CN105886451B - Single spore separation method of phytophthora capsici zoospores - Google Patents
Single spore separation method of phytophthora capsici zoospores Download PDFInfo
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- CN105886451B CN105886451B CN201610265019.8A CN201610265019A CN105886451B CN 105886451 B CN105886451 B CN 105886451B CN 201610265019 A CN201610265019 A CN 201610265019A CN 105886451 B CN105886451 B CN 105886451B
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Abstract
The invention relates to a single spore separation method of phytophthora capsici zoospores, which comprises the steps of inoculating phytophthora capsici on a V8 culture medium, sealing the culture medium by using a sealing film at the constant temperature of 25 ℃ for 5d, removing the sealing film, illuminating for 24h, adding sterile water for inducing at the temperature of 4 ℃ and releasing at the normal temperature for 25min respectively to obtain zoospore suspension, diluting the zoospore suspension to 1-2 visual fields of each (10 ×), adding 1m L into a V8 culture medium plate with antibiotics for uniform coating, standing at the normal temperature for 6h, searching for single germinated zoospores by an inverted microscope, cutting a square mark of 0.5cm by using an operating knife, transferring the mark into an antibiotic plate after confirming the single spores, and purifying to obtain single spores.
Description
Technical Field
The invention relates to the technical field of agricultural plant protection, in particular to a single spore separation method of phytophthora capsici zoospores.
Background
The Phytophthora capsici (Phytophthora blight) is a destructive disease caused by Phytophthora capsici (Phytophthora capsicii L), which damages crops such as tomatoes, eggplants, cucumbers, melons and the like besides roots, stems, branches, leaves and fruits of the peppers, often causes large-area dead plants, generally has a field dead plant rate of 20-30 percent and seriously reaches more than 90 percent, has great influence on yield and quality, reduces yield by more than 50 percent when serious and even causes destructive damage.
At present, the method for separating phytophthora capsici zoospore monospore is a method for separating phytophthora capsici zoospore monospore by referring to Yuan sea and Yayuqi (2002) basically, the method comprises the steps of preparing zoosporangia or zoospore suspension, sterilizing a cover glass on a carrying platform, placing the cover glass on a sterilization overload glass sheet, taking 10-15 mu L spore suspension, dripping one drop of zoosporangia or zoospores on the cover glass, and performing microscopic examination to confirm that only 1 zoosporangia or zoospores in the drop of suspension is the monospora strain.
The method reported by the research has the problems of large workload, insufficient accuracy, long consumed time, complex process and the like. Aiming at the separation of single spores of phytophthora capsici zoospores, small individual, light color, irregular movement in visual field and large separation difficulty, the method has strong operability, high accuracy, high speed, good separation effect and simple equipment.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for separating monospore of phytophthora capsici zoospore.
In order to achieve the above purpose, the present invention is realized by the following technical scheme.
(1) Preparation of V8 juice agar medium: centrifuging V8 juice (100% vegetable) (JINBAO V8, imported from U.S. and sold in supermarket) at 4,000rpm to remove precipitate, preparing 10% V8 juice agar culture medium, and wet-heat sterilizing for 20 min;
(2) production of phytophthora capsici zoosporangia: inoculating phytophthora capsici to the V8 juice agar medium plate prepared in the step (1), sealing the plate by using a sealing film (model PM-996, American Parafilm), culturing the plate in dark at 25 ℃ for 5d to promote hypha growth, then removing the sealing film, and transferring the plate to the light (5000 lx) to culture the plate at 23-25 ℃ for 24h to promote the generation of zoosporangia;
(3) preparing phytophthora capsici zoospore suspension, namely adding 15m L sterile water into the plate with a large amount of zoosporangia generated in the step (2), placing the plate in a refrigerator at 4 ℃ for 25min, placing the plate at room temperature (23-25 ℃) for 25min, pouring out the suspension (namely the phytophthora capsici zoospore suspension), and diluting the suspension with the sterile water to obtain the suspension with only 1-2 zoospores in each (10 ×) visual field under a microscope on average;
(4) preparing a phytophthora capsici zoospore plate, namely spreading 1m L spore suspension obtained in the step (3) on a plate with a V8 juice agar culture medium, and standing at room temperature (23-25 ℃) for 6 hours to promote the zoospore to germinate;
(5) separating phytophthora capsici zoospore single spore strain, namely placing an inverted microscope on an aseptic operation table, after ultraviolet sterilization is carried out for 30min, pouring the residual suspension liquid off a flat plate with zoospores in the step (4), placing the flat plate on an object stage, searching for only 1 germinated zoospore in a (10 ×) visual field, then sterilizing and cooling a scalpel on flame, cutting a square cut with the length of 0.5cm on a culture medium around a single zoospore (right above a lens of the inverted microscope), and confirming only 1 zoospore in the cut again;
(6) and (3) picking and purifying the phytophthora capsici zoospore single spore strain, namely taking out the culture medium small block which is confirmed to be provided with the single zoospore in the step (5) by using a scalpel, putting the culture medium small block into another V8 culture medium flat plate provided with penicillin (50 mu g/m L), then putting the culture medium flat plate into a thermostat, carrying out dark culture for 2d at 25 ℃, picking hypha on the edge, and purifying the hypha into a test tube to obtain the phytophthora capsici zoospore single spore strain.
The invention has the following remarkable advantages:
the invention provides a method for separating phytophthora capsici zoospores, aiming at the problems that phytophthora capsici zoospores are small and colorless, and do not move randomly in a visual field, are difficult to capture and separate easily. The traditional method for producing phytophthora capsici sporangium is a method for inducing phytophthora capsici to produce zoospores, the two patents need long time for obtaining the zoospores, the zoospores are brushed or an aseptic glass rod is used for coating a culture medium plate full of hyphae, more hyphae are contained in the zoospores, the zoospores cannot be filtered out, and the probability of obtaining single zoospores is reduced. Patent publication No. CN102154193A also induces Phytophthora capsici to produce a large amount of sporangia, but the produced sporangia cannot be brought into aseptic conditions and cannot be used for isolation of an zoospore single strain. Compared with other single spore separation methods, the method has the advantages of strong operability, high accuracy, high speed, good separation effect and simple equipment, and can be used for separating a large quantity of single spore strains of phytophthora capsici zoospores in a laboratory in a short time.
Detailed Description
The above and further features and advantages of the invention are explained in more detail below with reference to examples and test data.
Specific operation for separating single spore strain of phytophthora capsici zoospore
Phytophthora capsici (which can be isolated and cultured by itself or obtained from related reports: Semeru, Schumann, Yubo, Yuanlin, Chenzhuangquan. double screening of Phytophthora capsici biocontrol bacteria. plant protection bulletin, 2006,33(1): 41-46; Chenxianren, Lecapone, Pecheine, Xujinyou, Huimihui. Phytophthora capsici CRN coding gene cloning, transcription characteristics and interaction with host. Phytophthora capsici bulletin, 2015,45(4): 384-one 394); jinbao V8 (100% vegetable) juice is imported from America and sold in various supermarkets in China; sealing films (model PM-996, Parafilm, USA) are available from reagent companies, and the operation is as follows:
(1) obtaining phytophthora capsici zoospore mother liquor
Inoculating phytophthora capsici to be separated onto a V8 (100% vegetable) juice culture medium plate, sealing with a sealing film, culturing at 25 ℃ in the dark for 5 days to promote hypha growth, then removing the sealing film, transferring to the light (5000 lx) for culturing at 23 ℃ for 24 hours to promote the generation of zoosporangia, adding 15m L sterile water into the plate, placing in a refrigerator at 4 ℃ for 25 minutes, placing at room temperature (23-25 ℃) for 20-30 minutes, and pouring out a suspension (namely phytophthora capsici zoospore suspension).
(2) Germination and dilution of conidia
The suspension containing only 1-2 zoospores per field of the microscope (10 ×) was diluted with sterile water, and 1m of L spore suspension was spread on a plate containing V8 juice medium, left at room temperature (23-25 ℃) for 6h, and the remaining suspension was decanted.
(3) Single spore isolation of conidia
Placing the zoospores on an inverted microscope, observing only 1 germinated zoospore in each (10 ×) visual field, sterilizing and cooling the zoospores on flame by using a scalpel, cutting the zoospores into square small blocks with the length of 0.5cm by using a knife around the zoospores (namely above a lens of the inverted microscope), confirming only 1 germinated spore in the zoospores again, taking out the small blocks of the culture medium with single zoospores by using the scalpel, placing the small blocks of the culture medium into another V8 culture medium plate with penicillin (50 mu g/m L), placing the culture medium plate into a thermostat, culturing the culture medium plate in the dark at 25 ℃ for 3d, picking hyphae on the edge, and purifying the hyphae into a test tube to obtain the phytophthora capsici zoospores.
Example 1 measurement of mating types of Phytophthora capsici strains
The phytophthora capsici used in this example was the control strain a1 mating type (peyingkun, dawn, liri, lili. phytophthora capsici mating type analysis and distribution characteristics in qinghai province. northern horticulture 2014(18):
the experimental method is as follows:
(1) isolation of Phytophthora capsici
Collecting phytophthora capsici strains on a land where pepper is planted, taking a plurality of tissues at the junction of disease and health, treating the tissues for 1-2s by 70% alcohol, then placing the tissues into 0.1% mercuric chloride for treatment for 3min, changing and washing the tissues for 3 times by sterile water, drying the tissues, placing the dried tissues on a V8 juice agar medium plate with penicillin (50 microgram/m L), culturing the dried tissues in the dark at 26 ℃ for 2-3d, and purifying the obtained colonies for later use.
(2) Isolation of zoospore monospore
The specific operation for separating the zoospore single strains is shown in the specific operation for separating the single spores of phytophthora capsici, and 72 phytophthora capsici zoospore single strains are obtained in total.
(3) Determination of mating type of Phytophthora capsici
After 72 phytophthora capsici zoospore single spore strains and phytophthora capsici A1 mating types are propagated by using a V8 juice culture medium, 0.7cm of bacterial butterflies are punched by using a puncher, then different phytophthora capsici zoospores and A1 mating type bacterial butterflies are respectively transferred to a new V8 juice culture plate (the diameter is 9cm), the two bacterial butterflies are 3cm apart, and after being cultured in a dark opposite environment at 26 ℃ for 4 days, if oospores are observed through an inverted microscope, the strain to be tested is A2 mating type, and if no oospores are found, the strain is A1 mating type. The results are shown in Table 1.
Table 172 determination of the mating types of the Phytophthora capsici single strains
As can be seen from Table 1, the two mating types of Phytophthora capsici in the field were found, wherein 39 mating types were found for A1, 33 mating types were found for A2, and slightly dominant for A1.
Example 2 measurement of disease resistance of Capsicum species to Phytophthora capsici
(1) Cucumber planting
20 tested cucumber varieties are planted, 20 seeds are planted in each variety, 1 seed is planted in each culture pot (the diameter is 8cm), 5 seeds are blank control, the culture medium is black loam, the cucumber varieties are placed in a greenhouse (13-33 ℃), watering is carried out for 1 time every day, and inoculation is carried out after 15 days.
(2) Propagation of test strains
Selecting 6 single strains of phytophthora capsici zoospores (obtaining of the single strains is shown in the detailed operation of separating the single spores of the phytophthora capsici zoospores) to be respectively inoculated into a V8(100 percent vegetable) juice culture medium plate to be inoculated with the phytophthora capsici, sealing the plate by using a sealing film, culturing the plate in dark at 25 ℃ for 5d to promote the growth of hypha, then transferring the plate to the light (5000 lx) to remove the sealing film and culturing the plate at 23 ℃ for 24h to promote the generation of zoosporangia, then adding 15m L sterile water into the plate, placing the plate in a refrigerator at 4 ℃ for 25min, placing the plate at room temperature (20-23 ℃) for 20-30min, and pouring out suspension (namely phytophthora capsici
(3) Preparation and inoculation of spore suspension
Respectively preparing 6 phytophthora capsici zoospore suspensions into 10 by using a blood counting plate4Spores/m L, and after mixing in equal amounts, 3m L bacterial suspension was inoculated to each plant, 15 strains to each variety, and 5 control strains were not inoculated, and the investigation was performed 1 time every 1 week.
(4) Disease investigation
After phytophthora capsici infects cucumbers, the investigation grading standard is as follows:
0 is no symptom, 1 is small brown or water-soaked lesion at the stem base, 2 is large brown or water-soaked lesion at the stem base, 3 is full water-soaked and overflowed at the stem base of the plant, the plant part collapses, the leaves wither obviously, 4 is full water-soaked at the stem base of the plant, the plant collapses seriously and withers, and 5 is dead.
Wherein n is the total number of investigation times, XiSeverity of the ith survey, TiTime of the ith survey
(5) Analysis of results
The results are shown in table 1 by analyzing the resistance of 20 cucumber varieties to phytophthora capsici.
Table 120 pepper varieties determination of Mixed disease resistance to 6 Phytophthora capsici Single strains
As can be seen from Table 1, most varieties have poor disease resistance performance and are highly susceptible, and PI 561145 and Ames 28956 from the United states have good disease resistance performance, so that the method deserves further research on disease resistance mechanisms.
In a word, aiming at the problems that phytophthora capsici zoospores are small, irregularly move and colorless, and the difficulty of monospore separation is high, the method has the advantages of strong operability, high accuracy, high speed, good separation effect and simple equipment. The method is simple and easy to implement, and can separate a large quantity of phytophthora capsici zoospores in a laboratory in a short time, and then carry out mating type and pathogenicity determination, screening of resistance resources and the like.
Claims (3)
1. A single spore separation method of phytophthora capsici zoospores is characterized by comprising the following steps:
1) centrifuging V8 juice at 4000rpm in a centrifuge to remove precipitate, preparing 10% V8 juice agar culture medium, and performing moist heat sterilization;
2) inoculating Phytophthora capsici to a V8 juice agar medium plate, sealing with a sealing film, culturing at 25 deg.C in dark for 5d to promote hypha growth, removing the sealing film, and culturing at 23-25 deg.C for 24h under 50001x fluorescent lamp to promote the generation of zoosporangia;
3) adding 15ml of sterile water into the plate of the large-amount produced zoosporangium, placing in a refrigerator at 4 ℃ for 25min, placing at room temperature of 23-25 ℃ for culturing for 25min, and pouring out a suspension, namely a phytophthora capsici zoospore suspension;
4) diluting with sterile water to obtain a suspension containing only 1 zoospore per 10 × visual fields, spreading 1ml spore suspension on a plate containing V8 juice culture medium, and standing at room temperature of 3-25 deg.C for 6 hr;
5) placing an inverted microscope on a sterile operating platform, pouring out residual suspension after ultraviolet sterilization for 30min after placing a flat plate with zoospores for 6h, and placing the flat plate on an object stage to search for only 1-2 germinated zoospores in each 10 × visual field on average;
6) after sterilizing and cooling on flame by using a scalpel, cutting a square cut with the length of 0.5cm on a culture medium right above an inverted microscope lens around a single zoospore found in a 10 × visual field, and confirming only 1 zoospore in the cut again;
7) taking out the culture medium small block confirmed to be provided with 1 zoospore by using a scalpel, putting the culture medium small block into another V8 agar culture medium flat plate with 50ug/ml of penicillin, then putting the culture medium flat plate into a thermostat, carrying out dark culture at 25 ℃ for 3d, picking hyphae on the edge, purifying the hyphae into a test tube, and obtaining the phytophthora capsici zoospore single spore strain.
2. The method of claim 1, wherein: the operations in the method all require aseptic operations, which are carried out in an aseptic station.
3. Use of the method according to any one of claims 1-2 in a method for the monosporal isolation of phytophthora capsici zoospores.
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| CN107446824B (en) * | 2017-08-21 | 2019-12-17 | 湖北省农业科学院植保土肥研究所 | Single spore isolation method of erysiphe graminis |
| CN108410793B (en) * | 2018-04-27 | 2020-11-06 | 江西省农业科学院植物保护研究所 | Culture medium for inducing phytophthora capsici to produce spores, preparation method and application |
| CN108865966A (en) * | 2018-07-12 | 2018-11-23 | 福建农林大学 | A method of the separation single egg spore of phytophthora infestans |
| CN113061565B (en) * | 2021-04-12 | 2023-03-24 | 东北农业大学 | Rapid formation method of fusarium graminearum conidia |
| CN112941008B (en) * | 2021-04-19 | 2022-06-17 | 广西大学 | Separation method of botrytis cinerea |
| CN112941007B (en) * | 2021-04-19 | 2022-06-17 | 广西大学 | Single spore separation method of banana fusarium wilt |
| CN113174335B (en) * | 2021-04-19 | 2022-06-17 | 广西大学 | Separation method of banana colletotrichum gloeosporioides |
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| CN104357333A (en) * | 2014-10-21 | 2015-02-18 | 东北农业大学 | Fusarium oxysporum single spore isolation method for soybean root rot |
| CN105441375A (en) * | 2015-12-09 | 2016-03-30 | 福建省农业科学院植物保护研究所 | Method for generating sporangia and releasing zoospore by inducing phytophthora capsici |
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