CN108641967A - A kind of Ustilago maydis haploid strains UM02 and its application - Google Patents
A kind of Ustilago maydis haploid strains UM02 and its application Download PDFInfo
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Abstract
The invention discloses a kind of Ustilago maydis (Ustilago maydis) haploid strains UM02, deposit number is CGMCC No.15387.Pathogenicity is strong after Ustilago maydis haploid strains UM02 of the present invention is combined with the haploid strains that its mating type matches, and success ratio of inoculation is high, accurate to the anti-Ustilago maydis Resistance Identification result of corn variety, reliable, and consistency is good；Not only corn mushroom yield is high when for corn Mushroom production, and is wrapped in bract, convenient for harvesting and transport.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of Ustilago maydis haploid strains UM02 is further related to
The purposes of the bacterial strain.
Background technology
Ustilago maydis (or corn smut) be by Ustilago maydis (Ustilago maydis, or for jade
Rice smut, corn tumor smut etc.) caused by maize diseases, take place mostly in warm Arid Area, and spring sowing area compares summer sowing
Area's morbidity is serious, and general incidence is 5~10%, it is serious cause 30~80% production loss, be one urgently to be resolved hurrily
The problem of.
Other than causing damages to corn, the mycoceicidum formed on fruit ear after Ustilago maydis infection corn ear is also
It is a kind of ticbit, the Aztecs of Mexico eats the history that it has more than 600 years, is referred to as " Mexico's truffle ",
It is known as mould corn mushroom, corn crow in China, corn tumor smut, corn black mould, ear of maize packet, harvests head, Wu meter or black courages etc..It is beautiful
Rice mushroom can both eat raw, also can cooking eat, taste is very delicious.Other than with edible value, it also has high medicinal valence
Value, often edible energy supplementing qi and nourishing yin, invigorating qi for tranquilization, bowl spares removing toxic substances can prevent and treat liver system and gastrointestinal ulceration, and energy
Aid digestion and defaecation；It can also be used for the deficiency of blood or body fluid deficiency, mouth parched and tongue scorched or pyreticosis impairment of both QI and YIN are tired of the tired thirsty, mind not
Peace, insomnia and dreamful sleep person；It can be additionally used in weakness of the spleen and the stomach, burnout deficiency of food, gastral cavity abdomen is had a pain or the illnesss such as food, drug poisoning.In addition, beautiful
The protein content of rice mushroom belongs to one of highest in all mushroom families, than the protein content higher of corn itself,
Lysine content especially needed by human is very high.Therefore, corn mushroom is a kind of edible mushroom money of great market development potential
Source.Currently, corn mushroom is included in edible mushroom ranks by the U.S..Although China peasant also has the tradition for eating corn mushroom,
There are no being mass produced, it is only limitted to the fragmentary natually morbid corn mushroom in field.
The either identification of anti-Ustilago maydis or the production of corn mushroom of corn variety, is required for carrying out beautiful another name for Sichuan Province
The inoculation of broomcorn millet smut.Patent《A kind of corn Mushroom production method and product》(CN10540409593A) jade will be mixed with by disclosing
The aqueous solution of rice smut chlamydospore or basidiospore is injected into the method that corn ear top carries out inoculation production corn mushroom,
The method is although easy to operate, but due to being easy to be mixed into other pathogenic bacteria in chlamydospore and being not easy to find, so using corn
Smut chlamydospore aqueous solution is directly inoculated with, and entire plant is often made to rot to lead to test failure；Secondly as beautiful
Chinese sorghum Ustilago is bred in mycelium anastomosis mode, it is necessary to be mixed the affine basidiospore of two kinds of property and is inoculated into plant together
Strain could succeed in vivo, and to be basidiospore mixture after directly sprouting chlamydospore be made inoculation liquid to existing method connects
Kind, such mixture randomness is strong, might not can guarantee comprising the haploid strains that two kinds of property are affine in inoculation liquid, thus
The result of artificial bacteria inoculation is caused to there is very big uncertainty, the accuracy of experiment, consistency and repeatability are all very poor.Therefore, it selects
The Ustilago maydis haploid strains that selecting property is affine are the premises for successfully carrying out artificial infection.
Invention content
Present invention aims at a kind of Ustilago maydis haploid strains are provided, the haploid strains are affine with its property
Other haploid strains combine after have it is stronger pathogenic, can be used for Ustilago maydis resistance inoculated identification and corn mushroom
Mushroom produces.
The present invention second is designed to provide the preparation method of above-mentioned Ustilago maydis haploid strains inoculation mother liquor.
Third of the present invention is designed to provide a kind of Ustilago maydis inoculation liquid.
The present invention the 4th is designed to provide above-mentioned Ustilago maydis haploid strains and reflects in Ustilago maydis resistance
Purposes on fixed.
The present invention the 5th is designed to provide use of the above-mentioned Ustilago maydis haploid strains on corn Mushroom production
On the way.
The present invention the 6th is designed to provide purposes of the above-mentioned inoculation liquid on Ustilago maydis Resistance Identification.
The present invention the 7th is designed to provide purposes of the above-mentioned inoculation liquid on corn Mushroom production
The invention is realized by the following technical scheme：
The present invention provides a kind of Ustilago maydis (Ustilago maydis) haploid strains UM02, deposit number
For CGMCC No.15387.
The present invention also provides the preparation methods that above-mentioned Ustilago maydis haploid strains are inoculated with mother liquor, including walk as follows
Suddenly：
(1) by the UM02 inoculations of Cord blood on PDA plate culture medium, under 25 DEG C~28 DEG C, dark condition
Culture 1~2 day, obtains activated strains；
(2) activated strains for using gained in transfer needle picking step (1), are then uniformly coated on PDA plate culture medium,
It is cultivated 3~5 days under 25 DEG C~28 DEG C, dark condition, obtains inoculation thalline；
(3) preparation of haploid strains inoculation mother liquor：Thalline obtained by step (2) is trained from PDA plate with sterilizing glass slide
Support base on all scrape, and according to the ratio of the thalline 1L aqua sterilisas of (a diameter of 9cm of culture dish) on 1 culture dish tablet into
Row dilutes the inoculation mother liquor to get haploid strains UM02.
The constituent and its weight ratio of PDA plate culture medium described in above-mentioned preparation method step (1) or (2) be：
Potato 200g, glucose 20g, agar 20g, distilled water 1000ml.
The PDA plate culture medium is conventionally prepared, i.e., will be cut into small pieces after potato 200g peelings, be put into
It boils 20min in 800ml distilled water, after 4 layers of filtered through gauze, glucose 20g and agar 20g is proportionally added, it is fixed after boiling
Hold to 1000ml, 121 DEG C of sterilizing 20min.
Above-mentioned Ustilago maydis (Ustilago maydis) haploid strains UM02 is in Ustilago maydis Resistance Identification
On application.
Above-mentioned Ustilago maydis (Ustilago maydis) haploid strains UM02 answering on corn Mushroom production
With.
A kind of Ustilago maydis inoculation liquid contains above-mentioned Ustilago maydis (Ustilago in the inoculation liquid
Maydis) haploid strains UM02 and another Ustilago maydis haploid strains affine with its property.
The mating type of another Ustilago maydis haploid strains described in above-mentioned inoculation liquid is mfa2.
The inoculation mother liquor of haploid strains UM02 and another haploid strains affine with its property in the inoculation liquid
The volume ratio ratio being inoculated between mother liquor is 1：1.
The polysorbas20 for being also 0.01% containing percent by volume in above-mentioned inoculation liquid.
The basidiospore number of haploid strains UM02 described in above-mentioned inoculation liquid is 1 × 105-1×106A/ml.
The preparation method of above-mentioned inoculation liquid：Before use, according to 1：1 volume ratio is by haploid strains obtained by step (3)
The inoculation mother liquor equal proportion of the inoculation mother liquor of the UM02 another Ustilago maydis haploid strains affine with its property mixes, and presses
Polysorbas20, as Ustilago maydis inoculation liquid is added in the ratio for being 0.01% according to percent by volume.
Above-mentioned inoculation liquid using when pay attention to that thalline is mixed well or is filtered, otherwise inoculation when be easy to cause company
Continuous syringe blocks.
Application of the above-mentioned inoculation liquid on Ustilago maydis Resistance Identification.
Application of the above-mentioned inoculation liquid on corn Mushroom production.
The present invention has the advantage that and advantageous effect：(1) Ustilago maydis haploid strains UM02 of the present invention and property are used
The inoculation liquid for the haploid strains mixing gained that affine mating type is mfa2 is inoculated with corn, pathogenic strong, success ratio of inoculation
Height, success ratio of inoculation can be used for the jade of corn variety up to 80% in the big section 966 of corn variety of high sense boil smut
The production of rice gall smut Resistance Identification and corn mushroom；(2) with containing haploid strains UM02 of the present invention inoculation liquid into
Row Ustilago maydis Disease Resistance Identification is tested, and test result is accurate, reliable and stable, and repeatability and consistency are good；(3) with containing
The corn mushroom monomer weight of the inoculation liquid production of haploid strains UM02 of the present invention, yield is high, and convenient for harvesting and transport；(4)
The starting strain being transformed using haploid strains UM02 of the present invention as genetic engineering, can be used for the improvement of corn tumor smut, such as
The content etc. to human body beneficial ingredient such as polysaccharide, anthocyanidin in raising corn tumor smut.
Biological deposits
Ustilago maydis (Ustilago maydis) haploid strains UM02 of the present invention, certainly by the present inventor
Row screening obtains, and is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 2 7th, 2018,
Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, and deposit number is：CGMCC
No.15387。
Description of the drawings
Fig. 1 haploid strains screen and the identification electrophoresis pattern of mating type；Wherein No. 1, No. 2, No. 4, No. 5, No. 9 and 10
Number be failed single spore separation monoploid hybrid bacterial strain, No. 3, No. 6, No. 7, No. 8, No. 11 be haploid strains；Wherein No. 3 and
The mating type of No. 11 haploid strains is mfa1；The mating type of No. 6, No. 7 and No. 8 haploid strains is mfa2.
The mating type of Fig. 2 Ustilago maydis haploid strains UM02 detects；Wherein 1-3 is single times of Ustilago maydis
Body bacterial strain UM02；4 sprout isolate for the chlamydospore without single spore separation；5 be blank control.
The systematic growth tree graph of Fig. 3 Ustilago maydis haploid strains UM02.
Specific implementation mode
The present invention is made further explanation and description by the following examples, but the scope of the present invention is not constituted
Any restrictions.Unless otherwise instructed, the method described in following embodiment is conventional method, and the chemical agent can lead to
Cross commercial channel purchase.
The separation screening of 1 Ustilago maydis haploid strains of the present invention of embodiment and identification
It carries out as follows:
(1) it collects teleutospore and prepares teleutospore suspension：The corn tumor that in December, 2016 acquires natural air drying in Hainan is black
Powder mycoceicidum, the direct a small amount of teleutospore of picking is placed in the 2ml centrifuge tubes of sterilizing from corn tumor smut goitre, and weight is then added
The liquor natrii hypochloritis 1ml that percentage is 1% is measured, 1min is centrifuged under reverse mixing 2min, 12000rpm, supernatant, addition is gone to go out
Precipitation is finally diluted precipitation cleaning 3 times to get teleutospore suspension, final concentration of is 10 by bacterium water with sterile water3A spore
Son/ml.
(2) teleutospore is cultivated：The 200 μ l of teleutospore suspension obtained by step (1) are taken to be coated in PDA culture medium, at 25 DEG C
Lower culture 2 days, you can observe macroscopic tiny bacterium colony.
(3) single spore separation culture：With sterilizing toothpick picking, individually macroscopic single bacterium drops down onto in 1ml aqua sterilisas, uses whirlpool
It revolves oscillator and shakes mixing, take 200 μ l to be spread evenly across on PDA plate culture medium, cultivated 2 days at 25 DEG C, the single bacterium colony grown
As to be screened and identification sporidial colony.
(4) identification of haploid strains：The sites a of control genetic cross share 2 kinds of mating in usual Ustilago maydis
Type mfa1 and mfa2.One haploid strains is only possible to there are a kind of mfa genes, and in the affine haploid strains of two individual characteies
Mfa genes are inevitable different.Therefore design specific primer is for examining isolated haploid mating type and judging its that
This compatibility.Using the DNA of the basidiospore to be identified obtained by step (3) as template, with following Umfa1F, Umfa1R, Umfa2F
PCR amplification is carried out with Umfa2R primers, the primer sequence is：
Umfa1F：5'-CCTGGGAGGTTGACAAAGAA-3',
Umfa1R：5'-CCTTGAGACAAGCGAAGTCC-3'；
Umfa2F：5'-CCAACCACACACCCTCTTCT-3',
Umfa2R：5'-CTGCAATTGCTCTGGAAACA-3'.
Carry out PCR amplification by primer of Umfa1F and Umfa1R, gained amplified production size is 549bp, with Umfa2F and
Umfa2R is that amplified production size obtained by primer progress PCR amplification is 637bp；The reaction system (25 μ L) of PCR is：Umfa1F
(10 μm of ol/L) 0.5 μ L, Umfa1R (10 μm of ol/L) 0.5 μ L, Umfa2F (10 μm of ol/L) 0.5 μ L, Umfa2R (10 μm of ol/L)
0.5 μ L, Es Taq MasterMix (health is century) 12.5 μ L, 2 μ L of DNA profiling adds ddH2O to 25 μ L.The PCR reactions
Program is：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 40s, 50 DEG C of annealing 40s, 72 DEG C of extension 1min, totally 30 recycle；72 DEG C are prolonged
Stretch 10min.
As a result in 11 bacterial strains to be identified obtained (see Fig. 1), No. 3, No. 6, No. 7, No. 8 and No. 11 bacterial strains only amplify
One band, explanation are haploid strains, are retained；The amplified production size of wherein No. 3 and No. 11 bacterial strains is 500bp or so,
Its mating type is mfa1；The amplified production size of No. 6, No. 7 and No. 8 bacterial strains is 600bp or so, mating type mfa2；Remaining
1,2,4,5,9 and 10 be failed single spore separation monoploid Mixed Microbes, eliminated.
PCR amplification is carried out according to No. 3 bacterial strains of method pair described in step (4), one is as a result only amplified (see Fig. 2)
The DNA fragmentation of 500bp or so demonstrates again that it is haploid strains, which is named as by mating type mfa1
UM02。
The taxonomic identification of 2 haploid strains UM02 of the present invention of embodiment
(1) morphological feature of the bacterial strain：The spore of the bacterial strain is in corynebacterium, no diaphragm, 15-25 μm of length.
(2) cultural character of the bacterial strain：On PDA plate culture medium, bacterium colony is creamy white, and there is fold on surface, wetter
Profit, similar saccharomycete bacterium colony, but it is more more viscous than the bacterium colony of bacterium and saccharomycete when picking.
(3) Molecular Identification of haploid strains UM02 of the present invention：
Using the genomic DNA of UM02 bacterial strains as template, using fungi universal primer ITS1 and ITS4 to fungal gene group ITS
Section carries out PCR amplification, and the primer sequence is：
ITS1：5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4：5’-TCCTCCGCTTATTGATATGC-3’.
The PCR reaction systems (25 μ L), wherein：ITS1 (10 μm of ol/L) 0.5 μ L, ITS4 (10 μm of ol/L) 0.5 μ L,
Es Taq MasterMix (health is century) 12.5 μ L, 2 μ L of template add ddH2O to 25 μ L.The PCR response procedures：95℃
Pre-degeneration 5min, 95 DEG C of denaturation 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, 35 recycle；Last 72 DEG C of extensions 10min, 4
DEG C preserve.It takes 5 μ L amplified productions to carry out 1% agarose gel electrophoresis, PCR product size is detected under ultraviolet transilluminator, by PCR
Amplified production directly delivers the sequencing of bioengineering (Shanghai) limited liability company, obtains ITS sequence (the SEQ ID of bacterial strain UM02
NO.1).Sequencing result is subjected to Blast comparisons on the websites NCBI, as a result haploid strains UM02 of the present invention and maize are black
The sequence homology of powder bacterium reaches 99%, while utilizing MEGA5.2 software building phylogenetic trees, as a result UM02 (see Fig. 3) with
Ustilago maydis is aggregated to together, illustrates that bacterial strain UM02 belongs to Ustilago maydis (Ustilago maydis).
In summary analysis result is it is found that haploid strains UM02 of the present invention belongs to Ustilago maydis (Ustilago
Maydis), and it is different with existing Ustilago maydis bacterial strain, it is a kind of new Ustilago maydis haploid strains.
The preparation of embodiment 3 haploid strains UM02 of the present invention inoculation mother liquors
It carries out as follows:
(1) actication of culture：By the UM02 inoculations of Cord blood, in PDA plate culture medium, (preparation method is：By horse
It is cut into small pieces after the 200g peelings of bell potato, is put into 800ml distilled water and boils 20min, after 4 layers of filtered through gauze, glucose 20g is added
With agar 20g, 1000ml, 121 DEG C of sterilizing 20min are settled to after boiling) on, it cultivates 1 day, obtains under 25 DEG C, dark condition
Activated strains.
(2) prepared by thalline：It is uniformly coated on PDA with the UM02 activated strains of gained in the appropriate step of transfer needle picking (1)
On plating medium, is cultivated 3 days under 25 DEG C, dark condition, obtain UM02 thalline.
(3) it is inoculated with the preparation of mother liquor：It is with sterilizing glass slide that thalline obtained by step (2) is whole from PDA plate culture medium
It scrapes and is diluted with the ratio of 1L aqua sterilisas according to the thalline on 1 culture dish (a diameter of 9cm of culture dish) tablet, is configured to
The inoculation mother liquor of haploid strains UM02.In the inoculation mother liquor of the haploid strains UM02 obtained at this time basidiospore number be 1 ×
105-1×106A/ml.
Embodiment 4：The inoculation morbidity experiment of inoculation liquid containing haploid strains UM02
(1) No. 6, No. 7 and No. 8 haploid strains selected in embodiment 1 are prepared respectively according to method described in embodiment 3
Inoculation mother liquor.
(2) in use, the inoculation mother liquor of haploid strains UM02 prepared by embodiment 3 No. 6 affine with its property respectively,
The inoculation mother liquor of No. 7 and No. 8 haploid strains is respectively according to 1：1 volume ratio equal proportion mixing, and be according to percent by volume
Polysorbas20 is added in 0.01% ratio, is uniformly mixed, and inoculation liquid I, UM02 that UM02 is mixed with No. 8 haploid strains are made respectively
The inoculation liquid III that the inoculation liquid II, UM02 mixed with No. 7 haploid strains is mixed with No. 6 haploid strains.Pay attention to thalline
It mixes well or is filtered, otherwise be easy to causeing continuous syringe in inoculation blocks.
(3) on March 30th, 2017 in the experiment greenhouse of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, in flowerpot
The big section 966 of corn variety of the middle high sense gall smut of sowing, each 60 plants of processing sowing periodically water to seedling.Wait for jade
When 6~7 leaf phase of rice, with continuous syringe by inoculation liquid I, inoculation liquid II or inoculation liquid II prepared in step (2) from seedling
The piercing of stem side direction is inoculated with, inoculum concentration 2ml, 20 days Investigate incidences after inoculation.
As a result the haploid strains UM02 of the present invention that (is shown in Table 1) and No. 6, No. 7 or No. 8 affine haploid strains group splice graftings of property
Ustilago maydis incidence difference 26.3%, 80.0% and 66.7%, illustrates Ustilago maydis monoploid of the present invention after kind
Bacterial strain UM02 (No. 3 bacterial strains) can match with mating type (mating type mfa2) different haploid strains combine inoculation after can send out
Disease, and success ratio of inoculation (or incidence) is all higher；Secondly, incidence is poor between UM02 and the combination of different haploid strains
Different very big, the combination pathogenicity of wherein UM02 and No. 7 haploid strains are most strong, and incidence highest after inoculation can be used for corn tumor
Smut Resistance Identification and corn Mushroom production.In addition, this experiment, due to being to carry out in the greenhouse, plant growth is weaker,
Disease resistance is poor, so leading to manage incidence everywhere compared with incidence height in field experiment.
1 haploid strains UM02 of the present invention of table combines inoculation comparative test result with the affine different haploid strains of its property
Embodiment 5：Haploid strains UM02 of the present invention combines effect of inoculation contrast test with No. 7 haploid strains
(1) test material：Big section 966 (height sense Ustilago maydis)
(2) test process：
(1) 1 is handled：Inoculation liquid II (i.e. UM02 and No. 7 haploid strains combination) prepared by embodiment 4.
(2) 2 are handled：The Ustilago maydis teleutospore that in December, 2016 is acquired in Hainan is according to patent《A kind of corn
Mushroom production method and product》(201510937192.3) inoculation liquid prepared by the method described in.
(3) test method
On June 19th, (1) 2017 is sowed in the experimental plot of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, each to locate
2 rows are managed, row is 5 meters long, and 20-25 plants of often row sowing, 0.6 meter of line-spacing, field management is the same as general field management.Wait for -7 leaf of 6 leaf of corn
Phase starts to be inoculated with.
(2) basidiospore concentration in 2 inoculation liquids of processing 1 and processing is adjusted to 1 × 10 with aqua sterilisa5-1×106A/ml, makes
It is pierced into and is inoculated with from seedling stem side direction with continuous syringe, inoculum concentration 2ml.Investigated within 15-20 days after inoculation, when investigation with
Machine selects continuous 10 plants to be investigated as one group, investigates 3 groups altogether, records every group of morbidity number, calculates every group of incidence.
As a result the success ratio of inoculation (incidence) of (being shown in Table 2) processing 1 is apparently higher than processing 2, secondly, processing 1 each group morbidity
The variance of rate data is significantly less than processing 2, illustrates the monoploid to match with its mating type using haploid strains UM02 of the present invention
The mixed inoculation liquid inoculation incidence of bacterial strain is higher than the incidence of the smut monoploid mixture detached without monoploid, and
Morbidity is more stablized, and test repeatability, consistency are good, and test reliability is high.
2 haploid strains UM02 of the present invention of table combines inoculation incidence comparative test result with No. 7 haploid strains
| Grouping | Handle 1 incidence (%) | Handle 2 incidence (%) |
| 1 | 40 | 30 |
| 2 | 50 | 40 |
| 3 | 40 | 10 |
| Average value | 43.3 | 26.7 |
| Standard deviation | 4.7 | 12.5 |
Embodiment 6：The anti-black powder of corn tumor is carried out to corn variety using the inoculation liquid containing haploid strains UM02 of the present invention
Sick qualification test
(1) test material：
(1) permanent source 5, Dongdan 335 and middle list 909 (have not Ustilago maydis by what corn variety was authorized
With the corn variety of resistance)；
(2) neat 319 (the disease-resistant control self-mating systems of Ustilago maydis Resistance Identification)；
(3) 478 (the susceptible control self-mating systems of Ustilago maydis Resistance Identification) are tucked in.
(2) test method：
On June 23rd, (1) 2017 is sowed in the experimental plot of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, Mei Gepin
Kind 2 rows of plantation, row is 5 meters long, and 0.6 meter of line-spacing, field management is the same as general field management.
(2) it is inoculated in 6~7 leaf phase of corn, inoculation liquid II (UM02 and No. 7 list prepared according to embodiment 4 before inoculation
The combination of times body bacterial strain), it is pierced into and is inoculated with from seedling stem side direction using continuous syringe, every plant of inoculum concentration is 2ml.15 after inoculation~
It is investigated within 20 days, records the morbidity number of each kind, calculate incidence, and carry out disease-resistant level identification.
As a result (being shown in Table 3) carries out the black powder of anti-tumor using the inoculation liquid containing haploid strains UM02 of the present invention to corn variety
The Disease Resistance Identification test result of disease and the Disease Resistance Identification result of the kind in the corn variety authorization bulletin announced are complete
It is complete consistent, illustrate the disease resistance to the anti-gall smut of corn variety using the inoculation liquid containing haploid strains UM02 of the present invention
Qualification result is accurate, reliable, can be used for the inoculated identification of the anti-gall smut of corn variety.
Table 3 is using the inoculation liquid of the invention containing UM02 to the anti-gall smut Resistance Identification test result of different cultivars corn
Note：" HS " represents high sense, and " S " represents susceptible, and " MR " represents moderate resistance, and " R " represents disease-resistant
Embodiment 7：It is tested using the inoculation liquid production corn mushroom containing haploid strains UM02 of the present invention
(1) test material：Big section 966 (height sense gall smut)
(2) experimental design：
(1) 1 is handled：Inoculation liquid II prepared by embodiment 4 (UM02 is combined with No. 7 haploid strains).
(2) 2 are handled：The Ustilago maydis teleutospore that in December, 2016 is acquired in Hainan according to《A kind of corn mushroom
Production method and product》(201510937192.3) inoculation liquid prepared by the method.
(3) test method：
On May 5th, (1) 2017 is sowed, and 1 row, 3 repetitions are often handled.Row long 5m, line-spacing 60cm, spacing in the rows 25cm, field pipe
Reason is the same as general field management.The inoculation before corn capillament is extracted out.
(2) basidiospore concentration is 1 × 10 in 2 inoculation liquids of processing 1 and processing5-1×106A/ml, with continuous syringe from
Female tip of the spike is by filigree channel to fruit ear internal injection inoculation liquid 2ml.After inoculation is harvested for 16-19 days, and is counted
Incidence and corn mushroom yield.
Table 4 combines incidence and the examination of corn mushroom yield of inoculation with the UM02 of the present invention haploid strains affine with its property
Test result
As a result (be shown in Table 4) is with the incidence of the processing 1 of the inoculation liquid inoculation containing haploid strains UM02 of the present invention
52.9%, conversion per mu yield is 325.7kg；And be 40.2% with the incidence of the inoculation liquid of processing 2 inoculation, conversion per mu yield is
233.8kg, the incidence and per mu yield for handling 1 are all apparently higher than processing 2.Illustrate containing Ustilago maydis monoploid of the present invention
The combination of bacterial strain UM02 is pathogenic strong, and success ratio of inoculation is high, can be used for the production of corn tumor smut.And this experiment Inoculating date
Selection is before filigree extraction, and mostly entire fruit ear morbidity, not only yield is high, and is wrapped in inside bract, convenient for harvesting and transport.
Sequence table
<110>Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie
<120>A kind of Ustilago maydis monoploid UM02 and its application
<130> 2018S1211INH
<141> 2018-05-18
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 775
<212> DNA
<213> Ustilago maydis
<400> 1
tttttgatga accttttttt tctgaggtgt ggctcgcacc tgtccaacta aacctgagct 60
acctttttta tataacacgg ttgcatcggt cggtctgtcg aaaccagtag cgctcatagc 120
gagcagcgtc tggggaaaga cgggtcggcg cttcttacca acacttttga acactaggat 180
tggaaggaca aaaatcattt ttttgatgat ggaagcgact ggtaatgcgg tcgtctaaat 240
tgaaaaaaca acttttggca acggatctct tggttctccc atcgatgaag aacgcagcga 300
attgcgataa gtaatgtgaa ttgcagaagt gaatcatcga atctttgaac gcaccttgcg 360
ctcccggcag atttaatctg gggagcatgc ctgtttgagg gccgcgaatt gtttcgaacg 420
acagcttttt tttcttgttg agaaagagct ggcggatcgg tagtgagggt ctctgccatt 480
taccgtggct ccctcgaaat gcattagcgc atccattgga aggcggaaag acggacgaaa 540
gctcgagttt ttttgccctc gcttccctgc cgggttttga taatgtcagg acttcggagg 600
cgaagagagg gcgggagctg gacgcaacga cttttctgct gtttggcgtg cttctgaacc 660
ccgcccatgc ctctgcttta accacagagg aagggattta tttttcaatt catcggcctc 720
agattggtag gactacccgc tgaacttaag catatcaaaa aggcgggaga gaaaa 775
Claims (10)
1. a kind of Ustilago maydis (Ustilago maydis) haploid strains UM02, deposit number CGMCC
No.15387。
2. the preparation method of Ustilago maydis haploid strains UM02 inoculations mother liquor described in claim 1, it is characterised in that
Include the following steps：
(1) by the UM02 inoculations of Cord blood on PDA plate culture medium, 1 is cultivated under 25 DEG C~28 DEG C, dark condition
~2 days, obtain activated strains；
(2) activated strains for using gained in transfer needle picking step (1), are then uniformly coated on PDA plate culture medium, 25
DEG C~28 DEG C, cultivate 3~5 days under dark condition, obtain inoculation thalline；
(3) preparation of monoploid inoculation mother liquor：It is with sterilizing glass slide that thalline obtained by step (2) is flat from PDA before Field inoculation
It is all scraped on plate culture medium, and according to the ratio of the thalline 1L aqua sterilisas on 1 culture dish (a diameter of 9cm of culture dish) tablet
Example is diluted the inoculation mother liquor to get haploid strains UM02.
3. preparation method according to claim 2, it is characterised in that the PDA described in step (1) or (2) described in it is flat
The constituent and its weight ratio of plate culture medium be：Potato 200g, glucose 20g, agar 20g, distilled water 1000ml.
4. Ustilago maydis (Ustilago maydis) haploid strains UM02 described in claim 1 is in the black powder of corn tumor
Application on sick Resistance Identification.
5. Ustilago maydis (Ustilago maydis) haploid strains UM02 described in claim 1 gives birth in corn mushroom
Application in production.
6. a kind of Ustilago maydis inoculation liquid, it is characterised in that contain beautiful another name for Sichuan Province described in claim 1 in the inoculation liquid
Broomcorn millet smut (Ustilago maydis) haploid strains UM02 and another Ustilago maydis monoploid bacterium affine with its property
Strain；The wherein inoculation mother liquor of haploid strains UM02 and connecing for another haploid strains affine with its property in the inoculation liquid
Volume ratio ratio between kind mother liquor is 1：1.
7. inoculation liquid according to claim 6, it is characterised in that another Ustilago maydis haploid strains
Mating type is mfa2.
8. inoculation liquid according to claim 6, it is characterised in that be also containing percent by volume in the inoculation liquid
0.01% polysorbas20.
9. application of the inoculation liquid on Ustilago maydis Resistance Identification described in claim 6~8.
10. application of the inoculation liquid on corn Mushroom production described in claim 6~8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110501295A (en) * | 2019-08-15 | 2019-11-26 | 河北省农林科学院植物保护研究所 | A kind of method of indoor quickly screening prevention and treatment Ustilago maydis medicament |
Citations (1)
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|---|---|---|---|---|
| CN105409593A (en) * | 2015-12-15 | 2016-03-23 | 江苏省农业科学院 | Maize mushroom production method and products |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105409593A (en) * | 2015-12-15 | 2016-03-23 | 江苏省农业科学院 | Maize mushroom production method and products |
Non-Patent Citations (2)
| Title |
|---|
| EVELYN VOLLMEISTER等: "Fungal development of the plant pathogen Ustilago maydis", 《FEMS MICROBIOL REV》 * |
| 张美景等: "玉米黑粉菌单倍体菌株的分离及交配型a位点的鉴别", 《江苏农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110501295A (en) * | 2019-08-15 | 2019-11-26 | 河北省农林科学院植物保护研究所 | A kind of method of indoor quickly screening prevention and treatment Ustilago maydis medicament |
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