Ustilaginoidea virens isolation medium and preparation method thereof

Abstract

The invention relates to a Ustilaginoidea virens isolation culture medium and a preparation method thereof. Every 100 mL of the culture medium comprises 1.5-2.5 g of peptone, 1.0-2.0 g of cane sugar, 1.0-1.5 g of rice meal, 30-50 mL of rice hull water and 1.5-2.0 g of agar. The pH value of the culture medium is 6-7. The preparation method comprises the following steps: adding peptone, cane sugar and rice meal into a 250 mL triangular flask, adding rice husk water, supplementing water to 100 mL scale marks, adding agar, uniformly mixing, adjusting the pH value to 6-7 by using a sodium hydroxide aqueous solution with the concentration of 1% and a hydrochloric acid aqueous solution with the concentration of 1%, binding a bottle opening by using a sealing film, sterilizing in a 121 DEG C autoclave for 30 minutes, and cooling to room temperature. The Ustilaginoidea virens isolation medium is obtained. The culture medium has a good culture effect on Ustilaginoidea virens, is high in mycelial growth rate, and has very important significance on Ustilaginoidea virens etiology research and disease control.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a culture medium for separating smut pathogens in rice and a preparation method thereof. Background Art

[0002] False Smut of Rice (FSR) is a fungal disease of rice panicles caused by Ustilaginoidea virens (Cooke) Tak in the asexual stage of the subphylum Ascomycetes. It is widely distributed in more than 40 countries in the main rice producing areas of Asia, Africa, South America, Europe, etc., and is more serious in China, the Philippines, and Japan. With the large-scale promotion of high-yield and high-quality rice and the continuous increase in fertilizer application, false smut of rice has become more and more serious in my country, and is widely occurring in Liaoning, Heilongjiang, Hunan, Yunnan, Jiangsu, Zhejiang, Guangdong and other provinces. False smut of rice will increase the empty rate, reduce the thousand-grain quality, reduce rice yield, and bring huge losses to rice production. The genome sequence size of the rice false smut fungus is about 39.4Mbp, containing about 8426 protein-coding genes, with an average gene length of 1672bp, and each gene contains an average of 2.6 exons and 1.6 introns and 25% repeat sequences.

[0003] Studies have shown that sclerotia or thick-walled spores that overwinter in diseased residues or field soil are the main sources of initial infection. In the early stage of rice filling or flowering, the rice smut pathogen invades from the gap between the lemma and palea of ​​the grain, forming rice smut balls that are larger than the grain on the yellow-ripe rice ears. The center is a white fleshy mass composed of dense hyphae tissue, and the surface is covered with a large number of yellow or dark green thick-walled spores. The periphery is divided into three layers due to the different maturity of the hyphae that produce thick-walled spores. The outer layer of these three layers matures first and is dark green or olive green; the second layer is orange-yellow; and the third layer is yellow. The formation of rice smut balls not only leads to a reduction in rice yield, but also produces two types of fungal toxins that seriously affect the quality of rice. One type is Ustiloxins, which belongs to the cyclic polypeptide class of compounds, and the other is Ustilaginoidins, which belongs to the dinaphthopyrone class of compounds. It is reported that the two types of toxins produced by U. virens can prevent the polymerization of cell microtubules, interfere with the formation of the cytoskeleton, and inhibit cell mitosis. If humans or livestock accidentally ingest them, they will experience poisoning symptoms such as visceral lesions.

[0004] At present, the culture system of rice false smut fungus mostly adopts commercial culture medium on the market, such as PDA culture medium, potato sucrose agar (PSA) culture medium, and Tadashi Wakimoto culture medium, but the rice false smut fungus grows very slowly in these culture media. Due to the long artificial isolation and culture cycle of rice false smut fungus, low separation efficiency and easy contamination, the research work related to rice false smut etiology, pathogenic mechanism, and biological control is seriously restricted. Therefore, the development of the isolation culture medium of rice false smut fungus has become the primary link in solving the scientific research work of rice false smut, which has extremely important practical significance. Summary of the invention

[0005] Technical problem to be solved: In view of the shortcomings of the existing artificial separation and cultivation of rice false smut, such as long culture cycle, low separation efficiency and easy contamination, the present invention provides a rice false smut isolation culture medium and a preparation method thereof. The culture medium has good culture effect on rice false smut and fast mycelium growth rate, which is of great significance for the etiology research and disease prevention and control of rice false smut.

[0006] Technical solution: A culture medium for separating smut pathogens of rice, wherein each 100 mL of the culture medium comprises 1.5-2.5 g of peptone, 1.0-2.0 g of sucrose, 1.0-1.5 g of rice flour, 30-50 mL of rice husk water and 1.5-2.0 g of agar; and the pH value of the culture medium is 6-7.

[0007] The peptone described above is soy peptone.

[0008] The rice flour described above is polished round-grained rice flour.

[0009] The rice husk water is prepared by mixing rice husk and water, boiling for 30 minutes, filtering through double-layer gauze, and taking the supernatant; 20 mL of water is added for every 1 g of rice husk.

[0010] The preparation method of the above-mentioned rice smut pathogen isolation medium comprises the following steps: adding peptone, sucrose and rice flour into a 250mL triangular flask, adding rice husk water, replenishing the water to the 100mL mark, adding agar and mixing evenly, then adjusting the pH value to 6-7 with a 1% sodium hydroxide aqueous solution and a 1% hydrochloric acid aqueous solution, wrapping the bottle mouth with a sealing film, and sterilizing in a high pressure sterilizer at 121°C for 30 minutes to obtain a rice smut pathogen isolation medium.

[0011] The peptone in the culture medium of the invention is soybean peptone, which provides the nitrogen element required for the growth of the mycelium of rice false smut fungus.

[0012] The rice flour in the culture medium of the present invention is prepared by grinding polished round-grained rice with a mill, and the rice flour provides a carbon source, inorganic salts such as calcium, potassium, magnesium, phosphorus, and vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin E, and vitamin K for the growth of mycelium of smut fungus. The inorganic salts and vitamins are necessary for regulating the normal metabolism of microorganisms.

[0013] The rice husk water in the culture medium of the present invention provides trace elements such as calcium, magnesium, iron, copper, zinc and the like for the growth of rice false smut fungus hyphae, and plays a role in regulating the growth of microbial cells.

[0014] Beneficial effects: The rice smut pathogen isolation culture medium and preparation method thereof provided by the present invention have the following beneficial effects:

[0015] 1. The culture medium material of the present invention is easy to obtain, low in cost and simple to operate.

[0016] 2. The culture medium of the present invention has a good culture effect on rice false smut fungus, and the mycelium growth rate is fast, which is of great significance for the etiology research and disease prevention and control of rice false smut fungus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the mycelial growth rate of the optimized culture medium and the control culture medium. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below through specific implementation modes, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0019] The direct sources of the reagents used below are as follows:

[0020] Peptone was purchased from Qingdao Haibo Biological Co., Ltd.;

[0021] Sucrose was purchased from Tianjin Ruijinte Chemical Co., Ltd.;

[0022] Agar was purchased from Biotopped, Japan;

[0023] Sodium hydroxide was purchased from Nanjing Chemical Reagent Co., Ltd.;

[0024] Hydrochloric acid was purchased from Nanjing Chemical Reagent Co., Ltd.

[0025] This embodiment provides a method for preparing a culture medium for separating smut pathogens of rice, and the preparation steps are as follows:

[0026] Step 1: Grind the polished round-grained rice with a mill to prepare rice flour, mix the rice husk with water in a ratio of 5:100 (g:mL), boil for 30 minutes, filter with double-layer gauze, and take the supernatant to obtain rice husk water;

[0027] Step 2: Add 1.5-2.5 g of peptone, 1.0-2.0 g of sucrose, and 1.0-1.5 g of rice flour into a 250 mL triangular flask, add 30-50 mL of rice husk water, make up the water to the 100 mL mark, add 1.5-2.0 g of agar and mix well, then adjust the pH value to 6-7 with a 1% sodium hydroxide aqueous solution and a 1% hydrochloric acid aqueous solution, wrap the bottle mouth with a sealing film, and sterilize in a high pressure sterilizer at 121° C. for 30 min to obtain a rice smut pathogen isolation medium.

[0028] The optimization process and effect verification of the rice false smut pathogen isolation culture medium of the present invention are as follows.

[0029] 1. Optimization of culture medium for isolation of U.S. smut pathogen

[0030] Using L 16 (4 5 ) Orthogonal experiment was used to optimize the culture medium formula of rice false smut pathogen. The levels of orthogonal experiment factors are shown in Table 1 below, A: peptone, B: sucrose, C: rice flour, D: rice husk water, E: pH value, wherein peptone is soybean peptone, rice flour is prepared by grinding polished round-grained rice with a mill, rice husk water is prepared by mixing rice husk and water in a ratio of 5:100 (g:mL), boiling for 30 minutes, filtering with double-layer gauze, taking the supernatant, adding 2 g of agar, and repeating the experiment three times. Steam sterilize at 121℃ for 30min, pour the plates aseptically on a clean bench, inoculate rice false smut fungus in the center of the dish, culture at 28℃ for 21 days, measure the colony diameter by the cross method, repeat 10 times for each treatment, and use PDA as a control (peel 20g potatoes, cut into small pieces, put in a pot with 100mL of water, boil for half an hour, filter with double-layer gauze, take the filtrate, add 2g sucrose, 2g agar, and make up to 100mL with distilled water).

[0031] Table 1

[0032]

[0033] The results of the orthogonal test are shown in Table 2. As shown in Table 2, the effects of peptone, sucrose, rice flour, rice husk water, and pH value on mycelial growth rate are positively correlated with the range. The ranges of the six factors are E>B>A>D>C from large to small. It can be seen that pH value has the greatest impact on mycelial growth rate, followed by sucrose and peptone. 3 B 2 C 1 D 2 E 3 It is the best combination for the growth of rice false smut fungus mycelium, that is, the best proportion of the culture medium components is: 2.0g peptone, 1.5g sucrose, 1.0g rice flour, 40mL rice husk water, and pH value 6.5 per 100mL culture medium.

[0034] Table 2

[0035]

[0036]

[0037] 2. Verification of the effect of optimized culture medium

[0038] The separation medium was prepared by using 2.0 g of peptone, 1.5 g of sucrose, 1.0 g of rice flour, 40 mL of rice husk water, pH 6.5, supplementing distilled water to 100 mL, and adding 2 g of agar. The medium was steam sterilized at 121 ° C for 30 min, and the plates were aseptically poured on the clean bench. The rice smut pathogen was inoculated in the center of the culture dish and cultured at 28 ° C for 21 days. The colony diameter was measured by the cross method. PDA was used as the control. Each treatment was repeated 10 times. The mycelial growth rate after optimization was Figure 1 It can be seen that the mycelial growth rate of the optimized isolation culture medium was 2.8 mm / d, which was significantly higher than that of the control, while the mycelial growth rate of the control group was 2.1 mm / d.

[0039] The embodiments of the present invention are described in detail above. For those skilled in the art, according to the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A culture medium for separating smut pathogenic bacteria of rice, characterized in that: Each 100 mL of the culture medium contains 1.5-2.5 g of peptone, 1.0-2.0 g of sucrose, 1.0-1.5 g of rice flour, 30-50 mL of rice husk water and 1.5-2.0 g of agar; the pH value of the culture medium is 6-7.

2. A culture medium for separating smut pathogens of rice according to claim 1, characterized in that: The peptone is soy peptone.

3. A culture medium for separating smut pathogens of rice according to claim 1, characterized in that: The rice flour is polished round-grained rice flour.

4. The isolation medium for smut pathogen of rice according to claim 1, characterized in that: The rice husk water is prepared by mixing rice husk and water, boiling for 30 minutes, filtering through double-layer gauze, and taking the supernatant; 20 mL of water is added for every 1 g of rice husk.

5. The method for preparing a culture medium for separating smut pathogens of rice as claimed in claim 1, characterized in that The preparation steps are as follows: add peptone, sucrose and rice flour into a 250mL triangular flask, add rice husk water, make up the water to the 100mL mark, add agar and mix evenly, then adjust the pH value to 6-7 with a 1% sodium hydroxide aqueous solution and a 1% hydrochloric acid aqueous solution, wrap the bottle mouth with a sealing film, and sterilize in a high-pressure sterilizer at 121°C for 30 minutes to obtain a rice smut pathogen isolation medium.

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