CN108690811B - Maize smut haploid strain UM01 and application thereof - Google Patents
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Abstract
The invention discloses a maize Ustilago maydis haploid strain UM01 with the preservation number of CGMCC No. 15263. The maize smut haploid strain UM01 has strong pathogenicity after being combined with the haploid strain matched with the mating type, high inoculation success rate, accurate and reliable identification result of the maize anti-corn-tumor smut resistance of the maize variety and good consistency; when the method is used for producing the corn mushrooms, the corn mushrooms are high in yield and convenient to harvest and transport.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a maize smut haploid strain UM01 and application of the strain.
Background
Corn smut (or called corn smut) is a corn disease caused by corn smut (Ustilago maydis, or called corn smut, corn smut and the like), mainly occurs in warm and dry areas, and the spring sowing area is more serious than the summer sowing area, the general morbidity is 5-10%, and the serious can cause 30-80% of yield loss, which is a problem to be solved urgently.
In addition to causing damage to corn, the fungus gall formed on the corn ear after corn smut is infected with corn ear is a delicious food, and the azteck in mexico has been eating it for over 600 years and is called "mexico truffle", which is called corn mushroom, corn Usnea, corn smut, corn black mold, lollipop, Reda, black rice or black gall in China. The corn mushrooms can be eaten as raw materials or as fried dishes, and the taste is very delicious. Besides the edible value, the health-care food has extremely high medicinal value, and can supplement qi and nourish yin, supplement qi and tranquilize mind, tonify middle-jiao and detoxify, prevent and treat liver system and gastrointestinal tract ulcer, and help digestion and relax bowels after being eaten frequently; it can also be used for treating blood deficiency or body fluid deficiency, dry mouth, or fever, qi and yin impairment, vexation, fatigue, thirst, uneasiness, insomnia, and dreaminess; it can also be used for treating weakness of spleen and stomach, listlessness, anorexia, abdominal pain, food poisoning, etc. In addition, the protein content of corn mushrooms is among the highest of all mushroom families, is higher than that of corn itself, and particularly the content of lysine necessary for human body is very high. Therefore, the corn mushroom is an edible mushroom resource with great market development potential. Currently, corn mushrooms have been listed in the united states as edible mushroom ranks. Although farmers in China also have the tradition of eating the corn mushrooms, the method has not been carried out on a large scale and is only limited to sporadic and naturally-occurring corn mushrooms in the field.
The corn smut resistant identification of corn varieties and the production of corn mushrooms require the inoculation of maize smut. Patent "a method for producing maize mushroom and product" (CN10540409593A) discloses a method for producing maize mushroom by injecting the aqueous solution mixed with maize smut chlamydospore or basidiospore to the top of maize ear for inoculation, although the method is simple to operate, because other pathogenic bacteria are easily mixed in chlamydospore and are not easy to be found, the direct inoculation of maize smut chlamydospore aqueous solution can often cause the whole maize plant rot and the test fails; secondly, as the maize smut belongs to a heterogeneous combination mode for propagation, two kinds of basidiospores with affinity must be mixed and inoculated into a plant body together for success, but the existing method is to directly prepare the basidiospore mixture after the chlamydospores germinate into inoculation liquid for inoculation, the mixture has strong randomness and cannot necessarily ensure that the inoculation liquid contains haploid strains with affinity of two kinds, so that the result of artificial inoculation has great uncertainty, and the accuracy, the consistency and the repeatability of the test are poor. Thus, selectively compatible haploid strains of Ustilago zeae are a prerequisite for successful artificial inoculation.
Disclosure of Invention
The invention aims to provide a maize smut haploid strain, which has stronger pathogenicity after being combined with other haploid strains with affinity, and can be used for the resistance inoculation identification of the maize tumor smut and the production of maize mushrooms.
The second purpose of the invention is to provide a preparation method of the maize smut haploid strain inoculation mother liquor.
The third purpose of the invention is to provide a maize smut inoculation liquid.
The fourth purpose of the invention is to provide the application of the maize smut haploid strain in the identification of the resistance of the maize tumor smut.
The fifth purpose of the invention is to provide the application of the maize smut haploid strain in the production of maize mushrooms.
The sixth purpose of the invention is to provide the application of the inoculation liquid in the identification of the corn tumor smut resistance.
The seventh purpose of the invention is to provide the application of the inoculation liquid in the production of corn mushrooms
The invention is realized by the following technical scheme:
the invention provides a maize Ustilago maydis haploid strain UM01 with the preservation number of CGMCC No. 15263.
The invention also provides a preparation method of the maize smut haploid strain inoculation mother liquor, which comprises the following steps:
(1) inoculating the UM01 strain stored at low temperature on a PDA plate culture medium, and culturing at 25-28 ℃ for 1-2 days under a dark condition to obtain an activated strain;
(2) picking the activated strain obtained in the step (1) by using an inoculating needle, then uniformly coating the activated strain on a PDA (personal digital Assistant) plate culture medium, and culturing for 3-5 days at the temperature of 25-28 ℃ under the dark condition to obtain a thallus for inoculation;
(3) preparing a haploid strain inoculation mother solution: and (3) scraping all the thalli obtained in the step (2) from the PDA plate culture medium by using a sterilized glass slide, and diluting the thalli on 1 culture dish plate (the diameter of the PDA plate culture medium culture dish is 9cm) by using 1L of sterilized water to obtain the inoculation mother liquor of the haploid strain UM 01.
The PDA plate culture medium in the step (1) or (2) of the preparation method comprises the following components in percentage by weight: 200g of potato, 20g of glucose, 20g of agar and 1000ml of distilled water.
The PDA plate culture medium is prepared by conventional method, peeling 200g of potato, cutting into small pieces, boiling in 800ml of distilled water for 20min, filtering with 4 layers of gauze, adding 20g of glucose and 20g of agar, boiling, diluting to 1000ml, and sterilizing at 121 deg.C for 20 min.
The application of the maize smut (Ustilago maydis) haploid strain UM01 in the identification of maize tumor smut resistance.
The application of the maize Ustilago maydis haploid strain UM01 in production of maize mushrooms is provided.
A maize Ustilago esculenta seed inoculum, which contains the maize Ustilago esculenta haploid strain UM01 and another maize Ustilago esculenta haploid strain with affinity to the maize Ustilago esculenta haploid strain.
The mating type of another maize smut haploid strain described above in the inoculum solution was mfa 1.
The volume ratio of the mother liquid inoculated with the haploid strain UM01 to the mother liquid inoculated with the haploid strain with affinity to the mother liquid in the inoculation liquid is 1: 1.
the inoculation liquid also contains 0.01 percent of Tween 20 by volume percentage.
The haploid basidiospore number of UM01 in the inoculation liquid is 1 × 105-1×106One per ml.
The preparation method of the inoculation liquid comprises the following steps: according to the following steps of 1: 1, mixing the inoculation mother liquor of the haploid strain UM01 obtained in the step (3) with another maize smut haploid strain inoculation mother liquor with affinity in equal proportion, and adding Tween 20 according to the proportion of 0.01% by volume percentage to obtain the maize smut inoculation liquid.
When the inoculation liquid is used, the thalli are required to be fully mixed or filtered, otherwise, a continuous injector is easy to block during inoculation.
The inoculation liquid is applied to the identification of the corn tumor smut resistance.
The application of the inoculation liquid in the production of corn mushrooms.
The invention has the advantages and beneficial effects that: (1) the maize smut resistant strain can be used for identifying the resistance of the maize smut and for producing the maize smut, and the maize smut resistant strain is inoculated by an inoculation liquid obtained by mixing the maize smut haploid strain UM01 with the mating type of mfa1, the inoculation success rate is high, the inoculation success rate can reach 80 percent on a maize variety Vitaceae 966 with high maize smut sensitivity; (2) the inoculation liquid containing the haploid strain UM01 is used for carrying out the identification test of the disease resistance of the corn tumor smut, and the test result is accurate, stable and reliable and has good repeatability; (3) the corn mushroom produced by the inoculation liquid containing the haploid strain UM01 has high yield and is convenient to harvest and transport; (4) the haploid strain UM01 can be used as a starting strain for genetic engineering transformation to improve the variety of the maize tumor smut, such as increasing the content of beneficial components of the maize tumor smut, such as polysaccharide, anthocyanin and the like, to human bodies.
Biological preservation
The corn smut (Ustilago maydis) haploid strain UM01 is obtained by screening by the inventor of the invention, is stored in the common microorganism center of China Committee for culture Collection of microorganisms in 1 month and 16 days 2018, has the storage address of the institute of microbiology of China academy of sciences No. 3 of North Chen West Lu No.1 Hospital in the sunward area of Beijing, and has the storage number of: CGMCC No. 15263.
Drawings
FIG. 1 shows an electrophoretogram for selection of haploid strains and identification of mating types; wherein, No.1, No. 2, No. 4, No. 5, No. 9 and No. 10 are haploid mixed strains which are not successfully subjected to monospore isolation, and No. 3, No. 6, No. 7, No. 8 and No. 11 are haploid strains; wherein the mating types of haploid strains No. 3 and No. 11 are mfa 1; the mating types of haploid strains No. 6, 7 and 8 were mfa 2.
FIG. 2 mating type detection of Ustilago zeae haploid strain UM 01; wherein 1-3 is the haploid strain UM01 of the invention; 4 is chlamydospore germination isolate which is not monosporally separated; blank control 5.
FIG. 3 is a phylogenetic tree diagram of Ustilago zeae haploid strain UM01 of the present invention.
Detailed Description
The invention is further illustrated and described by the following examples, which do not limit the scope of the invention in any way. Unless otherwise specified, the methods described in the following examples are conventional methods, and the chemical agents are commercially available.
Example 1 isolation, screening and identification of haploid strains of Ustilago zeae of the invention
The method comprises the following steps:
(1) collecting the winter spores and preparing a winter spore suspension: collecting naturally air-dried corn nodule black powder galls in Hainan at 2016 for 12 months, directly picking a small amount of winter spores from the corn nodule black powder galls, placing the small amount of winter spores in a sterilized 2ml centrifuge tube, adding 1ml of sodium hypochlorite solution with the weight percentage of 1%, reversing and uniformly mixing for 2min, centrifuging at 12000rpm for 1min, removing supernatant, adding sterilized water to clean precipitates for 3 times, and finally diluting the precipitates with sterilized water to obtain a winter spore suspension with the final concentration of 103Spores per ml.
(2) And (3) culturing the winter spores: and (2) coating 200 mu l of the winter spore suspension obtained in the step (1) on a PDA culture medium, and culturing for 2 days at 25 ℃ to observe macroscopic tiny colonies.
(3) And (3) single spore isolation culture: picking single macroscopic single colony with a sterilizing toothpick into 1ml of sterilizing water, shaking and uniformly mixing with a vortex oscillator, uniformly coating 200 mu l of the single macroscopic single colony on a PDA (personal digital Assistant) plate culture medium, and culturing for 2 days at 25 ℃, wherein the grown single colony is the haploid basidiospore colony to be screened and identified.
(4) Identification of haploid strains: the a site controlling genetic mating in maize smut normally shares 2 mating types mfa1 and mfa 2. Only one mfa gene may be present in one haploid strain, whereas mfa genes in two sexually compatible haploid strains are necessarily different. Specific primers were therefore designed to examine the mating types of the isolated haploids and to judge their affinity for each other. Carrying out PCR amplification by using DNA of the haploid basidiospore to be identified obtained in the step (3) as a template and using the following primers Umfa1F, Umfa1R, Umfa2F and Umfa2R, wherein the primer sequences are as follows:
Umfa1F：5'-CCTGGGAGGTTGACAAAGAA-3'，
Umfa1R：5'-CCTTGAGACAAGCGAAGTCC-3'；
Umfa2F：5'-CCAACCACACACCCTCTTCT-3'，
Umfa2R：5'-CTGCAATTGCTCTGGAAACA-3'。
using Umfa1F and Umfa1R as primers to perform PCR amplification, wherein the size of the obtained amplification product is 549bp, and using Umfa2F and Umfa2R as primers to perform PCR amplification, and the size of the obtained amplification product is 637 bp; the reaction system for PCR (25. mu.L) was: 0.5. mu.L of Umfa1F (10. mu. mol/L), 0.5. mu.L of Umfa1R (10. mu. mol/L), 0.5. mu.L of Umfa2F (10. mu. mol/L), 0.5. mu.L of Umfa2R (10. mu. mol/L), 12.5. mu.L of Es Taq MasterMix (century kang), 2. mu.L of DNA template, and ddH2O to 25. mu.L. The PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 40s, annealing at 50 ℃ for 40s, and extension at 72 ℃ for 1min for 30 cycles; extension at 72 ℃ for 10 min.
As a result (see FIG. 1), among the 11 strains to be identified obtained, only one band, which indicates a haploid strain, was amplified in the strains No. 3, No. 6, No. 7, No. 8 and No. 11, and was retained; wherein the size of the amplification products of the strains No. 3 and No. 11 is about 500bp, and the mating type is mfa 1; the sizes of the amplification products of the strains No. 6, No. 7 and No. 8 are about 600bp, and the mating types are mfa 2; the rest 1, 2, 4, 5, 9 and 10 are haploid mixed bacteria which are not successfully separated by monospore and are eliminated.
The PCR amplification of strain No. 7 was carried out according to the method described in step (4), and as a result (see FIG. 2), only one DNA fragment of about 600bp was amplified, which was again confirmed to be a haploid strain whose mating type was mfa2, and this haploid strain was named UM 01.
Example 2 taxonomic identification of the haploid Strain UM01 of the invention
The morphological characteristics of the strain are as follows: the spore of the strain is in a short rod shape, has no septum and has a length of 15-25 mu m.
(II) culture characteristics of the strain: on PDA plate culture medium, the colony is milk white, has wrinkles on the surface, is wet, is similar to yeast colony, but is more viscous than bacterial and yeast colony when being picked.
(III) molecular identification of the haploid strain UM01 of the invention:
using genome DNA of UM01 strain as a template, and using fungus universal primers ITS1 and ITS4 to perform PCR amplification on the ITS segment of the fungus genome, wherein the primer sequences are as follows:
ITS1：5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4：5’-TCCTCCGCTTATTGATATGC-3’。
the PCR reaction system (25. mu.L), wherein: ITS1 (10. mu. mol/L) 0.5. mu.L, ITS4 (10. mu. mol/L) 0.5. mu.L, Es Taq MasterMix (kang century) 12.5. mu.L, template 2. mu.L, plus ddH2O to 25. mu.L. The PCR reaction procedure: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 45s, extension at 72 ℃ for 1min, and 35 cycles; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃. Taking 5 mu L of amplification product to carry out 1% agarose gel electrophoresis, detecting the size of the PCR product under an ultraviolet transilluminator, directly delivering the PCR amplification product to bioengineering (Shanghai) GmbH for sequencing to obtain the ITS sequence (SEQ ID NO.1) of the strain UM 01. The sequencing result is subjected to Blast comparison on an NCBI website, the sequence homology of the haploid strain UM01 and the maize smut reaches 99%, meanwhile, MEGA5.2 software is utilized to construct a phylogenetic tree, and the result (shown in figure 3) that the haploid strain UM01 and the maize smut are polymerized together indicates that the haploid strain UM01 belongs to the maize smut (Ustilago maydis).
From the above analysis results, it can be seen that the haploid strain UM01 of the present invention belongs to smut maize (Ustilago maydis), and is a new haploid strain of smut maize, different from the existing smut maize.
EXAMPLE 3 preparation of mother liquor for inoculation of the haploid Strain UM01 of the invention
The method comprises the following steps:
(1) activating strains: inoculating UM01 strain stored at low temperature on PDA plate culture medium (prepared by peeling potato 200g, cutting into small pieces, boiling in 800ml distilled water for 20min, filtering with 4 layers of gauze, adding glucose 20g and agar 20g, boiling to reach volume of 1000ml, sterilizing at 121 deg.C for 20min), and culturing at 25 deg.C in dark for 1 day to obtain activated strain.
(2) Preparing thalli: and (2) picking a proper amount of the UM01 activated strain obtained in the step (1) by using an inoculating needle, uniformly coating the activated strain on a PDA (PDA) plate culture medium, and culturing for 3 days at the temperature of 25 ℃ in the dark to obtain UM01 thalli.
(3) Preparation of an inoculation mother liquor: scraping all the thalli obtained in the step (2) from a PDA plate culture medium by using a sterilized glass slide, and diluting the thalli on a plate of a 1-culture dish (the diameter of the culture dish is 9cm) by using 1L of sterilized water to prepare an inoculation mother solution of a haploid strain UM 01. The obtained haploid strain UM01 has a basidiospore number of 1 × 105-1×106One per ml.
Example 4: inoculation test of an inoculum containing the haploid Strain UM01
The method comprises the following steps:
(1) the inoculation mother liquors of haploid strains No. 3 and No. 11 of example 1, of mating type mfa1, were prepared as described in example 3, respectively.
(2) The mother inoculum of the haploid strain UM01 prepared in example 3 was mixed with the mother inoculum of haploid strain No. 3 or 7, respectively, according to a ratio of 1: 1, adding tween 20 according to the volume percentage of 0.01 percent, and uniformly mixing to respectively prepare an inoculum I mixed by UM01 and a haploid strain No. 3 and an inoculum II mixed by UM01 and a haploid strain No. 11. The thallus is well mixed or filtered, otherwise, the continuous syringe is easy to block during inoculation.
(3) The Vitaceae 966 of the corn variety with high susceptibility to the smut is sown in flowerpots of a test greenhouse of the plant protection research institute of agriculture and forestry academy of sciences in Hebei province in 2017 for 30 months, 60 plants are sown in each treatment, and seedlings are watered regularly. When the corn grows to 6-7 leaf stage, the inoculation liquid I or the inoculation liquid II prepared in the step (2) is inserted into the seedling stem side direction by a continuous injector for inoculation, and the inoculation amount is 2 ml; incidence was investigated 20 days after inoculation.
Results (see table 1) the incidence rate of corn smut after the combined inoculation of the haploid strain UM01 and the haploid strain No. 3 is 80.0%, and the incidence rate after the combined inoculation of UM01 and the haploid strain No. 11 is 61.1%; the haploid strain UM01 (strain No. 7) of the invention is combined with different haploid strains with compatible mating type mfa1, the combination can cause diseases after inoculation, and the inoculation success rate (or incidence rate) is higher; secondly, the incidence rate of different combinations is greatly different, wherein the combination of UM01 and haploid strain No. 3 has the strongest pathogenicity, and the incidence rate after inoculation is the highest, so that the method can be used for the resistance identification of corn tumor smut or the production of corn mushrooms. In addition, the test is carried out in a greenhouse, the corn plants grow weakly and the disease resistance is poor, so the incidence of diseases of each treatment is high in field tests.
TABLE 1 inoculation test of an inoculum mixture of the haploid strain UM01 of the invention with a strain having affinity for it
Example 5: comparison test of combined inoculation effect of haploid strain UM01 and haploid strain with affinity
Test materials (one): vitaceae 966 (high-infection corn tumor smut)
(II) test treatment:
(1) treatment 1: inoculum I (i.e., UM01 and haploid strain No. 3 combination) prepared in example 4.
(2) And (3) treatment 2: an inoculum prepared from Farinia zeae (Paecilomyces maydis) tuber spore collected in Hainan at 2016 (12 months) by the method described in "A production method and product of Zea mays" (201510937192.3).
(III) test method
(1) And 6, 19 days in 2017, sowing the Vitaceae 966 on a test farm of plant protection research institute of agriculture and forestry academy of sciences in Hebei province, wherein 2 rows of seeds are sown in each treatment, the row length is 5 meters, 20-25 plants are sown in each row, the row spacing is 0.6 meter, and the field management is similar to that of the common field management. And (4) starting inoculation when the 6-7 leaf stage of the corn is started.
(2) The basidiospore concentration in the inoculum solution of treatment 1 and treatment 2 was adjusted to 1X 10 with sterilized water5-1×106Piece/ml, the inoculation was performed by piercing the seedling stem side with a continuous syringe, with an inoculum size of 2 ml. Investigation was conducted 15 to 20 days after inoculation, 10 consecutive strains were randomly selected as one group for investigation, 3 groups were investigated in total, the number of diseases in each group was recorded, and the disease incidence in each group was calculated.
The result (shown in table 2) shows that the inoculation success rate (or incidence rate) of the treatment 1 is obviously higher than that of the treatment 2, and the variance of incidence rate data of each group of the treatment 1 is obviously smaller than that of the treatment 2, which shows that the incidence rate of inoculation liquid inoculation after the haploid strain UM01 is mixed with the haploid strain matched with the mating type of the haploid strain is higher than that of a haploid mixture of maize smut bacteria without haploid separation, the incidence rate is more stable, the test repeatability and consistency are good, and the test reliability is high.
TABLE 2 results of comparative inoculum inoculation test of the haploid strain UM01 of the invention
| Grouping | Incidence of treatment 1 (%) | Incidence of treatment 2 (%) |
| 1 | 40 | 30 |
| 2 | 50 | 40 |
| 3 | 40 | 10 |
| Mean value of | 43.3 | 26.7 |
| Standard deviation of | 4.7 | 12.5 |
Example 6: corn anti-corn-tumor smut identification test on corn varieties by using inoculation liquid containing haploid strain UM01
Test materials (one):
(1) hengyuan No. 5, Dongsan 335, and Zhongdan 909 (maize varieties that have been approved by maize varieties for differential resistance to corn smut);
(2) qi 319 (maize melanoma resistance identification disease-resistant control inbred line);
(3) fluid seed 478 (corn tumor smut resistance identification susceptible contrast inbred line).
(II) test method:
(1) and sowing the seeds on a test farm of a plant protection research institute of agriculture and forestry academy of sciences in Hebei province in 23.6.7.6.6 months in 2017, wherein each variety is planted with 2 rows, the row length is 5 meters, and the row spacing is 0.6 meter.
(2) Inoculating the corn in 6-7 leaf stage with the inoculation liquid I prepared in example 4, and inoculating by puncturing the seedling from the stem side direction by using a continuous injector, wherein the inoculation amount of each plant is 2 ml. Investigating 15-20 days after inoculation, recording the morbidity of each variety, calculating the morbidity, and identifying the disease resistance grade.
Results (see table 3) the disease resistance identification test result of the corn variety with the inoculation solution containing the haploid strain UM01 of the invention for resisting the corn smut is completely consistent with the disease resistance identification result of the corn variety in the published approval bulletins of the corn variety, which shows that the resistance identification result of the corn variety with the inoculation solution containing the haploid strain UM01 of the invention for resisting the corn smut is accurate and reliable, and the method can be used for the inoculation identification of the corn new variety for resisting the corn smut.
TABLE 3 identification of resistance to smut of corn of various varieties by using the inoculation solution containing UM01 of the present invention
Note: "HS" stands for high sensitivity, "S" stands for sensitive disease, "MR" stands for moderate resistance, "R" stands for disease resistance
Example 7: production of maize mushrooms test (one) test material using an inoculum containing the haploid strain of the invention UM 01: vitaceae 966 (high-infection tumor smut)
(II) test design:
(1) treatment 1: inoculum I prepared in example 4 (inoculum prepared by mixing the UM01 inoculum mother liquor of the present invention and the mating type matched haploid strain # 3 inoculum mother liquor of the present invention in equal proportions).
(2) And (3) treatment 2: an inoculum prepared from Farinia maydis (Ustilago maydis) winter spore collected in Hainan at 2016 (12 months) by the method of "A production method and product of Zea mays" (201510937192.3).
(III) test method:
(1) seeding 5 months and 5 days in 2017, and repeating for 3 times at each treatment line of 1 line. The row length is 5m, the row spacing is 60cm, the plant spacing is 25cm, and the field management is the same as the common field management. Inoculating before the corn filament is extracted.
(2) The basidiospore concentration in the inoculation liquid of treatment 1 and treatment 2 was 1X 105-1×106And 2ml of inoculation liquid is injected into the interior of the ear from the top of the female ear through a filament channel by using a continuous injector. And (5) harvesting 16-19 days after inoculation, and counting morbidity and corn mushroom yield.
TABLE 4 incidence and corn mushroom production test results of inoculation with an inoculum containing the haploid strain UM01 of the invention
Results (see table 4) the incidence of treatment 1 inoculated with the inoculum containing the haploid strain UM01 of the invention was 52.9%, amounting to a yield per mu of 325.7 kg; while the morbidity rate of inoculation with the inoculation liquid of the treatment 2 is 40.2 percent, the equivalent yield per mu is 233.8kg, and the morbidity rate and the yield per mu of the treatment 1 are obviously higher than those of the treatment 2. The result shows that the maize smut haploid strain UM01 has high inoculation success rate and can be used for producing maize tumor smut. And the whole cluster is mostly attacked before the filament is extracted in the inoculation period, and the produced corn mushrooms are wrapped inside the corn bracts, so that the yield is high, and the harvesting and the transportation are convenient.
Sequence listing
<110> institute of plant protection of academy of agriculture, forestry and science of Hebei province
<120> maize smut haploid UM01 and application thereof
<130> 2018S1210INH
<141> 2018-05-18
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 778
<212> DNA
<213> Ustilago maydis
<400> 1
ggcattttga tgagcttttt tttctgaggt gtggctcgca cctgtccaac taaacctgag 60
ctaccttttt tatataacac ggttgcatcg gtcggtctgt cgaaaccagt agcgctcata 120
gcgagcagcg tctggggaaa gacgggtcgg cgcttcttac caacactttt gaacactagg 180
attggaagga caaaaatcat ttttttgatg atggaagcga ctggtaatgc ggtcgtctaa 240
attgaaaaaa caacttttgg caacggatct cttggttctc ccatcgatga agaacgcagc 300
gaattgcgat aagtaatgtg aattgcagaa gtgaatcatc gaatctttga acgcaccttg 360
cgctcccggc agatttaatc tggggagcat gcctgtttga gggccgcgaa ttgtttcgaa 420
cgacagcttt tttttcttgt tgagaaagag ctggcggatc ggtagtgagg gtctctgcca 480
tttaccgtgg ctccctcgaa atgcattagc gcatccattg gaaggcggaa agacggacga 540
aagctcgagt ttttttgccc tcgcttccct gccgggtttt gataatgtca ggacttcgga 600
ggcgaagaga gggcgggagc tggacgcaac gacttttctg ctgtttggcg tgcttctgaa 660
ccccgcccat gcctctgctt taaccacaga ggaagggatt tatttttcaa ttcatcggcc 720
tcagattggt aggactaccc gctgaactta agcatatcaa aaggggggga gaaaaaaa 778
Claims (10)
1. A kind of corn smut bacteria (A)Ustilago maydis) Haploid strain UM01 with preservation number of CGMCC No. 15263.
2. The method of preparing an inoculated mother liquor of Ustilago zeae haploid strain UM01 as claimed in claim 1, characterized in that it comprises the following steps:
(1) inoculating the UM01 strain stored at low temperature on a PDA plate culture medium, and culturing at 25-28 ℃ for 1-2 days under a dark condition to obtain an activated strain;
(2) picking the activated strain obtained in the step (1) by using an inoculating needle, then uniformly coating the activated strain on a PDA (personal digital Assistant) plate culture medium, and culturing for 3-5 days at the temperature of 25-28 ℃ under the dark condition to obtain a thallus for inoculation;
(3) preparing a haploid inoculation mother solution: before field inoculation, the thalli obtained in the step (2) are completely scraped from a PDA plate culture medium by using a sterilized glass slide, and the thalli are diluted according to the proportion of 1L of sterilized water on 1 culture dish plate with the diameter of 9cm, so that the inoculation mother liquor of the haploid strain UM01 is obtained.
3. The method according to claim 2, wherein the PDA plate medium in step (1) or (2) comprises the following components in parts by weight: 200g of potato, 20g of glucose, 20g of agar and 1000ml of distilled water.
4. Ustilago zeae (Ustilago zeae) of claim 1Ustilago maydis) Application of haploid strain UM01 in identification of corn tumor smut resistance.
5. Ustilago zeae (Ustilago zeae) of claim 1Ustilago maydis) Application of haploid strain UM01 in production of corn mushroom is provided.
6. A Ustilago zeae inoculum comprising the Ustilago zeae of claim 1(Ustilago maydis) Haploid strain UM01 and another maize smut haploid strain with affinity for it; wherein the volume ratio of the inoculation mother liquor of the haploid strain UM01 in the inoculation liquid to the inoculation mother liquor of another maize Ustilago esculenta haploid strain with affinity to the inoculation liquid is 1: 1.
7. the inoculum of claim 6 wherein said another haploid strain of Ustilago zeae has a mating type mfa 1.
8. The inoculum according to claim 6 further comprising Tween 20 in a volume percent ratio of 0.01%.
9. The use of the inoculum of claims 6-8 for the identification of resistance to corn tumor smut.
10. Use of the inoculum of claims 6-8 in the production of corn mushrooms.
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| CN104939043A (en) * | 2015-06-18 | 2015-09-30 | 广西大学 | Ustilago maydis sauce |
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| CN102204465A (en) * | 2011-04-20 | 2011-10-05 | 广州甘蔗糖业研究所 | Method for identifying sugarcane smut resistance |
| CN104939043A (en) * | 2015-06-18 | 2015-09-30 | 广西大学 | Ustilago maydis sauce |
| 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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