CN102807957B - Method for increasing phytophthora capsici oospore output and promoting oospore germination - Google Patents
Method for increasing phytophthora capsici oospore output and promoting oospore germination Download PDFInfo
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- CN102807957B CN102807957B CN201210284431.6A CN201210284431A CN102807957B CN 102807957 B CN102807957 B CN 102807957B CN 201210284431 A CN201210284431 A CN 201210284431A CN 102807957 B CN102807957 B CN 102807957B
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Abstract
The invention provides a method for increasing phytophthora capsici oospore output and promoting oospore germination. The method comprises the following steps: culturing phytophthora capsici strains in a dark place at the temperature of 25-28 DEG C by 10% of V8 vegetable juice; keeping phytophthora capsici oospores in the dark place for 15-45 days; and processing the oospores by 0.05-0.25% by mass of potassium permanganate, preparing an oospore suspension solution, coating the oospore suspension solution onto an S+L culture medium, and inducing oospore germination by alternately illuminating for 16 h by a daylight lamp or 8 h by a black light lamp. According to the method, an economic, simple and effective method is provided for inducing a large amount of phytophthora capsici oospores to be generated and germinated in a short time, and a guarantee is provided for the application of a large amount of phytophthora capsici single oospore groups to drug resistance or other genetics researches.
Description
Technical field
The present invention relates to a kind of method that improves Phytophthora capsici oospore output and promote Germination of Oospores.
Background technology
Epidemic disease on the Solanaceaes such as the capsicum epidemic disease being caused by Phytophthora capsici and tomato and pumpkin and ground family crop at present in Asia, America, Europe and Australia generally occurs, and in the most of provinces and cities of China, also has generation.Because capsicum epidemic disease spreads rapidly, destructive strong to host plant, from seedling stage to fruiting period, all can fall ill, serious harm plants stems stalk, blade and fruit finally cause dead seedling and decayed fruit and even total crop failure, in addition for this type of disease, lack the reasons such as disease-resistant variety and effective agricultural cultivation control measures, cause its harm to increase the weight of gradually and be difficult to control, become the important disease in China's vegetables production at present.
Phytophthora capsici belongs to heterothally cause of disease oomycetes, has A
1, A
2, in field, can there is syngenesis and produce oospore in two kinds of mating types.The oospore of Phytophthora capsici is a kind of dormancy sexual spore with heavy wall being produced by sexual generation, at occurring in nature, can resist poor environment, is the major way that Phytophthora capsici is survived the winter.Under natural condition, Phytophthora capsici oospore forms need to could be sprouted through dormancy after a while conventionally successively afterwards, but under indoor conditions, mutants which had oospore production amount that some wild type strains or induction obtain is few, germination rate is low, has brought difficulty to the correlative studys such as phytophthora genetics.Genetic analysis research requires Phytophthora capsici can produce fast a large amount of oospore under indoor cultivation condition, and can sprout simultaneously, to shorten the test period and to ensure the accuracy of result of study.
Research for the mould oospore production of epidemic disease and sprouting also has relevant report, but belong to its oospore production of Mtr mutant not of the same race together and sprout required condition different, and mainly concentrate on to the generation of oospore and the researchs of sprouting condition such as phytophthora infestans, ramie mould, soybean phytophthora, Phytophthora cactorums, about the domestic report that has no of research of Phytophthora capsici oospore at present.Setting up a kind of methods simple and easy, that induce a large amount of generations of Phytophthora capsici oospore and sprout efficiently, is this area technical problem urgently to be resolved hurrily, also for the correlative study of Phytophthora capsici genetics, lays a good foundation.
Summary of the invention
An object of the present invention is to provide a kind of method that improves Phytophthora capsici oospore output.
The method of raising Phytophthora capsici oospore output provided by the present invention, comprises the steps: to use 10%V
8vegetable juice culture is cultivated Phytophthora capsici bacterial strain, and culture condition is dark.
In said process, the time of described cultivation is 15 days-60 days, is specially 45 days-60 days; The temperature of described cultivation is 25 ℃-28 ℃, is specially 25 ℃.
Another object of the present invention is to provide a kind of method that promotes Phytophthora capsici Germination of Oospores.
The method of promotion Phytophthora capsici Germination of Oospores provided by the present invention, comprises the steps:
(1) Phytophthora capsici oospore is cultivated to 15-45 days under dark condition;
(2) the potassium permanganate treatment step that is 0.05%-0.25% with quality percentage composition (1) gained oospore;
(3) under the condition of step (2) gained oospore alternately being irradiated at 16h fluorescent lamp/8h black lamp, cultivate.
In said process, in described step (1), the time of cultivation is 15 days, 30 days or 45 days;
In described step (2), the quality percentage composition of described potassium permanganate is 0.1%-0.25%.
In said process, in described step (2), the quality percentage composition of described potassium permanganate is 0.1% or 0.25%;
In said process, in described step (2), the method for described processing is: described oospore is made to suspension, add potassium permanganate, supersound process 20-40s in suspension.
In said process, the time of supersound process is specially 30s, and ultrasonic power is 250W, and operating frequency is 40KHz.
In said process, in described step (3), the time of cultivation is 5 days.
In said process, in described step (3), the substratum using in described cultivation is S+L substratum.Concrete preparation method is: Yelkin TTS 0.1g, basic culture solution 10ml[MgSO
47H
2o 100mg, (NH
4) SO
4100mg, K
2hPO
460mg, CaCl
22H
2o 30mg, KH
2pO
430mg, ZnSO
47H
2o 3mg, distilled water 100ml], glucose 0.04g, agar powder 14g, distilled water is settled to 1L.
In said process, in step 1), cultivating substratum used is 10%V
8vegetable juice culture.
In said process, in described step (1) and step (3), temperature is 25 ℃-28 ℃, is specially 25 ℃.
In said process, in described step (1) before, comprise the steps: to prepare Phytophthora capsici oospore by method described in claim 1 or 2.
Under indoor conditions, induce Phytophthora capsici to produce a large amount of oospore, and the oospore producing can be sprouted at one time in a large number, to carrying out the biological heredity of phytophthora sexual progeny and Study on Variation and the correlative study of phytophthora genetics, all have great importance.The present invention for realize induce at short notice Phytophthora capsici oospore a large amount of produce and sprout a kind of economy, simple and easy and effective means are provided, for obtaining a large amount of Phytophthora capsici list oospore colony, for resistance or other genetics research, provide assurance.
Accompanying drawing explanation
Fig. 1 is Phytophthora capsici selfing cultural method.
Fig. 2 is Phytophthora capsici hybridization face-off cultural method.
Fig. 3 is Phytophthora capsici Germination of Oospores figure.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment Phytophthora capsici bacterial strain Sx-3 used, Sx-22, Hx-1 and Sx-2 document " Cui Xiaolan; Meng Qingxiao; Bi Yang etc. Phytophthora capsici is to the research of the responsive baseline of dimethomorph and indoor resistance mutants. Plant Pathology .2009; 39(6): 630-637 " in disclose, the acquisition of public Ke Cong China Agricultural University.
Following V
8juice all refers to V
8vegetables juice.
Embodiment 1, raising oospore output
One, the screening of substratum
10%V
8substratum: V
8vegetables juice 100ml, CaCO
30.2g, agar powder 14g, adding distil water is settled to 1L, high pressure steam sterilization, 121 ° of C, 20min, to be cooled during to 40 ~ 50 ° of C, pour in sterilizing culture dish standby.
SLA substratum: β-sitosterol 20mg, Yelkin TTS 500mg, CaCO
30.2g, aspartic acid 10mg, V
8vegetables juice 100ml, agar 2g and deionized water 900ml.Yelkin TTS is placed in to 50ml water, with refiner (500r/min) homogenate 3min, Yelkin TTS is fully broken up, add V
8juice, CaCO
3, agar, supply water to 1000ml, after β-sitosterol is dissolved with a small amount of (several milliliters) methylene dichloride, mix with substratum.Aspartic acid is with being sub-packed in separately after a small amount of water dissolution in triangular flask, and 121 ° of C sterilizing 20min treat that substratum is down to 50 ° of C left and right, and aspartic acid solution is added in substratum, shake up pour in sterilizing culture dish standby.
Tryptophane substratum: β-sitosterol 30mg, L-Trp 20mg, CaCl
2100mg, vitamins B
11mg, 10% V
8substratum 1000ml.β-sitosterol is first dissolved in a small amount of methylene dichloride, after add 10% V
8in substratum, 121 ° of C sterilizing 20min, L-Trp and vitamins B
1after mixing, be dissolved in a small amount of water, separately sterilizing adds before use when substratum is down to 50 ° of C left and right, shake up pour in sterilizing culture dish standby.
The lay eggs detection method (as shown in Figure 1) of spore output of Phytophthora capsici selfing: will pick up from the Phytophthora capsici bacterial strain Sx-3(A in China Shaanxi
2mating type), Sx-22(A
1mating type) be inoculated in respectively above-mentioned 10%V
8substratum, on SLA substratum or tryptophane culture medium flat plate, under 25 ° of C dark conditions, cultivate 5d, after removing the bacterium cake of vaccination, use the sterilizing triangular flask that bottleneck diameter is 4cm, the substratum that covers with mycelia is broken into the bacterium cake of diameter 4cm, bacterium cake is moved into the central authorities of 1% water agar plate in the supine mode of mycelia, on mycelia face, cover the polycarbonate membrane (47mm of a sterilizing, aperture 0.2 μ m), light this film of pressing makes it and bacterium cake close contact to drain bubble, subsequently the bacterium cake of onesize relative mating type bacterial strain is placed in to poly-carbon film top in the ventricumbent mode of mycelia, and be close to film and below bacterium cake, use sealed membrane to seal to be placed under 25 ° of C dark conditions and cultivate respectively 15d, 30d, after 45d and 60d, calculate the output of Phytophthora capsici oospore.Each processing all has 4 repetitions, and result is taken the mean.
Result is as table 1.
Table 1 Phytophthora capsici Sx-22 and Sx-3 selfing on different culture media produces the amount of oospore
Table note:
xnd, does not detect;
ythe statistical method of use Fisher compares the significance of difference of bacterial strain generation oospore amount under 0.05 level, and same letter representative does not have significant difference;
zuse Fisher statistical method to analyze the significance of difference that bacterial strain under 0.05 level produces the amount of oospore and generation on tryptophane substratum on other substratum ,+He – represent respectively significantly more than or are significantly less than.
Table 1 shows, the amount that Phytophthora capsici produces oospore on different culture media there are differences, and can find out 10%V during except 30d from cultivating the average oospore amount that different time two strain selfing bacterial strains produce
8on substratum, produce outside the amount there was no significant difference producing on the amount of oospore and tryptophane substratum 45 and 10%V during 60d
8the amount that produces oospore on vegetable juice culture is all significantly higher than other substratum.
Two, the screening of culture condition
The output of Phytophthora capsici hybridization oospore: by the pre-incubated Phytophthora capsici Hx-1(A that picks up from China Shaanxi and Hebei of process
1mating type), Sx-2(A
2mating type), Sx-3(A
2mating type) and Sx-22(A
1mating type) buying the bacterium cake of getting 5mm size adopts the direct matched pair technique method of cultivating that stands facing each other to be connected to 10%V
8on vegetable juice culture flat board (as shown in Figure 2), test design is pairing hybridization between two, be that Hx-1 * Sx-2, Hx-1 * Sx-3, Sx-22 * Sx-2 and Sx-22 * Sx-3 are hybridized cultivation, using sealed membrane to seal the rear dark and 24h illumination box of 24h that is placed in respectively under 25 ° of C cultivates, each is processed 4 times and repeats, respectively at calculating the output of oospore after 7d, 15d, 30d, 45d and 60d.Result is as table 2.
The lower 4 pairs of Phytophthora capsici cross combinations of the different culture condition of table 2 produce the amount of oospore
Table note: nd, does not detect; Same letter representative does not have significant difference; Different letter representation significant differences.
Table 2 shows, the oospore amount that four groups of cross combination produces under dark culturing condition will be significantly higher than the amount under illumination cultivation condition, illustrates that illumination meeting suppresses the generation of oospore.
The method of embodiment 2, promotion Phytophthora capsici Germination of Oospores
(1) preparation of substratum
S+L substratum: Yelkin TTS 0.1g, basic culture solution 10ml[MgSO
47H
2o 100mg, (NH
4) SO
4100mg, K
2hPO
460mg, CaCl
22H
2o 30mg, KH
2pO
430mg, ZnSO
47H
2o 3mg, distilled water 100ml], glucose 0.04g, agar powder 14g, distilled water is settled to 1L.High pressure steam sterilization, 121 ° of C, 20min.It is to be cooled that to 50 ° of C left and right, to add final concentration be 50 μ g/ml Rifampins (rifampicin), 50 μ g/ml penbritins (penicillin), 50 μ g/ml quintozene (PCNB, pentachloronitrobenzene) and 50 μ g/ml derosal (carbendazim) shake up, and pour in sterilizing culture dish standby;
(2) period is processed Phytophthora capsici oospore in different after-ripening: cross combination Sx-3 * Sx-22, face-off is incubated at 10%V
8on culture medium flat plate, be placed under 25 ° of C dark conditions and cultivate 5-7d, to be seen to having after a large amount of oospore productions, from now starting to be placed in, 25 ° of C dark conditions, place respectively 15d, 30d and 45d(processes latter stage of ripening);
(3) stimulation of the potassium permanganate solution of different concns to Phytophthora capsici oospore: by the oospore after processing through different latters stage of ripening, cut the substratum of bacterium colony intersection, add appropriate sterilized water and smash rear use suction filter pump with agitator, successively through 300 orders (aperture 54 μ m) and 500 orders (aperture is 31 μ m) cell sieve, remove substratum, mycelia in impurity and filtrate and sporocyst, sporangiophore, to adding respectively final concentration in remaining oospore suspension, be 0.05%, 0.1%, 0.25% and 0.4%(mass percent) potassium permanganate (kill residual mycelia and sporocyst, and the dormancy of breaking oospore), after ultrasonication 30s, (ultrasonic power is 250W, operating frequency is 40KHz), after the centrifugal 20min of 4000r/min, outwell supernatant, use sterilized water suspension washing precipitation 3 times, each centrifugal 15min of 4000r/min, finally use a small amount of sterilized water suspension oospore,
(4) sprouting of different culture condition induction Phytophthora capsici oospore: be applied on the S+L culture medium flat plate in step (1) after the oospore suspension preparing in step (3) is diluted to appropriate concentration, be placed under 25 ° of C, by the condition that 24h illumination, 24h dark and 16h fluorescent lamp/8h black lamp alternately irradiate, induce respectively the sprouting of oospore.The judging criterion of Germination of Oospores: inner protoplasma is cleared up completely, germ tube passes oogonium wall or goes out from handle director, the oospore of each sprouting can form 1 radical bud pipe at the most, germ tube can directly extend form mycelia also can be on top or bifurcation produce sporocyst (Fig. 3).
Table 3 latter stage of ripening 15d, Germination of Oospores rate (%) under different potassium permanganate concentration and not isogeneous induction sprouting condition
Table 4 latter stage of ripening 30d, Germination of Oospores rate (%) under different potassium permanganate concentration and not isogeneous induction sprouting condition
Table 5 latter stage of ripening 45d, Germination of Oospores rate (%) under different potassium permanganate concentration and not isogeneous induction sprouting condition
Table 3 ~ 5 show, in induction Phytophthora capsici Germination of Oospores process, the interaction between different latters stage of ripening, potassium permanganate concentration and illumination condition three factors has a significant impact germination rate.After oospore production, carry out the latter stage of ripening of 45d dark condition and process, after the potassium permanganate solution of use 0.25% concentration stimulates, through 8h black lamp/16h fluorescent lamp alternate treatment of 5d, germination rate can reach 12.5%.
Claims (8)
1. a method that promotes Phytophthora capsici Germination of Oospores, comprises the steps:
(1) Phytophthora capsici oospore is cultivated to 15-45 days under dark condition;
(2) the potassium permanganate treatment step that is 0.05%-0.25% with quality percentage composition (1) gained oospore;
(3) under the condition of step (2) gained oospore alternately being irradiated at 16h fluorescent lamp/8h black lamp, cultivate;
In described step (1) and step (3), culture temperature is 25 ℃-28 ℃.
2. method according to claim 1, is characterized in that: in described step (1), the time of cultivation is 15 days, 30 days or 45 days;
In described step (2), the quality percentage composition of described potassium permanganate is 0.1%-0.25%.
3. method according to claim 2, is characterized in that: in described step (2), the quality percentage composition of described potassium permanganate is 0.1% or 0.25%;
4. method according to claim 3, is characterized in that: in described step (2), the method for described processing is: described oospore is made to suspension, add potassium permanganate, supersound process 20-40s in suspension.
5. method according to claim 4, is characterized in that: in described step (3), the time of cultivation is 5 days.
6. method according to claim 5, is characterized in that: in described step (1) and step (3), culture temperature is 25 ℃.
7. according to arbitrary described method in claim 3-6, it is characterized in that: in described step (1) before, comprise the steps: to use 10%V
8vegetable juice culture is cultivated Phytophthora capsici bacterial strain and is obtained Phytophthora capsici oospore, and culture condition is dark; The time of described cultivation is 15 days-60 days; The temperature of described cultivation is 25 ℃-28 ℃.
8. method according to claim 7, is characterized in that: the described 10%V of using
8the time that vegetable juice culture is cultivated the cultivation of Phytophthora capsici bacterial strain is 45 days-60 days; The temperature of described cultivation is 25 ℃.
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| CN105441375B (en) * | 2015-12-09 | 2018-10-02 | 福建省农业科学院植物保护研究所 | Induction phytophthora blight of pepper generates sporangium and discharges the method for zoospore |
| JP2019530455A (en) | 2016-10-14 | 2019-10-24 | バイオプレパラティ、 スポル． エス．アール．オー | Biological antifungal liquid product containing microbial Psium origolandrum and method of manufacture |
| CN106434385B (en) * | 2016-10-31 | 2019-08-13 | 华南农业大学 | A kind of convenient method extracting peronophythora litchi egg spore from solid medium |
| 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 |
| CN108570490A (en) * | 2018-05-10 | 2018-09-25 | 中国农业科学院郑州果树研究所 | A kind of muskmelon blight Resistance Identification method |
| CN113373064B (en) * | 2021-07-14 | 2023-04-14 | 海南大学 | Method for inducing phytophthora litchi sporocyst to release zoospores |
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