CN108410793B - Culture medium for inducing phytophthora capsici to produce spores, preparation method and application - Google Patents
Culture medium for inducing phytophthora capsici to produce spores, preparation method and application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention belongs to the technical field of culture media, and discloses a culture medium for inducing phytophthora capsici to quickly generate a large amount of zoosporangiums, a preparation method and application. The culture medium takes orange juice as a main raw material, has proper pH value and rich nutrition, comprises various inorganic and organic nutrients such as saccharides, proteins, vitamin B1, vitamin C and the like, provides various nutrient substances required by the growth of the phytophthora capsici, and is more favorable for the formation of phytophthora capsici sporangia. By using the culture medium, a large amount of zoosporangia can be induced in a short time, and experiments prove that: the number of the zoosporangia generated by each square centimeter of hypha block within 24h is up to 3.19 ten thousand, thereby not only solving the problem that a large amount of sporangia is difficult to generate by a common solid culture medium, but also avoiding the problem that the zoosporangia generated by the induction of a water culture method is easy to be polluted.
Description
Technical Field
The invention belongs to the technical field of culture media, relates to a culture medium for inducing phytophthora capsici to produce spores, and more particularly relates to a culture medium for inducing phytophthora capsici to rapidly produce a large amount of zoosporangiums, a preparation method and an application.
Background
Phytophthora capsici (Phytophthora capsici) is a worldwide devastating disease caused by an infestation of Phytophthora capsici (Phytophthora capsici Leonian). Phytophthora capsici phytopathogen oomycetes generally live through the winter on diseased plant residues, soil and seeds by using oospores and chlamydospores, become a primary infection source of diseases in the next year, and are infected again by wind, rain, irrigation and other agricultural activities to cause the spread and damage of the diseases. Besides being harmful to pepper, phytophthora capsici also infects more than 20 important vegetable crops such as tomato, eggplant, cucumber, watermelon, pumpkin and the like. The disease is discovered in new Mexico in the United states for the first time in 1918, has occurred worldwide, and tends to be aggravated year by year, so that the economic benefit of the vegetable planting industry is seriously influenced, and huge economic loss is caused to the safe production of vegetables. Therefore, how to effectively and comprehensively manage the pepper phytophthora blight and reduce the economic loss caused by the pepper phytophthora blight to the maximum is an urgent technical problem to be solved.
The research and the determination of the epidemic rule of the pepper phytophthora blight are the precondition for establishing the comprehensive disease management measures, the research of the epidemic rule of the pepper blight needs to be carried out by means of a pathogenic bacteria artificial inoculation technology, the pure culture of target pathogenic bacteria is obtained through separation and purification, and then a large amount of artificial inoculants are obtained through the pure culture, so that the first step of the artificial inoculation is realized. In the research of pepper phytophthora blight, an artificial inoculum is zoospores, and the traditional method for obtaining the zoospores is to obtain sporangiums and then stimulate the sporangiums to release the zoospores, so that the final inoculum is obtained. The conventional method for producing sporangium by phytophthora capsici is a water culture method which utilizes a solid culture medium such as a potato agar culture medium, a corn flour culture medium, a carrot culture medium, an oat culture medium and the like to be cultured by illumination for a certain time or adds a hypha block into sterile water to induce and produce the sporangium.
Disclosure of Invention
Therefore, the invention aims to provide a culture medium capable of quickly and efficiently inducing phytophthora capsici to generate a large amount of zoosporangia.
Therefore, the invention provides a culture medium for inducing phytophthora capsici to produce spores, wherein the culture medium takes orange juice as a carbon source, and each liter of the culture medium contains 25-75 mL of orange juice.
Furthermore, each liter of the culture medium also contains 15-20 g of agar.
Further, the pH value of the culture medium is 5.5-6.5.
The invention also provides a preparation method of the culture medium for inducing phytophthora capsici to produce spores, which comprises the following steps:
and adding the agar into the orange juice, adding water to a constant volume, mixing and sterilizing to obtain the culture medium for inducing the phytophthora capsici to produce spores.
Further, the sterilization specifically comprises: sterilizing at 120-125 deg.C under 0.10-0.12 MPa for 15-25 min.
Further, the step of filtering the orange juice before adding the agar is also included.
Further, the filtration is performed by using a double-layer gauze.
The method for inducing the phytophthora capsici to produce sporangiums by using the culture medium for inducing the phytophthora capsici to produce spores or the culture medium for inducing the phytophthora capsici to produce spores, which is obtained by the preparation method of the culture medium for inducing the phytophthora capsici to produce spores, comprises the following steps of:
(1) inoculating phytophthora capsici mycelium blocks on the culture medium for inducing phytophthora capsici to produce spores, and placing in the dark at the temperature of 25-28 ℃ for culture until the culture medium is full of mycelia;
(2) and (2) placing the culture medium system in the step (1) under the illumination of 1600-2400 lx for continuous illumination culture for 1-2 days.
Further, the step (1) was incubated in the dark at 26 ℃ for 5 days.
Further, the step (2) is placed under the illumination of 2000lx for continuous illumination culture for 1-2 days.
The technical scheme of the invention has the following advantages:
1. the culture medium for inducing phytophthora capsici sporulation provided by the invention takes orange juice as a main raw material, has a proper pH value and rich nutrition, comprises various inorganic and organic nutrients such as saccharides, proteins, vitamin B1, vitamin C and the like, provides various nutrient substances required by the growth of phytophthora capsici and is more beneficial to the formation of phytophthora capsici sporangia. By using the culture medium, a large amount of zoosporangia can be induced in a short time, and experiments prove that: the number of the zoosporangia generated by each square centimeter of hypha block within 24h is up to 3.19 ten thousand, thereby not only solving the problem that a large amount of sporangia is difficult to generate by a common solid culture medium, but also avoiding the problem that the zoosporangia generated by the induction of a water culture method is easy to be polluted.
The orange juice is adopted as the main raw material, the material is easy to obtain, the cost is low, the operation is simple and convenient, the large-scale popularization and application are facilitated, and the practical application value is very high.
2. The preparation method of the culture medium for inducing phytophthora capsici to produce spores provided by the invention can be used for filtering and sterilizing the culture medium, so that the chance of polluting the culture medium is greatly reduced. The preparation process of the culture medium is simple, the repeatability is good, the effect is stable, and the method has important significance for developing the research of the pepper phytophthora blight.
3. The method for inducing the sporangium by using the culture medium for inducing the phytophthora capsici sporulation is simple, a large amount of zoosporangiums can be induced and generated by full illumination treatment, and compared with the traditional culture induction method, the induction period is greatly shortened.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the results of sporangia production by a common strain of Phytophthora capsici cultured in 2000lx of light for 48 hours on a medium according to example 2 of the present invention;
FIG. 2 is a graph showing the results of sporangium production by culturing a common strain of Phytophthora capsici on CA medium under 2000lx of light for 48 hours;
FIG. 3 is a graph showing the results of sporangium production by culturing in OA medium, a common strain of Phytophthora capsici for 48 hours at 2000 lx.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In the following examples, the phytophthora capsici strains are common strains and are isolated from field diseased tissues;
the culture dish, PDA culture medium, OA culture medium, CA culture medium, RA culture medium, hole puncher, sterilizer, distilled water, deionized water, incubator and the like required by the experimental operation are all conventional tools or materials in the laboratory.
Example 1
The embodiment provides a culture medium for inducing phytophthora capsici to produce spores, which comprises the following specific components: each 1L of the culture medium contains 25mL of orange juice and 15g of agar powder. The preparation method specifically comprises the following steps: taking a plurality of common sweet oranges, peeling, cutting into small pieces, squeezing, measuring 25mL of orange juice, filtering with double-layer gauze, adding 15g of agar powder into the obtained filtrate, adding distilled water to a constant volume of 1L, and fully stirring with a glass rod. Subpackaging according to the requirement, placing in a high-pressure steam sterilization pot, sterilizing at 121 ℃ under 0.103MPa for 20min, taking out, pouring into a sterilized culture dish with the diameter of 9cm, and preparing the culture medium for inducing phytophthora capsici to produce spores.
Example 2
The embodiment provides a culture medium for inducing phytophthora capsici to produce spores, which comprises the following specific components: each 1L of the culture medium contained 50mL of orange juice and 15g of agar powder. The preparation method is the same as that of example 1.
Example 3
The embodiment provides a culture medium for inducing phytophthora capsici to produce spores, which comprises the following specific components: each 1L of the culture medium contained 75mL of orange juice and 15g of agar powder. The preparation method is the same as that of example 1.
Comparative example 1
The comparative example provides a Potato Dextrose Agar (PDA) culture medium, which comprises the following specific components: each 1L of the medium contained 200g of peeled potato, 20g of glucose and 15g of agar powder. The preparation method specifically comprises the following steps: weighing 200g of peeled potatoes, cutting into small pieces, putting the small pieces into 1000mL of distilled water, boiling for 30min, filtering, adding 20g of glucose and 15g of agar powder into the obtained filtrate, fully stirring by using a glass rod, and fixing the volume to 1L. Subpackaging according to the requirement, placing in a high pressure steam sterilization pot, sterilizing at 121 deg.C under 0.103MPa for 20min, taking out, and pouring into a sterilized culture dish with a diameter of 9cm to obtain the PDA culture medium.
Comparative example 2
The comparative example provides an Oat (OA) culture medium, which comprises the following specific components: each 1L of the culture medium contains 30g of oatmeal and 15g of agar powder. The preparation method specifically comprises the following steps: weighing 30g of oatmeal, putting the oatmeal into 1000mL of distilled water, heating the oatmeal in a water bath at 60 ℃ for 1h, filtering, adding 15g of agar powder into the obtained filtrate, fully and uniformly stirring the mixture by using a glass rod, and fixing the volume to 1L. Subpackaging as required, placing in a high pressure steam sterilization pot, sterilizing at 121 deg.C under 0.103MPa for 20min, taking out, and pouring into a sterilized culture dish with diameter of 9cm to obtain the OA culture medium.
Comparative example 3
The comparative example provides a Carrot (CA) medium, which comprises the following specific components: each 1L of the culture medium contained 200g of carrot and 15g of agar powder. The preparation method specifically comprises the following steps: weighing 200g of carrot, cutting into small blocks, putting the small blocks into 1000mL of distilled water, boiling for 30min, filtering, adding 15g of agar powder into the obtained filtrate, fully and uniformly stirring by using a glass rod, and fixing the volume to 1L. Subpackaging as required, placing in a high pressure steam sterilization pot, sterilizing at 121 deg.C under 0.103MPa for 20min, taking out, and pouring into a sterilized culture dish with diameter of 9cm to obtain the CA medium.
Comparative example 4
The present comparative example provides a Rye (RA) medium, comprising the specific components: each 1L of medium contained 50g of rye seeds and 15g of agar powder. The preparation method specifically comprises the following steps: weighing 50g of rye seeds, soaking in 1000mL of deionized water for 24-36 h, placing in a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 30min, filtering, adding 15g of agar powder into the obtained filtrate, fully stirring by using a glass rod, and fixing the volume to 1L. Subpackaging according to the requirement, placing in a high pressure steam sterilization pot, sterilizing at 121 deg.C under 0.103MPa for 20min, taking out, and pouring into a sterilized culture dish with a diameter of 9cm to obtain the RA culture medium.
Experimental example 1
Inoculating phytophthora capsici strain to a PDA (potato dextrose agar) culture medium on a clean bench, and culturing at 27 ℃ in the dark for 4 days. Punching at the edge of the colony with a puncher with diameter of 6cm, placing 7 mycelium blocks in the center of different plate culture media, culturing in dark at 26 deg.C for 5 days, and continuously culturing in an illumination incubator with illumination of 2000 lx. After 24h and 48h, the sporangium production on each medium was observed and counted. The counting method comprises the following steps: randomly beating 5 hypha blocks on a plate culture medium by using a perforator with the diameter of 6cm, placing the hypha blocks in an aseptic culture dish, adding 30mL of distilled water into the aseptic culture dish, and slightly brushing the surfaces of the hypha blocks by using a disinfection brush pen so as to enable zoosporangia to fall in water; the mycelium was then filtered off with a double gauze to give a filtrate, 15. mu.L of which was pipetted onto a glass slide and the number of zoosporangia was observed and recorded under a microscope. The number of zoosporangia on each square centimeter of hypha block is calculated according to the area of the hypha block, each sample is observed repeatedly for 3 times, 3 plate samples are randomly selected for each culture medium for detection, and finally the average value is calculated to be the average sporulation quantity of the phytophthora capsici strain on the culture medium, and the result is shown in table 1.
TABLE 1 number of zoosporangia and significance of difference
Wherein: the significance of the difference is the production amount of the zoosporangium after the phytophthora capsici is cultured in each culture medium for 48 hours, the lower case letters indicate the significance level of the 5% level, the upper case letters indicate the significance level of the 1% level, and different letters indicate that the difference exists.
As can be seen from the above table 1, the spore yield of the phytophthora capsici on the spore production culture medium of the phytophthora capsici is obviously higher than that of the PDA culture medium, the OA culture medium, the CA culture medium and the RA culture medium in the levels of 1% and 5%, the induced spore production speed of the spore production culture medium of the phytophthora capsici is higher, a large number of sporangiums can be produced within 24 hours, and the requirement of a rapid inoculation test can be met.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (8)
1. A method for inducing phytophthora capsici to produce sporangiums by using a culture medium for inducing the phytophthora capsici to produce the spores is characterized in that,
the culture medium for inducing phytophthora capsici to produce spores takes orange juice as a carbon source, and each liter of the culture medium contains 25-75 mL of orange juice; the preparation method of the culture medium for inducing phytophthora capsici sporulation comprises the following steps: adding agar into the orange juice, adding water to a constant volume, mixing, and sterilizing to obtain the culture medium for inducing phytophthora capsici to produce spores;
a method for inducing phytophthora capsici to produce sporangia, comprising the steps of:
(1) inoculating phytophthora capsici mycelium blocks on the culture medium for inducing phytophthora capsici to produce spores, and placing in the dark at the temperature of 25-28 ℃ for culture until the culture medium is full of mycelia;
(2) and (2) placing the culture medium system in the step (1) under the illumination of 1600-2400 lx for continuous illumination culture for 1-2 days.
2. The method according to claim 1, wherein the step (1) is carried out in the dark at 26 ℃ for 5 days.
3. The method according to claim 1 or 2, wherein the step (2) is performed by continuous light culture under an illumination of 2000lx for 1-2 days.
4. The method according to claim 1, wherein the culture medium for inducing phytophthora capsici sporulation further comprises 15 to 20g of agar per liter.
5. The method according to claim 1 or 4, wherein the pH of the culture medium for inducing the sporulation of Phytophthora capsici is 5.5 to 6.5.
6. The method according to claim 1, characterized in that the sterilization is in particular: sterilizing at 120-125 deg.C under 0.10-0.12 MPa for 15-25 min.
7. The method as claimed in claim 1, wherein the method for preparing the culture medium for inducing phytophthora capsici sporulation further comprises filtering the orange juice before adding the agar.
8. The method of claim 7, wherein the filtering is performed using a double layer gauze filter.
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