CN115340954A - Method for culturing mycelium of ustilago flagelliforme and application of mycelium - Google Patents

Method for culturing mycelium of ustilago flagelliforme and application of mycelium Download PDF

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CN115340954A
CN115340954A CN202211008908.8A CN202211008908A CN115340954A CN 115340954 A CN115340954 A CN 115340954A CN 202211008908 A CN202211008908 A CN 202211008908A CN 115340954 A CN115340954 A CN 115340954A
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basidiospore
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崔国兵
邓懿祯
袁梅婷
尹凯
毕新萍
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South China Agricultural University
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Abstract

The invention discloses a method for culturing sugarcane smut mycelium, which comprises the following steps: s1, selecting activated sugarcane black pollen fungus basidiospores of different mating types for culturing to obtain sugarcane black pollen fungus basidiospore mother liquor; s2, centrifuging the sugarcane black pollen bacterium basidiospore mother liquor obtained in the step S1, discarding the supernatant, re-suspending with sterile water, centrifuging, discarding the supernatant, re-suspending with sterile water and adjusting the OD of the basidiospore liquid 600 Obtaining sugarcane smut fungus basidiospore liquid with different mating types when the content is 0.8-1.2; s3, uniformly mixing sugarcane smut basidiospore liquid of different mating types in equal volume, dripping the mixed liquid onto a PDA solid culture medium, and culturing for 6-8 days at 26-30 ℃ to obtain the primary mycelium of sugarcane smut. Hair brushThe nascent mycelium obtained by the Ming culture can make up the defects existing in the current research of sugarcane smut bacteria, and can be used for the research on environmental stress of the sugarcane smut bacteria, biocontrol bacteria screening and the like.

Description

Method for culturing mycelium of ustilago flagelliforme and application of mycelium
Technical Field
The invention relates to the technical field of microbial culture, in particular to a culture method and application of sugarcane smut mycelium.
Background
Sugarcane is one of important economic crops, and the planting area of the sugarcane accounts for more than 90% of the total planting area of the sugar crops. Sugarcane diseases, especially fungal diseases, seriously affect the field yield of sugarcane, with the damage caused by sugarcane smut being the most serious. Through identification, the pathogenic bacteria of the sugarcane smut are the basidiomycotina smut fungi of the genus Sporisorium scabrosoides (Ss). The life history of the sugarcane smut can be divided into 3 different stages: haploid yeast sample basidiospore, binuclear hypha and diploid winter spore. The haploid basidiospores do not have the capacity of infecting sugarcane, and dinuclear mycelium formed by sexual matching of haploids of different mating types has pathogenicity and can penetrate through plant tissues to infect. The infected hypha absorbs nutrient substances in the host body and gradually spreads upwards along with the upward growth of the growing points of the sugarcane plants, finally, black penis is drawn out from the sugarcane tips, and a large amount of winter spores are filled in the black penis. The germ and the spores can be remotely spread along with the media such as airflow, rainwater and the like, and can also fall into the soil to survive for a long time. The conidia germinate basidiomycetes in different lengths under proper environmental conditions, each basidiomycetes cell can generate 1 to a plurality of haploid basidiospores, and hyphae formed by sexual matching of heterotypic basidiospores can infect the sugarcane plants again to complete reinfection.
Because the basidiospore of the sugarcane smut is haploid and has no pathogenicity, the basidiospore of the sugarcane smut forms binuclear hyphae with infection capacity after sexual matching. Therefore, sexual coordination is one of the key processes of pathogenicity of sugarcane smut. In recent years, the complex and sexual coordination molecular regulation mechanism has been analyzed and is dependent on the cAMP and MAPK pathway signaling pathways. Specifically, the method comprises the following steps: the mating allele a site encodes pheromone which is recognized by its opposite sex receptor protein Pra1/2, transmitting signals into the cells via the receptor protein. The cAMP and MAPK pathways are transmitted to a global regulatory factor Prf1 step by step, so that the b site expression of mating allele is influenced, and the sexual coordination is completed. The smut of sugarcane is not easy to be effectively controlled because the smut of sugarcane has a long latent period from the sugarcane infection to the disease attack and the symptom before the disease attack is not easy to be detected. Therefore, the pathogenic mechanism of the sugarcane smut can be deeply researched, and a theoretical basis can be provided for effectively preventing and treating the sugarcane smut. For example, chinese patent CN108552205A discloses the use of farnesol in the prevention and treatment of smut, chinese patent CN106942230A discloses the use of farnesol in the prevention and treatment of smut, chinese patent CN105925498A discloses pseudomonas strain ST4 and its application in the prevention and treatment of sugarcane smut, and the research on sugarcane smut in the prior art is mainly to research basidiospores and binary type transformation thereof. Due to the lack of a culture method of cane penis smut mycelium, the related research of the cane penis smut mycelium infection as a direct path for infecting the sugarcane is not developed, so that a molecular mechanism influencing the growth and pathogenicity of the cane penis smut mycelium is rarely reported.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provides a method for culturing sugarcane smut mycelium, which can effectively separate and culture the sugarcane smut mycelium. Overcomes the defects of only researching basidiospore and binary type transformation of the conventional sugarcane smut bacteria, and is successfully applied to the research on aspects of environmental stress of the sugarcane smut bacteria, biocontrol bacteria screening and the like.
The second purpose of the invention is to provide the application of the separated primary mycelium of the sugarcane smut bacteria in environmental stress and biocontrol bacteria screening of the sugarcane smut bacteria.
The above purpose of the invention is realized by the following technical scheme:
a method for culturing sugarcane smut mycelium comprises the following steps:
s1, respectively picking activated sugarcane smut basidiospores with different mating types (+, -) for culturing to obtain sugarcane smut basidiospore mother liquor;
s2, centrifuging the sugarcane smut fungus basidiospore mother liquor obtained in the step S1, and discarding supernatant without adding any other materialResuspending the basidiospore in sterile water, centrifuging, discarding the supernatant, resuspending in sterile water, and adjusting the OD of basidiospore solution 600 When the mating type is 0.8 to 1.2, obtaining sugarcane smut basidiospore liquid with different mating types (+, -);
s3, uniformly mixing the sugarcane smut basidiospore solutions with different mating types (+, -) obtained in the step S2 in equal volume, dripping the mixed solution onto a solid culture medium, and culturing for 6-8 days at 26-30 ℃ to obtain the sugarcane smut primary mycelium.
The method comprises the steps of carrying out sexual matching on haploid basidiospores (namely heterotypic mating haploid) of the sugarcane smut fungi with different mating types (+, -), and culturing on a solid culture medium at a certain temperature for a specific time (6-8 days) to obtain the primary mycelium of the sugarcane smut fungi. The nascent mycelium can make up the defects of basidiospore and two-state transformation in the current research, and can be used for the subsequent experimental research in various aspects such as environmental stress of sugarcane smut, biocontrol bacteria screening and the like.
Preferably, the Hemerocallis sugarcane is a wild-type Hemerocallis sugarcane strain or a mutant strain of Hemerocallis sugarcane still having a two-state transformation ability.
Preferably, the culture in step S1 is YEPS medium.
Preferably, the culture conditions in step S1 are 26-30 ℃ and 180-220 rpm for 1-3 days.
Further preferably, the culture condition in step S1 is a culture at 200rpm at 28 ℃ for 2 days.
Preferably, the OD of step S2 600 Is 1.0.
Preferably, the solid medium in step S3 is PDA solid medium.
Preferably, the mixed liquid is dropped into the PDA solid culture medium in step S3, and then is dried by blowing, sealed and cultured.
Preferably, the culturing in step S3 is at 28 ℃ for 7 days.
Preferably, the sugarcane smut basidiospores with different mating types (+, -) in the step S1 are WT17 (+), WT18 (-).
The sugarcane smut primary mycelium obtained by the invention can be used for subsequent related tests, such as environmental stress, biocontrol bacteria screening and the like, and is not limited to the stress test related to the invention.
The invention also provides application of the sugarcane smut primary mycelium obtained by any one of the culture methods in environmental stress and biocontrol bacterium screening of the sugarcane smut.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for culturing sugarcane smut mycelium, which comprises the steps of carrying out sexual matching on haploid basidiospores of sugarcane smut with different mating types (+, -), and culturing on a PDA (PDA) solid culture medium for a specific time (6-8 days) at a certain temperature to obtain the sugarcane smut primary mycelium. The nascent mycelium can be used for subsequent tests to make up for the defects of basidiospore and two-state transformation in the current research, and can be used for the research of environmental stress of sugarcane smut, biocontrol bacteria screening and other aspects.
Drawings
FIG. 1 shows the colony morphology of mycelia of Hemerocallis sugarcane under different illumination conditions.
FIG. 2 shows the effect of environmental stress on the growth of hyphae of Hemerocallis sugarcanum under different treatment conditions.
FIG. 3 shows the effect of biocontrol bacteria on the growth of hyphae of sugarcane smut.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
PDA culture medium: adding 40g/L of PDA (purchased from Guangzhou Dingguo Biotechnology company) into water, mixing, sterilizing at 121 deg.C for 20 min, cooling, and storing.
YEPS medium: 20g/L of yeast extract powder, 10g/L of peptone and 20g/L of sucrose, adding water, mixing uniformly, sterilizing at 121 ℃ for 20 minutes, cooling and storing for later use.
The haploids of different mating types (+, -) sugarcane smut fungi adopted in the following embodiments of the invention are sugarcane smut fungi basidiospore WT17 (+), WT18 (-), and WT17 (+), WT18 (-) are haploids which are obtained by separating the germinated sugarcane smut disease winterspores, and are identified as "+" and "-", respectively, can be mutually identified and can be subjected to sexual coordination to generate binuclear hyphae.
Example 1 sugarcane Hemerocallis mycelium culture
(1) Respectively picking activated haploid basidiospores (wild type strains) of different mating types (+, -) of the sugarcane smut bacteria in a culture medium containing 2mL of YEPS, and culturing at 28 ℃ and 200rpm for 2 days to obtain sugarcane smut bacteria basidiospore mother liquor.
(2) Centrifuging the sugarcane penis and testes pollen basidiospore mother liquor obtained in the step (1) to remove supernatant, adding sterile water-suspended bacteria, centrifuging again, removing supernatant, adding sterile water-suspended bacteria and adjusting OD 600 And (5) obtaining sugarcane smut bacterium basidiospore solution with different mating types when the concentration is 1.0.
(3) And (3) isovolumetrically and uniformly mixing the basidiospore liquid of the sugarcane smut fungi of different mating types (+, -) obtained in the step (2), taking 2 mu L of the uniformly mixed basidiospore liquid, dripping the basidiospore liquid on a PDA (personal digital assistant) plate, drying by blowing, sealing, and culturing at 28 ℃ for 7 days to obtain the primary mycelium of the sugarcane smut fungi.
Example 2 sugarcane Hemerocallis mycelium culture
(1) Activated haploid basidiospores (wild strains) of different mating types (+, -) of sugarcane smut are respectively picked and cultured in a culture medium containing 2mL of YEPS at 30 ℃ and 180rpm for 1 day, and sugarcane smut basidiospore mother liquor can be obtained.
(2) Centrifuging the sugarcane penis and testes pollen basidiospore mother liquor obtained in the step (1) to remove supernatant, adding sterile water-suspended bacteria, centrifuging again, removing supernatant, adding sterile water-suspended bacteria and adjusting OD 600 And (5) obtaining sugarcane smut bacterium basidiospore solution with different mating types.
(3) And (3) isovolumetrically and uniformly mixing the sugarcane smut fungi basidiospore liquid with different mating types (+, -) obtained in the step (2), taking 2 mu L of the uniformly mixed basidiospore liquid, dropping the liquid on a PDA (personal digital assistant) plate, drying the plate, sealing the plate, and culturing the plate at 26 ℃ for 8 days to obtain the sugarcane smut fungi primary mycelium.
Example 3 sugarcane Hemerocallis mycelium culture
(1) Activated haploid basidiospores (wild strains) of different mating types (+, -) of sugarcane smut are respectively picked and cultured in a culture medium containing 2mL of YEPS at the temperature of 26 ℃ and the rpm of 220 for 3 days, and then sugarcane smut basidiospore mother liquor can be obtained.
(2) Centrifuging the sugarcane penis and testes pollen basidiospore mother liquor obtained in the step (1) to remove supernatant, adding sterile water-suspended bacteria, centrifuging again, removing supernatant, adding sterile water-suspended bacteria and adjusting OD 600 And (5) obtaining sugarcane smut basidiospore liquid with different mating types when the cross breeding is 0.8.
(3) And (3) isovolumetrically and uniformly mixing the basidiospore liquid of the sugarcane smut fungi of different mating types (+, -) obtained in the step (2), taking 2 mu L of the uniformly mixed basidiospore liquid, dripping the basidiospore liquid on a PDA (personal digital assistant) plate, drying by blowing, sealing, and culturing at 30 ℃ for 6 days to obtain the primary mycelium of the sugarcane smut fungi.
Comparative example 1
The method is basically the same as the example 1, and the only difference is that the condition of transferring the basidiospore bacterial liquid mixed in the step (3) to PDA plate culture is that the basidiospore bacterial liquid is cultured for 3 days at 28 ℃. As a result, only the transformation of the secondary form of Ustilago canicola was observed, and primary mycelium of Ustilago canicola could not be obtained.
Example 4 investigation of the Effect of light conditions on the colony morphology of Ustilago Sachalinensis
(1) Respectively picking activated haploid basidiospores (wild type strains) of different mating types (+, -) of the sugarcane smut bacteria in a culture medium containing 2mL of YEPS, and culturing at 28 ℃ and 200rpm for 2 days to obtain sugarcane smut bacteria basidiospore mother liquor.
(2) Centrifuging the sugarcane penis and testes pollen basidiospore mother liquor obtained in the step (1) to remove supernatant, adding sterile water-suspended bacteria, centrifuging again, removing supernatant, adding sterile water-suspended bacteria and adjusting OD 600 And (5) obtaining sugarcane smut bacterium basidiospore solution with different mating types when the concentration is 1.0.
(3) And (3) isovolumetrically and uniformly mixing the basidiospores of the sugarcane smudge bacteria with different mating types (+, -) obtained in the step (2), taking 2 mu L of the uniformly mixed basidiospore bacteria liquid, dripping the basidiospore bacteria liquid on a PDA (personal digital assistant) plate, drying by blowing, sealing, and culturing at 28 ℃ for 7 days to obtain the sugarcane smudge bacteria primary mycelium.
(4) The cultured primary mycelium is carefully cut from the colony by a sterile double-sided blade, the primary mycelium is transferred to a new PDA for culture by a sterile toothpick, and the culture is continued for 21 days at 28 ℃, so that the colony of the mycelium of the sugarcane smut can be obtained. The colony morphology of the sugarcane smut can be influenced by the illumination condition, and the influence of continuous illumination and continuous darkness on the colony morphology of the sugarcane smut is shown in figure 1, which shows that the colony morphology change of the sugarcane smut mycelium can be researched by utilizing the primary mycelium of the sugarcane smut.
Example 5 Effect of different treatments on the growth of hyphae of sugarcane smut
(1) Activated haploid basidiospores (wild strains) of different mating types (+, -) of sugarcane smut are respectively picked and cultured in a culture medium containing 2mL of YEPS at the temperature of 28 ℃ and the rpm of 200 for 2 days, and then sugarcane smut basidiospore mother liquor can be obtained.
(2) Centrifuging the sugarcane penis and testes pollen basidiospore mother liquor obtained in the step (1) to remove supernatant, adding sterile water-suspended bacteria, centrifuging again, removing supernatant, adding sterile water-suspended bacteria and adjusting OD 600 And (5) obtaining sugarcane smut basidiospore liquid with different mating types when the strain is 1.0.
(3) And (3) isovolumetrically and uniformly mixing the basidiospores of the sugarcane smut fungi of different mating types (+, -) obtained in the step (2), taking 2 mu L of the uniformly mixed basidiospore bacterial liquid, dropping the 2 mu L of the uniformly mixed basidiospore bacterial liquid on a PDA (personal digital assistant) plate, drying the PDA plate, sealing the PDA plate, and culturing the PDA plate at 28 ℃ for 7 days to obtain the primary mycelium of the sugarcane smut fungi.
(4) The primary hyphae were transferred to PDA medium, and a preliminary environmental stress test was performed on the hyphae by adding different treatment reagents (glucose and NaCl treatment is used as an example in the present invention, no additional reagent is added as a control, and the culture conditions are light-dark alternation (24 h/24 h)) in advance (FIG. 2).
Example 6 Effect of biocontrol bacteria on the growth of hyphae of Hemerocallis Sacchari
(1) Respectively picking activated haploid basidiospores (wild type strains) of different mating types (+, -) of the sugarcane smut bacteria in a culture medium containing 2mL of YEPS, and culturing at 28 ℃ and 200rpm for 2 days to obtain sugarcane smut bacteria basidiospore mother liquor.
(2) Centrifuging the sugarcane penis and testes pollen basidiospore mother liquor obtained in the step (1) to remove supernatant, adding sterile water-suspended bacteria, centrifuging again, removing supernatant, adding sterile water-suspended bacteria and adjusting OD 600 And (5) obtaining sugarcane smut basidiospore liquid with different mating types when the strain is 1.0.
(3) And (3) isovolumetrically and uniformly mixing the basidiospores of the sugarcane smut fungi of different mating types (+, -) obtained in the step (2), taking 2 mu L of the uniformly mixed basidiospore bacterial liquid, dropping the 2 mu L of the uniformly mixed basidiospore bacterial liquid on a PDA (personal digital assistant) plate, drying the PDA plate, sealing the PDA plate, and culturing the PDA plate at 28 ℃ for 7 days to obtain the primary mycelium of the sugarcane smut fungi.
(4) The primary hyphae were transferred to PDA medium, and the inhibition of hyphae by biocontrol bacteria was observed on the hyphae circumferential points of biocontrol bacteria having inhibitory effects on filamentous fungi (FIG. 3).
In conclusion, the invention provides a method for culturing mycelium of ustilago pizoffii, which comprises the steps of carrying out sexual matching on haploid basidiospores of ustilago pizoffii with different mating types (+, -), and culturing on a PDA solid culture medium at a certain temperature for a specific time (6-8 days) to obtain the nascent mycelium of ustilago pizoffii. The sugarcane smut primary mycelium can be used for the research of the sugarcane smut environment stress, the biocontrol bacteria screening and other subsequent tests, and can make up the defect that the current research only can aim at the basidiospore and binary type transformation of the sugarcane smut.

Claims (10)

1. A method for culturing sugarcane smut mycelium is characterized by comprising the following steps:
s1, selecting activated sugarcane black pollen fungus basidiospores of different mating types for culturing to obtain sugarcane black pollen fungus basidiospore mother liquor;
s2, centrifuging the sugarcane smut fungus basidiospore mother liquor obtained in the step S1, discarding supernatant, re-suspending with sterile water, centrifuging, discarding supernatant, re-suspending with sterile water and adjusting OD of basidiospore liquid 600 When the mating type is 0.8 to 1.2, obtaining sugarcane smut fungus basidiospore liquid with different mating types;
s3, uniformly mixing the sugarcane smut basidiospore solutions with different mating types in equal volumes, dripping the mixed solution onto a solid culture medium, and culturing at 26-30 ℃ for 6-8 days to obtain the primary mycelium of the sugarcane smut.
2. The method of claim 1, wherein the Hemerocallis Sacchari is a wild-type Hemerocallis Sacchari strain or a mutant strain of Hemerocallis Sacchari still having a two-state transition ability.
3. The culture method according to claim 1, wherein the culture in step S1 is carried out using YEPS medium.
4. The culture method according to claim 1 or 3, wherein the culture conditions in step S1 are 26 to 30 ℃ and 180 to 220rpm for 1 to 3 days.
5. The culture method according to claim 4, wherein the culture conditions in step S1 are 28 ℃ and 200rpm for 2 days.
6. The culture method according to claim 1, wherein the OD in step S2 is 600 Is 1.0.
7. The culture method according to claim 1, wherein the solid medium in step S3 is PDA solid medium.
8. The culture method according to claim 1, wherein the mixed solution is dropped onto the solid culture medium in step S3, and then dried by blowing, sealed and cultured.
9. The method according to claim 1, wherein the culture in step S3 is carried out at 28 ℃ for 7 days.
10. The use of the primary mycelium of Hemerocallis praecox obtained by the culture method according to any one of claims 1 to 9 in environmental stress and biocontrol bacteria screening of Hemerocallis praecox.
CN202211008908.8A 2022-08-22 2022-08-22 Method for culturing mycelium of ustilago flagelliforme and application of mycelium Pending CN115340954A (en)

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杜瑜欣等: "前体调控对Bacillus sp. 108菌株发酵合成抑制甘蔗鞭黑粉菌活性物质产量的影响", 南方农业学报, vol. 50, no. 4, pages 755 - 760 *

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