CN114075513A - Hypha coating method for producing large number of sexual generations of rice blast germs - Google Patents
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Abstract
The invention provides a hypha coating method for producing a large number of sexual generations of rice blast fungus, which comprises the following steps of 1) preparing hypha suspension of different mating types of the rice blast fungus; 2) coating hypha suspensions of different mating types on an oat culture medium in proportion; 3) culturing at 12-27 deg.C for 5-15 days to obtain large amount of ascospores and ascospores of Pyricularia oryzae. The invention overcomes the defects of small total amount, instability, large difference among samples, inconsistent maturity of sexual offspring and the like of sexual generation ascospores generated by the traditional confrontation culture method in the prior art, can obtain a large number of sexual generations of rice blast germs in a short time, has high ascocarp and ascospore forming speed and consistent maturity, and can be used for quantitative comparison of sexual reproductive capacity of strains, sexual structure observation, nucleic acid extraction and gene expression quantitative analysis.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a novel method, which relates to a method for culturing and preparing sexual generations of rice blast germs, and the preparation method is a Hypha coating method (HC method).
Background
The rice blast is one of the most important diseases on rice, and seriously affects the yield of the rice. Pyricularia oryzae, also known as Pyricularia oryzae, Latin name Pyricularia oryzae, belongs to Fungi, Ascomycota, Sordariomycetes, Magnaporthales, Hypocreales, Pyricularia. Pyricularia oryzae can infect leaves, stem nodes, branch stalks, ear necks and grains of rice, and can infect more than 50 kinds of gramineous plants such as barley, wheat, millet and the like in addition to rice. At present, screening and breeding disease-resistant rice varieties are the most important measures for preventing and treating rice blast. However, the continuous variation of the rice blast germs in the field causes the continuous appearance of highly pathogenic strains to cause the change of the group structure, so that the field resistance of the disease-resistant variety can be lost after the disease-resistant variety is planted for 3 to 5 years. Therefore, the variability of rice blast germs is the root cause of the difficulty in controlling rice blast.
Reproduction is the formation of new individuals with all the typicalities of the species, and the reproductive organs produced by vegetative growth for a certain period of time are called propagules. The propagation of fungi is mainly converted from vegetative organs, some fungi are bred to a certain stage, the whole vegetative body is converted into a reproductive body, various spores are generated during the propagation of the fungi as a propagation unit, and the propagation form is divided into asexual form and sexual form.
The asexual propagation of fungi refers to the propagation of new individuals without the combination of two individual cells or sexual organs, and the asexual spores are directly generated on the trophosome of the fungi and the differentiated special structure thereof and are similar to tubers and bulbs of higher plants in nature. Common asexual spores include zoospores, sporocysts, conidia and chlamydospores, and the asexual spores of ascomycetes and basidiomycetes are mainly conidia and are the most common spore forms in fungi. The rice blast fungus is Pyricularia oryzae (Pyricularia oryzae) in a non-sexual state. Under natural conditions, the infection process of rice blast germs is mainly completed by asexual conidia. In recent 20 years, the research on the molecular mechanism of the asexual spore generation and infection process of rice blast fungus has made great progress, and a large number of genes involved in sporulation, formation of adherent cells, infection and expansion have been identified and analyzed.
Fungal sexual reproduction refers to a mode of reproduction by the combination of two individual cells or sexual organs. Spores produced by sexual reproduction are called sexual spores. The sexual reproduction process is characterized by nuclear binding, and is realized by the binding of active or inactive gametes, gametophytes and thalli. The mating of gametophytic sacs, which are sexual organs of fungi and have isomorphism or dysmorphism, is one of the common cell binding modes of ascomycetes. When mating, one nucleus is transferred into the counterpart's gametophytic via the seminiferous duct (silk), and the former is rapidly digested, and the seminiferous duct of ascomycetes is generated by the female (female gametophytic capsule). The ascomycetes are of the type of sexual spores, mainly ascospores. The ascospore is formed by combining two allotype gametophytes, male organs and an ascogenesis body, a plurality of filamentous branched ascogenesis filaments grow on the ascogenesis body, the cells at the top ends of the ascogenesis filaments stretch and bend to form hooks, then ascogenesis mother cells are formed, the ascogenesis is developed, the amphoteric cell nucleuses in the ascogenesis body are combined and then split, generally 8 or 4 or 2 endogenous ascospores with haploid cell nucleuses are formed. Ascospores are produced in the ascospores, which are enveloped by the ascoshells, which is a major characteristic of ascomycetes. Under laboratory conditions, the rice blast fungus can also undergo sexual reproduction, the sexual state of which is Magnaporthe oryzae. The sexual reproduction mode of rice blast fungus belongs to the heterozygote, and two haploid strains carrying different mating type genes (MAT1-1 or MAT1-2) are required to be fused to generate sexual offspring. In 1982, Kato et al successfully mated rice blast fungus strains under artificial culture conditions and formed ascospores. The mature ascospores of Pyricularia oryzae have spherical shells with long beaks, and the spherical parts contain a large number of ascomycetes. The ascomycetes are long rods and contain 8 longitudinally arranged ascospores. Sexual reproduction is more prone to variation than asexual reproduction for various organisms, so that offspring has genetic diversity and stronger adaptability, and is an important driving force for genetic recombination. The prior art considers that the rice blast fungus originates in southeast Asia, and certain areas in southeast Asia can have a sexual reproduction mode. However, at present, it has not been confirmed whether the sexual generation of Pyricularia oryzae can be generated under natural conditions, and the contribution of the sexual generation of Pyricularia oryzae to genetic variation and population pathogenic variation of Pyricularia oryzae is not clear. Therefore, the research on the sexual reproduction process and mechanism of the rice blast fungus has important significance for comprehensively understanding the life history of the fungus and explaining the variation source of the fungus. However, the study of the sexual reproduction of rice blast germs is much less than the study of the infection mechanism, and a necessary research method is also lacked.
The establishment of a stable method for producing a large number of sexual offspring is a necessary prerequisite for the study of sexual reproduction. The traditional rice blast germ sexual reproduction method adopts the steps that strains with opposite mating types are cross-inoculated on an artificial culture medium, and after the strains grow until bacterial colonies are connected, the culture is continued for about 20 days, so that an ascochyta zone can be generated at the junction of the strains. The method can produce mature asco-shells and ascospores, but the bandwidth of the asco-shells is only 2-3mm, the total amount of the asco-shells and the ascospores is small, and further research, especially molecular biological research is inconvenient, because the obtained sexual spore amount cannot support the sample amount required by molecular biological research such as nucleic acid sequencing, proteome analysis and the like. Meanwhile, the method has the defects of instability, large difference among samples, inconsistent maturity of sexual offspring and the like.
Disclosure of Invention
The present invention provides a novel method for sexual generation culture of Pyricularia oryzae, namely a Hypha coating method (HC method): the method comprises the following steps:
1) hypha suspensions of different mating types of Pyricularia oryzae were prepared.
2) Hypha suspensions of different mating types were spread proportionally on oat medium.
3) Culturing at 12-27 deg.C for 5-15 days to obtain large amount of ascospores and ascospores of Pyricularia oryzae.
Preferably, the rice blast fungus different mating type strains are inoculated on a CM solid plate with the length of 9CM, and are cultured for 7 days in the dark and in the whole black at the temperature of 28 ℃; or inoculating to CM liquid culture medium, and shake culturing at 28 deg.C in dark for 3d to obtain mycelium suspension.
Preferably, the hypha suspensions of different mating types are mixed in equal proportion in step (1).
More preferably, the mycelium suspension is obtained by taking 100g of mycelium after shake culture, adding a steel ball of 3mm and 400 microliters of ddH2O, and shaking for 90s at 60 HZ.
Preferably, the culture temperature in step (3) is 20 ℃.
Preferably, the culture in step (3) is performed under full light, and more preferably, the light intensity is 15000 lux.
The invention solves the defects of small total amount, instability, large difference among samples, inconsistent maturity of sexual offspring and the like of sexual generation ascospores generated by a traditional confrontation culture method in the prior art, and provides a new method for cultivating the sexual generation of rice blast germs, namely a hypha coating method (HC method). .
Drawings
FIG. 1: different mating type hyphae were spread to produce a sexual generation map. The mycelium segments are quickly germinated after being coated on OMA, and the new mycelium is uniformly distributed on the surface of the culture medium in day 2, and the mycelium of the two strains begins to contact; hypha continuously grows until the surface of the culture medium is completely covered by the 5 th day; after 10 days of culture, scattered small spots, namely the ascocarp shell, can be seen on the surface of the bacterial colony; the seed capsule shell gradually increases and grows to 15 days, and can be densely distributed on the surface of the culture medium. The speed of forming the ascocarp by combining different strains is slightly different, but a large amount of ascocarp can be formed after about 15 days of culture and is fully distributed on the surface of a culture medium. The culture was continued to 20d, and no significant change in the number and size of the ascoshells occurred.
FIG. 2: a comparison of the shells of the ascomycetes formed by the hypha coating method (HC method) and the conventional counter method (TC method) is shown. The ascocarp formed by the conidia coating method is normal in size, shape and distribution.
FIG. 3: statistical chart of the number of formed ascocarp cultured by hypha coating method (HC method) for 15 days and conventional confrontation method (TC method) for 25 days.
FIG. 4: statistical graphs of the numbers of ascospores formed by 15 days of Hypha Coating (HC) culture and 25 days of Traditional Confrontation (TC) culture.
FIG. 5: real-time fluorescent quantitative PCR mating type gene detection map. RNA extracted from an ascocarp formed by Guy11+ TH3 strain HC method is taken as a template, the expression of MAT1-2-1 gene can be effectively detected, and the result is in line with the expectation (MAT1-2-1 gene exists in TH3, and does not exist in Guy 11).
Detailed Description
The hypha coating method of the present invention is also called hypa coating method, HC method; the traditional confrontation culture method (traditional cross), abbreviated as TC method.
Example one preparation of a suspension of Magnaporthe grisea hyphae sexual generations
1. Strains and culture conditions
The cultivation of rice blast (Pyricularia oryzae) strain Guy11(MAT1-1), TH16(MAT1-1), 2539(MAT1-2), TH3(MAT1-2) was carried out on Complete Medium (CM). Oat Medium (OMA) was used to induce sexual generations from different mating type strains.
CM medium formula (1L)
The reagents are weighed according to the corresponding amount and then the volume is determined to the corresponding volume, the PH is adjusted to 6.5 by NaOH, 15g agar powder is added into each liter of solid culture medium, and the solid culture medium is sterilized by high-pressure steam at 121 ℃ for 15 min.
1000×Vitamin solution(100ml):
Dissolving the above reagents in distilled water, diluting to 100ml, and storing in refrigerator at 4 deg.C.
1000×Trace Elements(100ml):
Dissolving the above reagents in distilled water, diluting to 100ml, and storing in a refrigerator at 4 deg.C in dark place.
The formula of the oat culture medium comprises:
20g of oat, 2.5g of sucrose, 10g of agar powder and deionized water are prepared into 500ml of culture medium.
2. Formation of sexual generations
The hypha coating method (HC method) was as follows, all operations being performed on a clean bench:
(1) inoculating each strain on a CM solid plate of 9CM, and culturing at 28 ℃ for 7d in full darkness; or inoculating to CM liquid culture medium, and shake culturing at 28 deg.C in dark for 3d to prepare mycelium.
(2) OMA medium was prepared, autoclaved, and prepared in 9cm plates. Brushes, filters, funnels, filter papers, applicators, and the like are autoclaved.
(3) Mycelia of different strains cultured for 7d on solid CM or 3d in liquid CM were collected.
(4) Taking 100g of each bacterial strain hypha, putting the bacterial strains into a wall breaking pipe according to the combination of Guy11/2539, Guy11/TH3, TH16/2539 and TH16/TH3, adding a steel ball of 3mm, 400 mu L of ddH2O and 60HZ, oscillating for 90s, and repeating 3 times for each group.
(5) 400 μ L of the hyphae were pipetted into a shaking bath and placed on an OMA plate, which was spread evenly with a sterile spreader and incubated at 20 ℃ for 15 days under full light (15000 lux).
(6) After 15d, the production of the ascocarp shell on the OMA plate was observed and counted, and the formation and structure of the ascocarp and ascospore inside the ascocarp shell were observed under a microscope.
The mycelium segments are quickly germinated after being coated on OMA, and the new mycelium is uniformly distributed on the surface of the culture medium in day 2, and the mycelium of the two strains begins to contact; hypha continuously grows until the surface of the culture medium is completely covered by the 5 th day; after 10 days of culture, scattered small spots, namely the ascocarp shell, can be seen on the surface of the bacterial colony; the seed capsule shell gradually increases and grows to 15 days, and can be densely distributed on the surface of the culture medium. The speed of forming the ascocarp by combining different strains is slightly different, but a large amount of ascocarp can be formed around 15 days of culture and is fully distributed on the surface of a culture medium (figure 1). The culture was continued to 20d, and no significant change in the number and size of the ascoshells occurred. Under the same culture conditions, both mating type strains require 25-30d to produce mature ascochyta using the TC method. Therefore, the HC method is more advantageous in the generation time of the ascocarp.
The HC method produces the ascocarp shell densely distributed on the whole culture dish, while the TC method can only produce the ascocarp shell band distributed in a band shape on a strain boundary line. The numbers of the ascospores and the ascospores formed by 15d culture by the HC method and 25d culture by the TC method are counted, and the results show that the numbers of the ascospores (figure 3) and the ascospores (figure 4) produced on each culture dish by the HC method are nearly 100 times that of the TC method. The observation under the stereoscope shows that the shells formed by the HC method and the TC method have no obvious difference in size, form distribution and distribution (figure 2).
3. Extraction quantitative PCR detection of total RNA
HC method produces a large amount of ascocarp after 15 days of culture on OMA medium. The collected ascocarp is scraped by a sterilizing blade to obtain about 100mg of ascocarp, total RNA is extracted by a kit (TIANGEN kit), and the quality and integrity of the RNA are tested by electrophoresis detection and absorbance value measurement. And carrying out reverse transcription by taking the extracted total RNA as a template, and then carrying out real-time fluorescent quantitative PCR analysis.
In order to better evaluate whether the HC method can obtain high-purity RNA, the RNA of the ascocarp shells obtained by the HC method and the TC method is extracted by the same method and then subjected to electrophoresis verification. As a result, it was revealed that RNA of progeny produced by mating rice blast fungi could be efficiently extracted by both the TC method and the HC method. The quality of the progeny RNA obtained by HC method was further verified by real-time fluorescent quantitative PCR after reverse transcription of the extracted RNA into cDNA (fig. 5). The result shows that the expression of MAT1-2-1 gene can be effectively detected by taking RNA extracted from the ascocarp formed by Guy11+ TH3 strain HC method as a template, and the result is in line with the expectation (MAT1-2-1 gene exists in TH3, and does not exist in Guy 11). These experimental facts indicate that HC method can produce enough sexual offspring to provide enough RNA for detecting gene expression without adverse effect on the expression of genes related to sexual age.
Thus, the HC method of our invention provides a method for producing large amounts of sexual progeny of Pyricularia oryzae. By using the method, a large amount of uniform and stable sexual offspring can be generated more quickly; the generated sexual offspring can provide enough DNA or RNA samples for gene expression detection and further molecular biology research, and the defects of the method for researching the sexual reproduction mechanism of the rice blast fungus are made up. In addition, the use of hyphal fragments as initial cultures is more advantageous for sexual generations of strains with low conidium production.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (6)
1. A method for coating hypha capable of producing a large number of sexual generations of rice blast fungus, which is characterized in that: the method comprises the following steps:
1) preparing hypha suspensions of rice blast germs of different mating types;
2) coating hypha suspensions of different mating types on an oat culture medium in proportion;
3) culturing at 12-27 deg.C for 5-15 days to obtain large amount of ascospores and ascospores of Pyricularia oryzae.
2. The method of applying conidia to produce a large number of sexual generations of Pyricularia oryzae according to claim 1, wherein: and (3) mixing hypha suspensions of different mating types in the step (1) in equal proportion.
3. According to claim 1The conidium coating method for producing a large number of sexual generations of Pyricularia oryzae is characterized in that: the mycelium suspension is prepared by taking 100g of mycelium after shaking culture, adding a steel ball with the diameter of 3mm and 400 microliters of ddH2O, 60HZ was obtained by shaking for 90 s.
4. The method of applying conidia to produce a large number of sexual generations of Pyricularia oryzae according to claim 1, wherein: the culture temperature in step (3) was 20 ℃.
5. The method for coating conidia that produce large numbers of sexual generations of Pyricularia oryzae according to claim 1, wherein the culture conditions in step (3) are: and (5) full light culture.
6. The method of applying conidia that produce a large number of sexual generations of Pyricularia oryzae according to claim 5, wherein: the illumination intensity was 15000 lux.
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| WO2023035857A1 (en) * | 2021-09-08 | 2023-03-16 | 浙江省农业科学院 | Conidia coating method for producing large number of sexual generations of pyricularia oryzae |
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| CN102876577A (en) * | 2012-09-28 | 2013-01-16 | 中国林业科学研究院热带林业研究所 | Plate culture spreading method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023035858A1 (en) * | 2021-09-08 | 2023-03-16 | 浙江省农业科学院 | Hypha coating method for producing sexual generations of magnaporthe oryzae in quantity |
| WO2023035857A1 (en) * | 2021-09-08 | 2023-03-16 | 浙江省农业科学院 | Conidia coating method for producing large number of sexual generations of pyricularia oryzae |
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