Phytophthora nicotianae culturing method

A cultivation method and technology of Phytophthora nicotianae, applied in the field of microbial cultivation, can solve the problems of cultivation failure, contamination by miscellaneous bacteria, large differences in matrix components, etc., and achieve the effects of low cost, reduced pollution and convenient production.

Inactive Publication Date: 2015-09-30
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] In the process of fungus cultivation, due to the large difference in substrate composition between plate culture and solid culture, when Phytophthora nicotiana mycelium is transferred from plate culture to solid culture, the mycelium usually needs an adaptation period of 2 to 3 days. The internal hyphae cannot quickly occupy the matrix and provide opportunities for other bacteria to grow, which can easily lead to bacterial contamination and culture failure

Method used

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preparation example Construction

[0027] In the technical solution of the present invention, the preparation method of the wheat kernel filtrate culture medium may include: soaking the wheat kernels with hulls in water for 8-12 hours and then filtering them with gauze to obtain the filtrate and the filtered kernels with hulls, and adding to the filtrate Agar is sterilized to obtain the wheat kernel filtrate medium;

[0028] Among them, the volume ratio of wheat kernels to water is 1: (1 to 1.6), preferably 1: (1.2 to 1.4), and the filtrate obtained at this ratio can provide sufficient nutrients for the growth of the strain.

[0029] The preparation method of the wheat kernel medium may include: mixing the filtered hulled wheat kernels and straw kernels, and sterilizing them to prepare the wheat kernel medium; wherein the volume of the filtered wheat kernels and straw kernels The ratio is (70~90):(10~30), preferably 70:30. The advantage is to increase the air permeability, which can make the hyphae quickly overgrow ...

Embodiment 1

[0035] Preparation of culture medium: Weigh 150 mL of shelled oats, add 150 mL of distilled water, and soak for 8 hours to fully swell the oats, filter with gauze to obtain oat filtrate and filtered oats with shells. Measure 100 mL of oat filtrate, add 1.7 g of agar to it, and sterilize with moist heat at 121°C for 30 minutes to prepare an oat filtrate medium. Measure 70 mL of the filtered oats with shell, add 30 mL of fresh pea stalks with a particle size of 3 mm and a water content of 50%, and sterilize with moist heat at 121°C for 30 minutes to prepare an oat culture medium.

[0036] Strain activation: Take the preserved Phytophthora nicotianae strain, inoculate it on an oat filtrate medium plate, culture it at 26°C for 3 days, then subculture once under the same conditions;

[0037] Plate culture: inoculate the activated mycelium block on an oat filtrate medium plate, and cultivate it at 26°C for 10 days to obtain a pure white mycelium block;

[0038] Solid culture: take a surfa...

Embodiment 2～7

[0040] The method steps of Examples 2-7 are the same as that of Example 1, except that the experimental conditions and the parameters of the experimental materials are different. The specific differences are shown in the following table.

[0041] Table 1. Medium preparation

[0042]

[0043] Table 2. Cultivation of Phytophthora Nicotiana strains

[0044]

[0045] In Examples 1-7, after 7-10 days of solid culture, the pure white hyphae were covered with the whole wheat kernel medium, and the bacterial valleys that met the requirements for the subsequent determination of tobacco resistance were obtained, and they were not contaminated by other bacteria. Valley inoculum was successfully cultured.

[0046] It should be noted that, after the soaked wheat kernels are soaked in water, the filtered filtrate is used to make the wheat kernel filtrate culture medium, and the filtered wheat kernels are used to make the wheat kernel culture medium. Therefore, the wheat kernel filtrate culture medi...

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Abstract

The embodiment of the invention discloses a phytophthora nicotianae culturing method. The method includes the steps that plate culturing is conducted, phytophthora nicotianae strain is inoculated to a kernel filter liquor culture medium, culturing is conducted for 7 days to 10 days at the temperature of 26 DEG C to 28 DEG C, and sclertium obtained through plate culturing is obtained; solid culturing is conducted, the sclertium obtained through the plate culturing is inoculated into a kernel culturing medium, culturing is conducted for 7 days to 10 days at the temperature of 26 DEG C to 28 DEG C, and strains are obtained; the kernel filter liquor culturing medium is prepared in the mode that shelled wheat is soaked into water with the volume being 1-1.6 times that of the shelled wheat for 8 hours to 12 hours, filtering is conducted through a yarn screen, filtering liquor and filtered shelled wheat are obtained, (17-20) g / L of agar is added to the filtering liquor, and sterilization is conducted; a kernel culturing medium is prepared in the mode that the filtered shelled kernels are mixed with straw grains, the volume ratio between the shelled kernels and the straw grains is (70-90):(10-30), and sterilization is conducted. According to the phytophthora nicotianae culturing method, phytophthora nicotianae culturing is shortened by 5 days to 13 days compared with common phytophthora nicotianae culturing, and the culturing media are simple in formula, convenient to prepare and low in cost.

Description

Technical field [0001] The present invention relates to the technical field of microbial cultivation, in particular to a cultivation method of Phytophthora tabacum. Background technique [0002] Tobacco black shank (tobacco black shank) is the second largest type of disease in tobacco production caused by Phytophtora nicotianae, after viral diseases. The disease is serious and widely distributed in temperate, subtropical and tropical regions. At present, the best way to control this disease is to screen tobacco plants resistant to tobacco black shank, and the screening of disease-resistant plants requires resistance testing. [0003] At present, the commonly used inoculums of Phytophthora nicotianae in the determination of resistance to tobacco black shank include mycelial mass, mycelial liquid, zoospore liquid, and fungus valley. The field inoculation generally uses Mushroom Valley. The so-called bacterial valley is an inoculum obtained by directly inoculating the hyphae mass o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12R1/645
CPCC12N1/14
Inventor 方敦煌肖炳光曾建敏陈学军
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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