank is respectively：KX758542, KY000557, KY000558,
ITS designs primer sequence for KX758542, and LSU designs primer sequence for KY000557, and TUB design primer sequences are
KY000558, primer gene order is as shown in sequence table.
Identify that the isolated bacterial classification of the present invention is Chinese sorghum Phoma sp (Phoma according to morphology and molecular biology result
sorghina).
The Chinese sorghum Phoma sp (Phoma sorghina) that the present invention is isolated is used for the preventing and treating for starching grass to safflower, concrete anti-
Effect experiment is as follows：
Spore concentration 1 × 106-1×109Spore/ml, or 1 × 106The mycelium suspended liquid of -1.0 × 109cfu/mL, spray
Mist process.
1) pure culture biscuits involvng inoculation for taking diameter 5mm is beaten in PDB fluid nutrient mediums from the colony edge of culture 5d, cultivate 7 Hes
14d, takes zymotic fluid spraying treatment safflower slurry grass；
2) compound concentration is 1 × 106-1.0×109The mycelium suspended liquid of cfu/mL processes safflower sauce grass, and per plant sprays
10mL..
3) after testing, to the careless preventive effect of safflower slurry more than 80%.
Fatal rate and pathogenic situation to other weeds is as follows：
Pathogenic 0 represent not pathogenic；1 necrosis for representing 1-25%；2 represent 26-50% necrosis；3 represent 51-
75% necrosis；4 represent 76-99% necrosis；5 represent entirely extremely.
Compared with prior art, the invention has the beneficial effects as follows：The invention provides a kind of safflower slurry grass from morbidity
On isolate the method for Chinese sorghum Phoma sp (Phoma sorghina), and the form by the Morphological Identification bacterium on PDA is special
Levy：Pycnidia size 150-220 μm, conidium size 3.5-8.5 × 2.2-3.0 μm, by molecular biology identification,
DNA is extracted and ITS, LSU and TUB is sequenced, and the combining form characterization bacterium is Chinese sorghum Phoma sp (Phoma sorghina)；
The condition of culture wide adaptability of the bacterium, can grow between pH4-9 and temperature 15-35 DEG C, and culture medium cost is inexpensively easily obtained,
Spore can be sprouted between 15-35 DEG C, to most of crop safety, further, by starching careless preventive effect experiment to safflower,
Detection draws the bacterium in concentration 1 × 106-109Careless preventive effect is starched during conidia/ml to safflower more than 80%, it was demonstrated that from falling ill
The prevention and control field that the Chinese sorghum Phoma sp for obtaining can be efficiently applied to starch safflower grass is extracted in safflower slurry grass.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of spirit or essential attributes without departing substantially from the present invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment is only wrapped
Contain an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical scheme in each embodiment can also form those skilled in the art through appropriately combined
Understandable other embodiment.
Claims (3)
1. a kind of from safflower starch grass on separate Chinese sorghum Phoma sp method, it is characterised in that comprise the following steps：
1) separate：
1.1) the safflower slurry grass that collection is fallen ill, first rinses surface with running water；5 × 5mm of size tissues are cut in the strong intersection of disease
Some pieces；
1.2) by step 1.1) in the tissue of shearing immerse 30s in alcohol successively, immerse the sodium hypochlorite that mass fraction is 5% molten
45s in liquid, is finally rinsed 3 times with aqua sterilisa, and is placed in PDA culture medium dark culturing 5 days in 28 DEG C of incubators；
1.3) by step 1.2) in the bacterium colony of different shape that obtains of culture be switched in new PDA culture medium and cultivated
Purifying, and repeat purifying 2-3 time；
2) Morphological Identification：By step 1.3) in the card punch of bacterium colony diameter 0.5cm that purify lay bacteria cake and be placed on PDA and train
On foster base, colony characteristicses are observed daily in dark culturing 7 days in 28 DEG C of incubators, including form, color, quality, mycelia and point
Zygoblast form；
3) molecular biology identification：During kit extraction step sample 2), the DNA of bacterium colony, first by ITS universal primers, is carried out
Glue reclaim sequencing is carried out after PCR, PCR, and sequencing result submits NCBI to, and No. Genbank is respectively：KX758542；It is sequenced according to ITS
As a result it is Phoma (Phoma sp.), primer LSU and TUB is designed according to the conserved sequence of the category and designs 2 pairs of primers, carried out
Glue reclaim sequencing is carried out after PCR, PCR, and sequencing result submits NCBI to, and No. Genbank is respectively：KY000557、KY000558.
2. according to claim 1 from safflower starch grass on separate Chinese sorghum Phoma sp method, it is characterised in that
The step 2) in, colonial morphology is white, radial, densification；The transparent nothing of mycelia every；Conidium is monospore, ellipse
Shape；According to step 2) in Morphological Identification and step 3) in molecular biology identification, draw the bacterial classification be deuteromycetes
(Fungi Imperficti), the mould mesh of shell (Sphaeropsicaceae), Phoma (Phoma sp.) Chinese sorghum Phoma sp
(Phoma sorghina), with the safflower slurry grass that the conidial suspension of detached pure culture bacterium is seeded to Seedling Stage, connects
After kind, there is obvious water soaking mode spot in 2d, rear blade move back green turn white, cut incidence tissue and be separately cultured again, gained point
Consistent with former inoculation pathogen from thing, complete the checking of Ke's Huo Shi rules, it was demonstrated that separator is the pathogenic bacteria of safflower slurry grass；
Study its transformant and find that the bacterium, to sugarcane, paddy rice, wheat, seeding corn and other crops safety, is not caused to eleusine indica and lady's-grass
Characteristic of disease, safflower slurry grass are pathogenic strong, to show the bacterium have larger potential development into weeding biocontrol microorganisms；
Cultural method：The bacterium can be cultivated with following media, and induces generation conidium,
1.PDA culture mediums：Potato 200g, glucose 20g, agar 15g, water 1L
2. safflower slurry grass soaks juice culture medium：Safflower slurry blade of grass piece (dry, crush, cross 200 mesh sieves) 200g, glucose 20g,
Agar 15g, water 1L
3. maize powder medium：Corn flour 30g, glucose 20g, agar 15g, water 1L
4. oat medium：Oatmeal 30g, glucose 20g, agar 15g, water 1L.
3. the height for separating on grass that the method for Chinese sorghum Phoma sp is obtained is starched from safflower according to the arbitrary described utilization of claim 1-2
The application of fine strain of millet Phoma sp, it is characterised in that
1) by Chinese sorghum Phoma sp 28 DEG C of condition bottom fermentations of PDB culture mediums 7 or 14 days, zymotic fluid spraying treatment safflower slurry is taken
Grass；
2) conidium or the mycelium aqueous solution can be used to process safflower slurry blade of grass piece, concentration 1 × 106-1×109conidia/
Ml, or conidium is mixedly configured into suspension with all kinds of adjuvants.
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| CN201611117036.3A CN106497800A (en) | 2016-12-07 | 2016-12-07 | Starch method and the mould application that Chinese sorghum Phoma sp is separated on grass from safflower |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961782A (en) * | 2021-01-29 | 2021-06-15 | 湖南省烟草公司长沙市公司 | Pediococcus kaoliang, microbial inoculum and herbicide containing same and application thereof |
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| CN102382776A (en) * | 2011-12-09 | 2012-03-21 | 西南大学 | Small spore phoma microspora for controlling conyza sumatrensis |
| CN102657208A (en) * | 2012-05-30 | 2012-09-12 | 广西新晶科技有限公司 | Phytocide composition containing ethoxysulfuron and preparation method of phytocide composition |
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2016
- 2016-12-07 CN CN201611117036.3A patent/CN106497800A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382776A (en) * | 2011-12-09 | 2012-03-21 | 西南大学 | Small spore phoma microspora for controlling conyza sumatrensis |
| CN102657208A (en) * | 2012-05-30 | 2012-09-12 | 广西新晶科技有限公司 | Phytocide composition containing ethoxysulfuron and preparation method of phytocide composition |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961782A (en) * | 2021-01-29 | 2021-06-15 | 湖南省烟草公司长沙市公司 | Pediococcus kaoliang, microbial inoculum and herbicide containing same and application thereof |
| CN112961782B (en) * | 2021-01-29 | 2023-11-10 | 湖南省烟草公司长沙市公司 | Epicoccocus sorghum, microbial inoculum and herbicide containing same and application thereof |
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Application publication date: 20170315 |