CN108034629B - Application of CA culture medium in culturing strawberry brown leaf spot pathogen sporulation - Google Patents
Application of CA culture medium in culturing strawberry brown leaf spot pathogen sporulation Download PDFInfo
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- CN108034629B CN108034629B CN201810054501.6A CN201810054501A CN108034629B CN 108034629 B CN108034629 B CN 108034629B CN 201810054501 A CN201810054501 A CN 201810054501A CN 108034629 B CN108034629 B CN 108034629B
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- culture medium
- leaf spot
- brown leaf
- culturing
- spot pathogen
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- C—CHEMISTRY; METALLURGY
- 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
Abstract
The application relates to an application of a CA culture medium in culturing sporulation of strawberry brown leaf spot bacteria, wherein the formula of the CA culture medium is as follows: 100g to 400g of carrot juice and 13 g to 25 g of agar, in a total volume of 1000 ml.
Description
Technical Field
The application relates to application of a CA culture medium in culturing sporulation of strawberry brown leaf spot bacteria.
Background
Strawberry brown leaf spot pathogen is an important pathogenic bacterium causing strawberries, peonies and the like, and the existing research mainly adopts a PDA culture medium to culture the strawberry brown leaf spot pathogen, but the spore yield is generally very low, and the problem that the spore yield speed of the strawberry brown leaf spot pathogen is slow and the related research is not developed is solved.
Disclosure of Invention
In order to solve the problems in the prior art, one of the applications provides an application of a CA culture medium in culturing strawberry brown leaf spot pathogen (Pilidium concavum) spore production, wherein the formula of the CA culture medium is as follows: 100 to 400g of carrot juice and 13 to 22 g of agar, in a total volume of 1000 ml.
In one embodiment, the formulation of the CA medium is as follows: 200 to 400g of carrot juice and 19 to 20g of agar, in a total volume of 1000 ml.
The second application provides a method for culturing strawberry brown leaf spot pathogen (Pilidium concavum) to produce spores, which comprises the following steps: activating strawberry brown leaf spot pathogen in a PDA culture medium, inoculating the activated strawberry brown leaf spot pathogen to the PDA culture medium, culturing for 5d to 7d, beating a bacterial cake with the diameter of 5 +/-3 mm on the same circumference of the edge of a bacterial colony, then inoculating the bacterial cake to a flat plate of the CA culture medium in any one of the applications, and culturing for 7d to 9d in the dark at the temperature of 25 +/-2 ℃.
In one embodiment, the strawberry brown leaf spot pathogen is activated in PDA culture medium, inoculated on PDA culture medium for 6 days, and beaten into fungus cake with diameter of 5mm on the same circumference of colony edge, and then inoculated on the plate of CA culture medium in any one of the applications, and cultured in dark at 25 ℃ for 8 days.
The beneficial effect of this application:
through screening of a large number of culture medium formulas, the application unexpectedly finds that the formula of the CA culture medium is particularly suitable for sporulation of strawberry brown leaf spot pathogen (Pilidium concavum). Therefore, the problem that related researches are not enough to be carried out due to the slow spore production speed and the low spore production amount of the strawberry brown leaf spot pathogen in the prior art is solved.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
The experimental strains are strains which are obtained from Beijing strawberry through conventional tissue separation, single-spore picking and PDA slant preservation in earlier experimental research, are marked as BJ-4, BJ-10, BJ-12, BJ-15, BJ-17, BJ-18, BJ-20, BJ-21, BJ-25 and BJ-26, are determined to be strawberry brown leaf spot pathogen (Pilidium concavum) through Huada gene sequencing and Blast comparison in GenBank of rDNA ITS sequences, and can be obtained from Beijing academy of agriculture. The isolation procedure was carried out according to the references kaivan KARIMI, MAHDI ARZANLOU, ASADOLLAH BABABAI-AHARI and ILARIA PERTIT, biological and molecular characterization of Pilidinium lythri, engineering strain pathogram in Iran. phytopathogram Mediterranea, (2016)55,3, 366-.
Example 1
Preparation of PDA culture medium: peeling 200g potato, cutting into pieces, decocting for 30 min to soft and soft, filtering with four layers of gauze, adding 20g glucose and 20g agar powder, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and moist heat for 20min, cooling to 60 deg.C, and making into plate.
Example 2
Preparation of YDA culture medium: 5g of yeast powder, 20g of glucose and 20g of agar powder, fixing the volume in 1L of water, carrying out high-pressure damp-heat sterilization at 121 ℃ for 20min, cooling to about 60 ℃, and preparing into a flat plate.
Example 3
SPDA medium: peeling 200g potato, cutting into pieces, decocting for 30 min to soft and rotten, filtering with four layers of gauze, adding 20g glucose and 20g agar powder, adding 120ml fresh strawberry juice, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and moist heat for 20min, cooling to 60 deg.C, and making into flat plate.
Example 4
YPDA medium: peeling 200g potato, cutting into pieces, boiling for 30 min to soft, filtering with four layers of gauze, adding 20g glucose, 20g agar powder, and 5g yeast powder, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and moist heat for 20min, cooling to 60 deg.C, and making into flat plate.
Example 5
CPDA medium: peeling 200g potato and 100g carrot, cutting into pieces, decocting for 30 min to soft and rotten, filtering with four layers of gauze, adding 20g glucose and 20g agar powder, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and moist heat for 20min, cooling to 60 deg.C, and making into flat plate.
Example 6
CA culture medium: squeezing 200g radix Dauci Sativae, filtering, adding 20g agar into the filtrate, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and wet heat for 20min, cooling to 60 deg.C, and making into plate.
Wherein, the carrot juicing operation is as follows: washing carrot, peeling, cutting into blocks, and squeezing juice with a juicer.
Example 7
CA culture medium: squeezing 100g radix Dauci Sativae, filtering, adding 20g agar into the filtrate, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and humidity for 20min, cooling to 60 deg.C, and making into plate.
Carrot juicing was performed as in example 6.
Example 8
CA culture medium: squeezing 400g radix Dauci Sativae, filtering, adding 20g agar into the filtrate, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and wet heat for 20min, cooling to 60 deg.C, and making into plate.
Carrot juicing was performed as in example 6.
Example 9
TPDA medium: peeling 200g potato, cutting into pieces, boiling for 30 min to soft, filtering with four layers of gauze, adding 20g glucose, 20g agar powder, and 2g tryptone, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and humidity for 20min, cooling to 60 deg.C, and making into flat plate.
Example 10
2P2DA medium: peeling 400g of potato, cutting into pieces, boiling for 30 minutes until the potato is soft and rotten, filtering with four layers of gauze, adding 40g of glucose, 20g of agar powder and 2g of tryptone into 1L of water, carrying out high-pressure moist heat sterilization at 121 ℃ for 20min, cooling to 60 ℃, and preparing into a flat plate.
Example 11
BSPDA culture medium: peeling 200g potato, cutting into pieces, decocting with 100g bean sprout for 30 min to soft and rotten, filtering with four layers of gauze, adding 35g glucose and 20g agar powder, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and moist heat for 20min, cooling to 60 deg.C, and making into flat plate.
Example 12
BSDA medium: boiling 100g bean sprout for 30 min to soft, filtering with four layers of gauze, adding 50g glucose and 20g agar powder, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and damp heat for 20min, cooling to 60 deg.C, and making into plate.
Example 13
P2DA medium: peeling 200g potato, cutting into pieces, decocting for 30 min to soft and soft, filtering with four layers of gauze, adding 40g glucose and 20g agar powder, dissolving in 1L water, sterilizing at 121 deg.C under high pressure and moist heat for 20min, cooling to 60 deg.C, and making into plate.
Example 14
V8 medium: v8 vegetable juice 200ml, adding calcium carbonate 3g and agar powder 20g, dissolving in 1L water, sterilizing at 121 deg.C for 20min, cooling to 60 deg.C, and making into tablet.
V8 vegetable juice was purchased from Jinbao Tang corporation (Campbell SOUP COMPANY) at V8100% vegetable juice.
Example 15
Activating strawberry brown leaf spot pathogen to be tested in a PDA culture medium, inoculating the activated strawberry brown leaf spot pathogen to a freshly prepared (0-14 days) PDA culture medium for culturing for 6 days, beating a bacterial cake with the diameter of 5mm on the same circumference of the edge of a bacterial colony, then respectively inoculating the bacterial cake to a plate of the PDA culture medium, YDA culture medium, SPDA culture medium, YPDA culture medium, CPDA culture medium, CA culture medium, TPDA culture medium, 2P2DA culture medium, BSPDA culture medium, BSDA culture medium, P2DA culture medium and V8 culture medium, and carrying out dark culture at 25 ℃ for 8 days. And respectively radially beating a certain number of bacterial cakes from the central bacterial receiving position of the culture plate outwards by adopting a puncher with the diameter of 5mm, recording the number of the beaten bacterial cakes, then placing the bacterial cakes into a centrifugal tube added with 10mL of sterile water (added with 0.1% Tween-20), adding 5-8 pre-sterilized glass beads, oscillating for 2min to elute spores, calculating the number of the spores by using a blood counting cell plate, repeating for 3 times every treatment, calculating the average spore yield per square centimeter, and performing statistical analysis by adopting a minimum significant difference method in SPSS software. The whole experiment was repeated 2 times. The results are shown in tables 1 to 2.
As can be seen from tables 1 and 2, the rapid sporulation rates of the strawberry brown leaf spot bacterial strains BJ-4, BJ-10, BJ-12, BJ-15, BJ-17, BJ-18, BJ-20, BJ-21, BJ-25 and BJ-26 on the CA culture medium are obviously different from those on other culture media, which shows that the CA culture medium has the function of promoting the rapid sporulation of strawberry brown leaf spot pathogenic bacteria, and the CA can be obtained to have the good effect of promoting the rapid sporulation of the strawberry brown leaf spot pathogenic bacteria.
TABLE 1
TABLE 2
Claims (4)
- Use of CA medium in culturing strawberry brown leaf spot bacteria (A)Pilidium concavum) The application in sporulation, wherein the formula of the CA culture medium is as follows: 100g to 400g of carrot juice, 13 g to 22 g of agar, and the balance of water, in a total volume of 1000 ml.
- 2. The use according to claim 1, wherein the formulation of the CA medium is as follows: 200 to 400g of carrot juice, 18 to 20g of agar, and the balance of water, in a total volume of 1000 ml.
- 3. Use according to claim 1 or 2, characterized in that it comprises the following steps: activating strawberry brown leaf spot pathogen in PDA culture medium, inoculating to PDA culture medium, culturing for 5 d-7 d, beating bacterial cake with diameter of 5 + -3 mm on the same circumference of colony edge, inoculating to plate of CA culture medium, and culturing at 25 + -2 deg.C in dark for 7 d-9 d.
- 4. The use of claim 3, wherein the strawberry brown leaf spot pathogen is activated in PDA culture medium, inoculated on PDA culture medium for 6 days, and beaten on the same circumference of colony edge to obtain bacterial cake with diameter of 5mm, and then inoculated on the plate of CA culture medium, and cultured in dark at 25 ℃ for 8 days.
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