阅读说明：本技术 一种高效休止浓缩植物病原卵菌游动孢子的方法及其应用 (method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores ) 是由 方敦煌 肖炳光 童治军 陈学军 曾建敏 张谊寒 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种高效休止浓缩植物病原卵菌游动孢子的方法,所述的高效休止浓缩植物病原卵菌游动孢子的方法包括游动孢子悬浮液制备、休止游动孢子和浓缩休止孢步骤,具体包括：将植物病原卵菌接种培养制备植物病原卵菌游动孢子悬浮液；休止游动孢子：将植物病原卵菌游动孢子悬浮液置于离心管中,在瞬时超重和失重交替处理条件下形成休止孢；将休止孢进行低温离心浓缩得到目标物休止孢悬浮液。本发明采用瞬时超重和失重的交替处理方法快速休止游动孢子,从根本上克服了现有技术中休止效率低的难题,同时大幅度提升了休止游动孢子的侵染活力,且本发明制备方法稳定性好,效率高,可实现大规模制备,满足规模化实验接种体、测试体等的需求。(The invention discloses a method for efficiently stopping and concentrating zoospores of plant pathogenic oomycetes, which comprises the steps of preparing zoospore suspension, stopping zoospores and concentrating the stopped spores, and specifically comprises the following steps: inoculating and culturing the plant pathogenic oomycetes to prepare a plant pathogenic oomycete zoospore suspension; resting zoospores: placing the plant pathogenic oomycete zoospore suspension into a centrifugal tube, and forming resting spores under the conditions of instantaneous overweight and weightlessness alternative treatment; and carrying out low-temperature centrifugal concentration on the resting spores to obtain a target resting spore suspension. The method adopts an instantaneous overweight and weightlessness alternative treatment method to rapidly stop the zoospores, fundamentally overcomes the problem of low stopping efficiency in the prior art, greatly improves the infection activity of the resting zoospores, has good stability and high efficiency, can realize large-scale preparation, and meets the requirements of large-scale experimental inoculants, test bodies and the like.)

1. a method for efficiently stopping and concentrating zoospores of plant pathogenic oomycetes is characterized by comprising the steps of preparing zoospore suspension, stopping zoospores and concentrating the stopped spores, and specifically comprises the following steps:

A. Preparation of zoospore suspension: inoculating and culturing the plant pathogenic oomycetes to prepare a plant pathogenic oomycete zoospore suspension;

B. Resting zoospores: placing the plant pathogenic oomycete zoospore suspension into a centrifugal tube, and forming resting spores under the conditions of instantaneous overweight and weightlessness alternative treatment;

C. And (3) concentrating the resting spores: and carrying out low-temperature centrifugal concentration on the resting spores to obtain a target resting spore suspension.

2. The method for high-efficiency resting concentration of zoospores of plant pathogenic oomycetes according to claim 1, characterized in that the inoculation culture comprises hypha culture and zoospore induction culture.

3. The method for high-efficiency resting concentration of zoospores of plant pathogenic oomycetes according to claim 2, characterized in that the hypha culture is carried out by inoculating the plant pathogenic oomycetes into an OA culture medium and culturing in the dark.

4. the method for high ~ efficiency resting concentration of zoospores of plant pathogenic oomycetes according to claim 3, characterized in that the dark culture is carried out at a temperature of 24 ~ 26 ℃ for 7 ~ 10 days.

5. the method for efficient resting concentration of zoospores of plant pathogenic oomycetes according to claim 2, characterized in that the zoospores are induced and cultured by cutting hyphae into small pieces to obtain hypha blocks, inoculating the hypha blocks into a 10% V8 juice liquid culture medium, culturing in the dark for 3 ~ 4 days, pouring out the culture solution, washing the hypha blocks with sterilized water for 3 ~ 5 times, changing the sterilized water for 1 ~ 3 times every day, continuously inducing the generation of zoosporangia for 2 ~ 3 days, treating at 5 ~ 8 ℃ for 20 ~ 30min, taking out, placing at room temperature for 15 ~ 30min, inducing the zoospores to release the zoospores, and filtering to remove the hyphae, wherein the filtrate is the target plant pathogenic oomycetes spore suspension.

6. the method for high ~ efficiency resting concentration of zoospores of plant pathogenic oomycetes according to claim 5, characterized in that the temperature of dark culture is 24 ~ 26 ℃.

7. the method for efficiently suspending and concentrating the zoospores of plant pathogenic oomycetes according to claim 1, wherein the alternate treatment of instantaneous overweight and weightlessness is to place the suspension of the zoospores of plant pathogenic oomycetes in a centrifuge tube and place the suspension on a pendulum with a pendulum length of 20 ~ 30cm, and the pendulum swings left and right to realize the instantaneous overweight and weightlessness state to cause flagellum to fall off to form the cyclosporine.

8. the method for efficiently suspending and concentrating the zoospores of the plant pathogenic oomycetes according to claim 7, which is characterized in that the swing range of the left ~ right swing is 30 ~ 45 degrees, and the swing time is 15 ~ 30 min.

9. the method for high ~ efficiency resting and concentrating zoospores of plant pathogenic oomycetes according to claim 1, characterized in that the low ~ temperature centrifugal concentration is to centrifuge the resting spores at a low temperature of 4 ~ 6 ℃ and 2500 ~ 3000r/min for 15 ~ 30min, pour out and suck the supernatant, and resuspend to obtain a suspension of the target resting spores.

10. an application of the method for efficiently resting and concentrating zoospores of plant pathogenic oomycetes according ~ any one of claims 1 ~ 9, which is characterized in that the method for efficiently resting and concentrating zoospores of plant pathogenic oomycetes is applied ~ preparation of high-infection activity resting spores.

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application thereof.

Background

the method is characterized in that hyphae of plant pathogenic oomycetes can be directly formed or developed into zoosporangia in various shapes, protoplasts in the zoosporangia are divided into a plurality of small blocks, the small blocks are gradually changed into circles, zoospores are formed by surrounding a film, the zoospores are kidney ~ shaped, pear ~ shaped or spherical, one or two flagella are provided, the flagella fall off to form resting spores after swimming in water for a period of time, the concentration of the prepared zoospores cannot meet the requirements of experiments due to the influence of operation links such as solid culture hyphae, liquid culture induced sporulation, low ~ temperature treatment release of the zoospores and the like when the zoospores are prepared in a laboratory, the concentration can be improved by concentration, the concentration can be improved, the concentration is usually realized by the steps of enabling the zoospores to form resting spores and then centrifuging at low temperature, removing supernatant, the concentration of the micro liquid can be improved after resuspension, the concentration of the zoospores can be improved after 7 ~ 9 hours, the natural infection is long, the activity of the zoospores is generally greatly reduced, natural resting spores are rarely adopted in the laboratory, the resting spores, the existing vortex oscillation technology can promote the development of the resting spores to be improved, and the efficiency of the tobacco is usually higher than 50 percent.

Disclosure of Invention

the first purpose of the invention is to provide a method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes; the second purpose is to provide the application of the method for efficiently suspending and concentrating the zoospores of the plant pathogenic oomycetes.

The first purpose of the invention is realized by that the method for efficiently stopping and concentrating the zoospores of the plant pathogenic oomycetes comprises the steps of preparing a zoospore suspension, stopping the zoospores and concentrating the stopped spores, and specifically comprises the following steps:

A. Preparation of zoospore suspension: inoculating and culturing the plant pathogenic oomycetes to prepare a plant pathogenic oomycete zoospore suspension;

B. Resting zoospores: placing the plant pathogenic oomycete zoospore suspension into a centrifugal tube, and forming resting spores under the conditions of instantaneous overweight and weightlessness alternative treatment;

C. and (3) concentrating the resting spores: and carrying out low-temperature centrifugal concentration on the resting spores to obtain a target resting spore suspension.

The second purpose of the invention is realized by the application of the method for efficiently stopping and concentrating the zoospores of the plant pathogenic oomycetes in preparing the high-infection activity staurosporium.

The method adopts an instantaneous overweight and weightlessness alternative treatment method to rapidly stop the zoospores, fundamentally overcomes the problem of low stopping efficiency in the prior art, greatly improves the infection activity of the resting zoospores, has good stability and high efficiency, can realize large-scale preparation, and meets the requirements of large-scale experimental inoculants, test bodies and the like.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.

The method for efficiently stopping and concentrating zoospores of plant pathogenic oomycetes comprises the steps of preparing zoospore suspension, stopping zoospores and concentrating the stopped spores, and specifically comprises the following steps:

A. Preparation of zoospore suspension: inoculating and culturing the plant pathogenic oomycetes to prepare a plant pathogenic oomycete zoospore suspension;

B. Resting zoospores: placing the plant pathogenic oomycete zoospore suspension into a centrifugal tube, and forming resting spores under the conditions of instantaneous overweight and weightlessness alternative treatment;

C. And (3) concentrating the resting spores: and carrying out low-temperature centrifugal concentration on the resting spores to obtain a target resting spore suspension.

The inoculation culture comprises hypha culture and zoospore induction culture.

The hypha culture is to inoculate plant pathogenic oomycetes into an OA culture medium for dark culture to obtain hypha.

the dark culture is carried out for 7 ~ 10 days at the temperature of 24 ~ 26 ℃.

the zoospore induction culture comprises the steps of cutting hyphae into small pieces to obtain hypha blocks, inoculating the hypha blocks into a 10% V8 juice culture medium, culturing for 3 ~ 4 days in the dark, pouring out a culture solution, washing the hypha blocks with sterile water for 3 ~ 5 times, changing the sterile water for 1 ~ 3 times every day, continuously inducing the generation of zoosporangium for 2 ~ 3 days, treating for 20 ~ 30min at a low temperature of 5 ~ 8 ℃, taking out, placing at room temperature for 15 ~ 30min, inducing the zoosporangium to release zoospores, and filtering to remove the hypha, wherein a filtrate is a target plant pathogenic oomycete zoospore suspension.

the temperature of dark culture is 24 ~ 26 ℃.

the instantaneous overweight and weightlessness alternate treatment is to place the plant pathogenic oomycete zoospore suspension in a centrifuge tube, place the centrifuge tube on a pendulum with the pendulum length of 20 ~ 30cm, and swing left and right to realize the instant overweight and weightlessness state to cause flagellum to fall off to form resting spores.

the swing range of the left degrees ~ right swing is 30 degrees ~ 45 degrees, and the swing time is 15 degrees ~ 30 min.

and the low ~ temperature centrifugal concentration is to centrifuge the resting spores at the low temperature of 2500 ~ 3000r/min at 4 ~ 6 ℃ for 15 ~ 30min, pour out and suck the supernatant, and resuspend to obtain the target resting spore suspension.

the application of the method for efficiently stopping and concentrating the zoospores of the plant pathogenic oomycetes is the application of the method for efficiently stopping and concentrating the plant pathogenic oomycetes in preparing the high-infection activity zoospores.

the invention is further illustrated by the following specific examples: