CN108410793A - A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application - Google Patents
A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application Download PDFInfo
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- CN108410793A CN108410793A CN201810391163.5A CN201810391163A CN108410793A CN 108410793 A CN108410793 A CN 108410793A CN 201810391163 A CN201810391163 A CN 201810391163A CN 108410793 A CN108410793 A CN 108410793A
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Abstract
The invention belongs to culture medium technical field, discloses a kind of induction phytophthora blight of pepper and quickly generate the culture medium of a large amount of zoosporangiums, preparation method and application.For the culture medium using orange juice as primary raw material, pH value is suitable, full of nutrition, including a variety of inorganic and organic nutrient substance, such as carbohydrate, protein and vitamin B1, vitamin C provide all kinds of nutriments needed for phytophthora blight of pepper growth, are more advantageous to the formation of phytophthora blight of pepper sporangium.Using the culture medium, a large amount of zoosporangium can induce in a short time, experiment proves：The zoosporangium quantity that interior mycelia block every square centimeter generates for 24 hours is up to 3.19 ten thousand, has both solved the problems, such as that general solid medium is difficult to generate a large amount of sporangiums, in turn avoids being easy contaminated problem using water culture induction generation zoosporangium.
Description
Technical field
The invention belongs to culture medium technical fields, are related to a kind of culture medium of induction phytophthora blight of pepper production spore, more specifically,
It is related to a kind of induction phytophthora blight of pepper and quickly generates the culture medium of a large amount of zoosporangiums, preparation method and application.
Background technology
Capsicum epidemic disease (Phytophthora capsici) is a kind of worldwide destructive disease, is by capsicum
Caused by phytophthora (Phytophthora capsici Leonian) infects.Phytophthora blight of pepper platymiscium cause of disease oomycetes, disease
Opportunistic pathogen is usually overwintering in diseased plant residuum, in soil and on seed with egg spore and chlamydospore, becomes the generation of coming year disease
First aggression source, after the onset disease portion generate sporangium, sporangium by wind, rain, pour water and other farming activities are infected again,
Cause the sprawling and harm of disease.In addition to endangering capsicum, phytophthora blight of pepper also infects tomato, eggplant, cucumber, watermelon, pumpkin etc.
More than 20 kinds of important vegetable.The disease from 1918 for the first time New Mexico of the U.S. find, in worldwide
Occur, and have the tendency that aggravating year by year, has seriously affected the economic benefit of vegetable cultivation industry, caused to the safety in production of vegetables
Huge economic loss.Therefore, how integrated management effectively to be carried out capsicum epidemic disease and to maximize reduction economical caused by it
Loss is a technical problem to be solved urgently.
It studies and clear capsicum epidemic disease natural occurrence is the precondition for formulating disease integrated management measure, research disease
Evil regularty of epidemic need to be by pathogen artificial inoculation technique, and the pure culture of target pathogenic bacteria is obtained by isolating and purifying, then by
Pure culture obtains the first step that a large amount of artificial infection body is artificial infection.In the research of capsicum epidemic disease, artificial infection body is
Zoospore, the method that tradition obtains zoospore are to obtain sporangium, then discharge zoospore by stimulation sporangium, to
Obtain final inoculum.The conventional method that phytophthora blight of pepper generates sporangium is trained using solid medium such as potato agar
The illumination cultivation by certain time such as base, maize powder medium, carrot culture medium, oat medium is supported, or mycelia block is added
Induction generates the water culture of sporangium in sterile water, the former produces spore time length and hardly results in a large amount of sporangiums, although the latter
A large amount of zoosporangiums can be quickly obtained, but complex for operation step, and easily by other living contaminants in operating process.
Invention content
Therefore, the present invention is intended to provide a kind of energy rapidly and efficiently inducing phytophthora blight of pepper generates the training of a large amount of zoosporangiums
Support base.
For this purpose, the present invention provides the culture medium that a kind of induction phytophthora blight of pepper produces spore, the culture medium is with orange juice
Carbon source contains 25~75mL of orange juice in every liter of culture medium.
Further, every liter of culture medium also contains 15~20g of agar.
Further, the pH of the culture medium is 5.5~6.5.
The present invention also provides the preparation method that a kind of above-mentioned induction phytophthora blight of pepper produces the culture medium of spore, including it is as follows
Step：
The agar is added in the orange juice, adds water constant volume, sterilizing is to get the induction phytophthora blight of pepper after mixing
Produce the culture medium of spore.
Further, described sterilize is specially：Sterilize 15~25min at 120~125 DEG C, 0.10~0.12MPa.
Further, it further includes being filtered processing to the orange juice to be added before the agar.
Further, the filtering is filtered using double gauze.
Above-mentioned induction phytophthora blight of pepper produces the culture medium of spore or produces the culture medium of spore according to above-mentioned induction phytophthora blight of pepper
The method that the culture medium induction phytophthora blight of pepper for the induction phytophthora blight of pepper production spore that preparation method obtains generates sporangium, including such as
Lower step：
(1) by phytophthora blight of pepper inoculated by hypha block on the culture medium that the induction phytophthora blight of pepper produces spore, be placed in 25~
28 DEG C of dark culturings, until mycelia covers with culture medium；
(2) it is continuous illumination culture 1~2 day under 1600~2400lx the culture medium system of step (1) to be placed in illuminance
.
Further, the step (1) is placed in 26 DEG C of dark culturings 5 days.
Further, it is continuous illumination culture 1~2 day under 2000lx that the step (2), which is placed in illuminance,.
Technical scheme of the present invention has the following advantages that：
1. the culture medium of induction phytophthora blight of pepper production spore provided by the invention, using orange juice as primary raw material, pH value is suitable,
It is full of nutrition, including a variety of inorganic and organic nutrient substance, such as carbohydrate, protein and vitamin B1, vitamin C, it provides peppery
All kinds of nutriments needed for the growth of green pepper phytophthora, are more advantageous to the formation of phytophthora blight of pepper sporangium.It is short using the culture medium
It can induce a large amount of zoosporangium in phase, experiment proves：The zoosporangium that mycelia block every square centimeter generates in for 24 hours
Quantity is up to 3.19 ten thousand, has both solved the problems, such as that general solid medium is difficult to generate a large amount of sporangiums, has in turn avoided utilizing water
The induction of training method generates zoosporangium and is easy contaminated problem.
Use orange juice for primary raw material, materials are easy, at low cost, easy to operate, are convenient for large-scale promotion application, have
Very high actual application value.
2. the preparation method of the culture medium of induction phytophthora blight of pepper production spore provided by the invention, is filtered culture medium and goes out
Bacterium greatly reduces the contaminated chance of culture medium.Culture medium preparation process is simple, reproducible, effect stability, for carrying out
The research of capsicum epidemic disease is of great significance.
3. the method for the culture medium inducing spore capsule of induction phytophthora blight of pepper production spore using the present invention is simple, sufficient light
It can induce according to processing and generate a large amount of zoosporangium, more traditional culture abductive approach is compared, and induction duration shortens significantly.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
Fig. 1 is that the common bacterial strain of phytophthora blight of pepper cultivates 48 on the culture medium of the embodiment of the present invention 2 under 2000lx illumination
Hour generates the result figure of sporangium；
Fig. 2 is that the common bacterial strain of phytophthora blight of pepper cultivates 48 hours generation sporangiums on CA culture mediums under 2000lx illumination
Result figure；
Fig. 3 is to cultivate 48 hours to generate sporangium under 2000lx illumination on the common bacterial strain OA culture mediums of phytophthora blight of pepper
Result figure.
Specific implementation mode
Technical scheme of the present invention will be clearly and completely described below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
There is the every other embodiment obtained under the premise of making creative work, shall fall within the protection scope of the present invention.In addition, below
Involved technical characteristic as long as they do not conflict with each other can be mutual in described different embodiments of the present invention
In conjunction with.
In following embodiment, Phytophthora capsici bacteria strain is common bacterial strain, isolated from the diseased tissues of field；
Culture dish, PDA culture medium, OA culture mediums needed for experimental implementation, CA culture mediums, RA culture mediums, card punch, sterilizing
Pot, distilled water, deionized water, incubator etc. are laboratory conventional tool or material.
Embodiment 1
A kind of culture medium of induction phytophthora blight of pepper production spore is present embodiments provided, specific ingredient is：Per 1L culture mediums
In contain 25mL orange juices and 15g agar powders.Preparation method is specially：Several common sweet oranges are taken, removes the peel, is cut into small pieces, are squeezed
Juice measures 25mL orange juices, is filtered with double gauze, and 15g agar powders are added in gained filtrate, distilled water is added to be settled to 1L, uses
Glass rod fully stirs evenly.It is placed in after packing in high-pressure steam sterilizing pan as needed, sterilize 20min at 0.103MPa, 121 DEG C,
Taking-up is poured into sterilized a diameter of 9cm culture dishes, and the culture medium of the induction phytophthora blight of pepper production spore is made.
Embodiment 2
A kind of culture medium of induction phytophthora blight of pepper production spore is present embodiments provided, specific ingredient is：Per 1L culture mediums
In contain 50mL orange juices and 15g agar powders.Preparation method is the same as embodiment 1.
Embodiment 3
A kind of culture medium of induction phytophthora blight of pepper production spore is present embodiments provided, specific ingredient is：Per 1L culture mediums
In contain 75mL orange juices and 15g agar powders.Preparation method is the same as embodiment 1.
Comparative example 1
This comparative example provides a kind of potato dextrose agar (PDA) culture medium, and specific ingredient is：Per 1L culture mediums
In contain 200g peeled potatoes, 20g glucose and 15g agar powders.Preparation method is specially：Weigh 200g peelings Ma Ling
Potato is put into after boiling 30min in 1000mL distilled water after being cut into small pieces and filters, and 20g glucose, 15g fine jades are added in gained filtrate
Cosmetics is settled to 1L after fully being stirred evenly with glass rod.Be placed in after packing in high-pressure steam sterilizing pan as needed, 0.103MPa,
Sterilize 20min at 121 DEG C, and taking-up is poured into sterilized a diameter of 9cm culture dishes, and the PDA culture medium is made.
Comparative example 2
This comparative example provides a kind of oat (OA) culture medium, and specific ingredient is：Contain 30g oats in per 1L culture mediums
Piece and 15g agar powders.Preparation method is specially：Weigh 30g oatmeals, be put into 1000mL distilled water, at 60 DEG C water-bath add
It is filtered after hot 1h, 15g agar powders is added in gained filtrate, 1L is settled to after fully being stirred evenly with glass rod.Packing postposition as needed
In high-pressure steam sterilizing pan, sterilize 20min at 0.103MPa, 121 DEG C, and sterilized a diameter of 9cm cultures are poured into taking-up
In ware, the OA culture mediums are made.
Comparative example 3
This comparative example provides a kind of carrot (CA) culture medium, and specific ingredient is：In per 1L culture mediums recklessly containing 200g
Radish and 15g agar powders.Preparation method is specially：200g carrots are weighed, is put into 1000mL distilled water and boils after being cut into small pieces
It is filtered after boiling 30min, 15g agar powders is added in gained filtrate, 1L is settled to after fully being stirred evenly with glass rod.As needed after packing
It is placed in high-pressure steam sterilizing pan, sterilize 20min at 0.103MPa, 121 DEG C, and sterilized a diameter of 9cm trainings are poured into taking-up
It supports in ware, the CA culture mediums is made.
Comparative example 4
This comparative example provides a kind of rye (RA) culture medium, and specific ingredient is：Contain 50g ryes in per 1L culture mediums
Seed and 15g agar powders.Preparation method is specially：Weigh 50g rye seeds, in 1000mL deionized waters impregnate 24~
After 36h, it is placed in high-pressure steam sterilizing pan and is filtered after 121 DEG C of sterilizing 30min, 15g agar powders are added in gained filtrate, use glass rod
1L is settled to after fully stirring evenly.It is placed in after packing in high-pressure steam sterilizing pan, sterilizes at 0.103MPa, 121 DEG C as needed
20min, taking-up are poured into sterilized a diameter of 9cm culture dishes, and the RA culture mediums are made.
Experimental example 1
On superclean bench, by phytophthora blight of pepper inoculation to PDA culture medium, 27 DEG C of dark culturing 4d.With straight
Diameter is that the card punch of 6cm punch at colony edge, takes 7 ferfas silk block to be respectively placed in different plating medium central, 26 DEG C
Dark culturing 5d is subsequently placed in the illumination box that illuminance is 2000lx and continuously cultivates.For 24 hours, each culture medium is observed after 48h
The production of upper sporangium and counting.Method of counting is：It is beaten at random on plating medium with diameter 6cm card punch and takes 5
Mycelia block is placed in sterile petri dish, and 30mL distilled water is added thereto, is gently scrubbed mycelia block surface with disinfection writing brush type brush, is made
Zoosporangium is obtained to fall off in water；Then mycelium is filtered off with double gauze, obtains filtrate, drawn 15 μ L filtrates and be placed in load
On slide, and the quantity of zoosporangium is observed and recorded under the microscope.Bacterium every square centimeter is found out according to mycelia block area
The quantity of zoosporangium, each sample repeated observation 3 times, each culture medium take 3 flat board samples to be examined at random on silk block
It surveys, the average sporulation quantity finally averaged as Phytophthora capsici bacteria strain on the culture medium, the results are shown in Table 1.
The quantity and the significance of difference of 1 zoosporangium of table
Wherein：The significance of difference is the yield of phytophthora blight of pepper zoosporangium after each medium culture 48 hours,
The horizontal level of signifiance of lowercase letter 5%, capitalization indicate that the 1% horizontal level of signifiance, different letters indicate exist
Significant difference.
From in upper table 1 as can be seen that phytophthora blight of pepper the present invention phytophthora blight of pepper product spore culture medium on sporulation quantity
It is all remarkably higher than PDA culture medium, OA culture mediums, CA culture mediums and RA culture mediums, and the capsicum of the present invention in 1% and 5% level
Phytophthora product spore culture medium induction production spore speed, can generate a large amount of sporangiums, can meet the need of quick inoculation test for 24 hours
It wants.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of culture medium of induction phytophthora blight of pepper production spore, which is characterized in that the culture medium is using orange juice as carbon source, every liter
Contain 25~75mL of orange juice in the culture medium.
2. culture medium according to claim 1, which is characterized in that every liter of culture medium also contains 15~20g of agar.
3. culture medium according to claim 1 or 2, which is characterized in that the pH of the culture medium is 5.5~6.5.
4. a kind of preparation method of the culture medium of induction phytophthora blight of pepper production spore as claimed in claim 1 or 2, which is characterized in that
Include the following steps：
The agar is added in the orange juice, adds water constant volume, sterilizes after mixing and produces spore to get the induction phytophthora blight of pepper
Culture medium.
5. preparation method according to claim 4, which is characterized in that the sterilizing is specially：120~125 DEG C, 0.10
Sterilize 15~25min under~0.12MPa.
6. preparation method according to claim 4 or 5, which is characterized in that it further includes to the orange to be added before the agar
Sub- juice is filtered processing.
7. preparation method according to claim 6, which is characterized in that the filtering is filtered using double gauze.
8. application claim 1-3 any one of them induction phytophthora blight of pepper produces the culture medium of spore or according to claim 4-7
The training for the induction phytophthora blight of pepper production spore that the preparation method of the culture medium of any one of them induction phytophthora blight of pepper production spore obtains
Support the method that base induction phytophthora blight of pepper generates sporangium, which is characterized in that include the following steps：
(1) phytophthora blight of pepper inoculated by hypha block is placed in 25~28 DEG C on the culture medium that the induction phytophthora blight of pepper produces spore
Dark culturing, until mycelia covers with culture medium；
(2) it is continuous illumination culture 1~2 day under 1600~2400lx the culture medium system of step (1) to be placed in illuminance.
9. according to the method described in claim 8, it is characterized in that, the step (1) is placed in 26 DEG C of dark culturings 5 days.
10. method according to claim 8 or claim 9, which is characterized in that it is under 2000lx that the step (2), which is placed in illuminance,
Continuous illumination culture 1~2 day.
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Cited By (2)
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| CN113373064A (en) * | 2021-07-14 | 2021-09-10 | 海南大学 | Method for inducing phytophthora litchi sporangium to release zoospores |
| CN117126256A (en) * | 2023-08-16 | 2023-11-28 | 中国农业大学 | Function of PH domain and START domain on plant pathogenic oomycete oxidized sterol binding protein and application thereof |
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| CN113373064A (en) * | 2021-07-14 | 2021-09-10 | 海南大学 | Method for inducing phytophthora litchi sporangium to release zoospores |
| CN117126256A (en) * | 2023-08-16 | 2023-11-28 | 中国农业大学 | Function of PH domain and START domain on plant pathogenic oomycete oxidized sterol binding protein and application thereof |
| CN117126256B (en) * | 2023-08-16 | 2024-06-11 | 中国农业大学 | Function of PH domain and START domain on plant pathogenic oomycete oxidized sterol binding protein and application thereof |
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