CN105441375B - Induction phytophthora blight of pepper generates sporangium and discharges the method for zoospore - Google Patents
Induction phytophthora blight of pepper generates sporangium and discharges the method for zoospore Download PDFInfo
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Abstract
Sporangium is generated the invention discloses induction phytophthora blight of pepper and discharges the method for zoospore.Induction liquid, induction generation sporangium and the induction release zoospore that this method includes phytophthora blight of pepper activation, prepares generation sporangium.Induction liquid used in the present invention is the imported with original packaging V8 vegetable juice and 0.2 0.3g/LCaCO of volumetric concentration 10% 15%3Solution, the continuous illumination culture under white fluorescence when inducing Phytophthora capsici to generate sporangium.The method of the present invention is easy, quick, pollution-free, can be used for the biological activity determination of phytophthora blight of pepper Pathogenic Tests and fungicide to phytophthora blight of pepper.
Description
Technical field
The invention belongs to field of microbial culture technology, and in particular to induction phytophthora blight of pepper generates sporangium and discharges trip
The method of zoospore.
Background technology
Capsicum epidemic disease is the crushing fungal disease generally occurred in a kind of world wide, first in 1918 in the U.S.
New Mexico is found, causes the phytophthora blight of pepper of capsicum epidemic diseasePhytophthora capsiciLeonian is Leon
Leonian is named in nineteen twenty-two, it belongs to pathogenic oomycetes.Phytophthora capsici is invalid in soil with egg spore or chlamydospore
It is overwintering in body, the several months can be survived even more for a long time, host range is wide, mainly infects capsicum, tomato, eggplant, cucumber, pumpkin
Etc. a variety of important vegetables.Capsicum epidemic disease open country and protecting field can occur, and the prevalence of the disease causes capsicum loss serious, and one
As diseased plant rate be 15-30%, up to 80% or more when serious.Since nineteen eighty, capsicum epidemic disease is sent out in China vegetable cultivation region
The course of disease is subsisted exacerbation, and the economic benefit of vegetable cultivation industry has been seriously affected, and is caused to the vegetable safety production in China huge
Economic loss.Therefore, phytophthora blight of pepper by various countries phytopathologist and geneticist height extensive concern.
Under natural conditions, Phytophthora capsici is mainly infected by zoospore from the root of plant or basal part of stem, artificial to cultivate
When, cultural hypha, induction generation sporangium, low-temperature treatment can be passed through and promote Zoospore liberation, it is more to obtain
Zoospore.Tradition culture revulsion：Mycelia is placed in oat medium or rye culture medium or V8 juice culture solutions, activation culture
After longer period of time, soil extract or Pi Shi liquid are added, is induced under illumination condition and generates sporangium, through low-temperature treatment
After generate zoospore.This method induces phytophthora to generate sporangium to need 10 days or more, and the time is long；Meanwhile soil extract
Without stringent sterilization, Pi Shi culture solution moist heat sterilizations, effect that induction generates zoosporangium is unstable, is also easy pollution.Also
There is other abductive approach that can also generate a large amount of zoospore：Rye medium culture mycelia covers with tablet, with sterile L-type glass
After rod coating smears the culture medium flat plate for covering with mycelia, it is placed in 28 DEG C of light irradiation biochemical incubators and cultivates for 24 hours with the production of inducing spore capsule
It is raw, then 1h in 4 DEG C of refrigerators is moved to after the sterile water of 18mL is added in the culture medium flat plate for having generated sporangium, capsicum can be induced
Phytophthora generates zoospore, but the above method is comparatively laborious, and also to pass through filtering could obtain available zoospore.In addition,
Fresh host plant material revulsion also can induce and generate sporangium, zoospore be generated after low-temperature treatment, but this method is held
It is subject to the pollution of bacterium, and the more difficult acquisition of vegetable material.
Invention content
The shortcomings that it is an object of the invention to overcome the prior art and deficiency provide induction phytophthora blight of pepper and generate sporangium
And the method for zoospore is discharged, it is used for the bioactivity of phytophthora blight of pepper Pathogenic Tests and fungicide to phytophthora blight of pepper
The researchs such as measurement.
The purpose of the invention is achieved by the following technical solution：Induction phytophthora blight of pepper generates sporangium and discharges travelling spore
The method of son, includes the following steps：
(1) phytophthora blight of pepper activates：Phytophthora blight of pepper is inoculated on carrot culture medium and carries out activation culture.
(2) the induction liquid for generating sporangium is prepared：Imported with original packaging V8 vegetable juice will be bought in the market with deionized water to prepare
It is spare after sterilizing at the juice of volumetric concentration 10%-15%；Prepare 0.2-0.3g/LCaCO3Solution, it is spare after sterilizing.
(3) induction generates sporangium：The phytophthora blight of pepper that step (1) has activated is taken to be inoculated into the 10%- of step (2) preparation
In 15% V8 vegetable juice, 25 DEG C of dark culturing 2d；Then V8 vegetables juice is use up, is cleaned once, is changed with sterile deionized water
0.2-0.3g/LCaCO prepared by step (2)3Solution, in the lower 25 DEG C of continuous light of the white fluorescence of intensity of illumination 700-800Lux
According to culture 2d, you can induction generates sporangium.
(4) induction release zoospore：By 4 DEG C of placement 15min of phytophthora blight of pepper of the step (3) by induction, then set
In 25 DEG C, a large amount of zoospore can release after 30min.
Activation culture described in step (1) is 25 DEG C of dark culturing 2d.
Water described in step (2) is sterile deionized water.
The condition of sterilizing described in step (2) is preferably 121 DEG C of moist heat sterilization 30min.
Described in step (3) induction generate sporangium method be preferably：The phytophthora blight of pepper for taking step (1) to activate
Edge mycelia block is placed on sterile culture dish, mycelia block be 1cm × 1cm, 20 pieces, be added step (2) prepare volumetric concentration
15% V8 vegetable juice makes the just dipped mycelia block of juice, 25 DEG C of dark culturing 2d；Then V8 vegetable juice is confided all, with going for sterilizing
Ionized water cleaning is primary, then adds 0.3g/LCaCO3Solution makes just dipped mycelia block, is placed in the white day of intensity of illumination 800Lux
25 DEG C of continuous illumination culture 2d in light lamp illumination box, you can induction generates sporangium.
The carrot culture medium is that silk is cut into the peeling of 200g fresh carrots, and water 800ml is added to boil 1h, 4 layers of gauze mistake
It filters off and removes residue, agar 15g, heating makes agar melt, constant volume 1000ml, pH=6.0.
The present invention has the following advantages and effects with respect to the prior art:
(1) quickly：The method of the present invention can obtain the sporangium and travelling spore of a large amount of phytophthora blight of pepper in 4.5d
Son, more traditional culture abductive approach are compared, and induction duration shortens.
(2) pollution-free：The addition in the present invention pollution-free source in operation, the sporangium and zoospore induced
It does not pollute.
(3) induction liquid can be spare with long-term preservation：0.2-0.3g/L CaCO used in the present invention3By sterilization treatment,
Room temperature preservation can be set.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Specifications of raw materials
V8 vegetable juice：Campbell Soup Company, the U.S..
15% V8 vegetable juice：With deionized water 850mL and V8 juice beverage 150mL mixings, 121 DEG C of moist heat sterilizations
It is spare after 30min.
CaCO3：Sigma companies, the U.S..
0.3g/L CaCO3：Weigh the CaCO of 0.3g3, it is dissolved in 1000mL deionized waters, 121 DEG C of moist heat sterilization 30min
It is spare afterwards.
Other experimental implementations required culture dish, pipettor, knife blade, autoclave, refrigerator, is gone carrot culture medium
Ionized water, illumination box etc. are laboratory conventional tool or material.
1 phytophthora blight of pepper standard of embodiment is sequenced bacterial strain and generates sporangium and zoospore
(1) on superclean bench, by phytophthora blight of pepper (strain number：Reference culture is sequenced in LT1534) it is inoculated into recklessly
On radish culture medium, 25 DEG C of dark culturing 2d.
(2) on superclean bench, the mycelia block of 0.3 × 0.3cm is extracted with knife blade at colony edge, takes 20
Ferfas silk block is placed in the sterile petri dish of a diameter of 11cm, the V8 vegetable juice 30mL that volumetric concentration is 15% is added, 25 DEG C black
Light culture 2d；Then V8 vegetable juice is use up, is cleaned once with sterile deionized water, then change sterile 0.3g/L
CaCO315mL is placed in the illumination box of the white fluorescence of intensity of illumination 800Lux, 25 DEG C of continuous illumination culture 2d.
(3) in 4 DEG C of refrigerators, 15min will be placed by the phytophthora blight of pepper of above-mentioned Fiber differentiation；Then it places it in
In 25 DEG C of incubators, liquid is collected after 30min.
(4) it is that 10 μ L liquid-transfering guns draw the liquid of 5 μ L as on blood count glass slide, in 10 times of mirror visuals field to use range
The lower quantity for calculating zoospore.Sampling 3 times, calculates the average of zoospore in 5 μ L liquid, is then convert into every milliliter
The concentration of zoospore suspension.The zoospore number discharged after measured is up to 7. 32 × 105A spore/mL, release rate 90% with
On.
Embodiment 2 induces the Phytophthora capsici bacteria strain detached in fresh chilli epidemic disease disease sample to generate sporangium and travelling spore
Son
(1) the capsicum epidemic disease sick sample for taking fresh morbidity, with scissors the strong intersection clip size of disease be about 5mm ×
The tissue block of 5mm.
(2) on superclean bench, tissue block is placed in 75% alcohol and soaks 30s, then soaked in 5% liquor natrii hypochloritis
Bubble rinsing l min；Then it is cleaned 3 times with sterile deionized water.After blotting water in sterilizing filter paper, it is transferred into carrot training
It supports on base tablet, 25 DEG C of dark culturing 3d.
(3) after growing bacterium colony, on superclean bench picking colony color be white edge mycelia block, to its into
Row mycelia purifies, you can obtains phytophthora blight of pepper.
(4) on superclean bench, the mycelia block of 1cm × 1cm is extracted with knife blade at colony edge, takes 20 pieces
Mycelia block is placed in the V8 vegetable juice that volumetric concentration is 10%, makes the just dipped mycelia block of fruit juice liquid, 25 DEG C of dark culturing 2d；So
After confide all liquid, cleaned with sterile deionized water and once change to sterilized 0.2g/L CaCO3Just dipped mycelia block, is placed in
The illumination box of the white fluorescence of intensity of illumination 700Lux, 25 DEG C of continuous illumination culture 2d.
(5) the above-mentioned bacterial strain by Fiber differentiation is placed in 15min in 4 DEG C of refrigerators, is then placed into 25 DEG C of incubators
In, liquid is collected after 30min.It is that 10 μ L liquid-transfering guns draw the liquid of 5 μ L as on blood count glass slide, at 10 times with range
The quantity of zoospore is calculated under the mirror visual field.Sampling 3 times, calculates the average of zoospore in 5 μ L liquid, is then convert into
The concentration of every milliliter of zoospore suspension.After measured, the zoospore number 5.31 × 10 of release5A spore/mL, release rate
90% or more.
Claims (2)
1. induction phytophthora blight of pepper generates sporangium and discharges the method for zoospore, it is characterised in that：
(1) phytophthora blight of pepper activates：Phytophthora blight of pepper is inoculated on carrot culture medium and carries out activation culture；
(2) the induction liquid for generating sporangium is prepared：V8 vegetable juice is configured to the juice of volumetric concentration 10%-15% with deionized water
Liquid, it is spare after sterilizing；
(3) induction generates sporangium：The phytophthora blight of pepper edge mycelia block that step (1) has activated is taken to be placed in sterile culture dish
On, mycelia block be 1cm × 1cm, 20 pieces, be added step (2) prepare volumetric concentration 15% V8 vegetable juice, so that juice is just soaked
Cross mycelia block, 25 DEG C of dark culturing 2d；Then V8 vegetable juice is confided all, is cleaned once with the deionized water of sterilizing, then adds 0.3g/
LCaCO3Solution makes just dipped mycelia block, be placed in the white fluorescence illumination box of intensity of illumination 800Lux 25 DEG C it is continuous
Illumination cultivation 2d, you can induction generates sporangium；
(4) induction release zoospore：By 4 DEG C of placement 15min of phytophthora blight of pepper of the step (3) by induction, it is subsequently placed in
25 DEG C, it can release a large amount of zoospore after 30min.
2. induction phytophthora blight of pepper according to claim 1 generates sporangium and discharges the method for zoospore, feature
It is：The carrot culture medium is that silk is cut into the peeling of 200g fresh carrots, adds water 800ml to boil 1h, 4 layers of filtered through gauze are gone
Except residue, agar 15g, heating makes agar melt, constant volume 1000ml, pH=6.0.
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| CN105886451B (en) * | 2016-04-26 | 2020-07-10 | 东北农业大学 | Single spore separation method of phytophthora capsici zoospores |
| CN106636302A (en) * | 2016-12-27 | 2017-05-10 | 福建省农业科学院植物保护研究所 | Method for evaluating pepper phytophthora blight resistance of crops |
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| 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 |
| CN110819583B (en) * | 2018-08-10 | 2021-07-06 | 云南农业大学 | Spore-producing culture method of phytophthora sojae |
| CN109825443A (en) * | 2019-02-12 | 2019-05-31 | 安徽农业大学 | It is a kind of induce Phytophthora capsici Germination of Oospores culture medium and its application |
| CN110564667B (en) * | 2019-09-25 | 2023-02-28 | 云南省烟草农业科学研究院 | Method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
| CN111172182B (en) * | 2019-12-06 | 2021-09-24 | 长江大学 | Phytophthora capsici PcMPK12 gene and vector and application thereof |
| CN113373064B (en) * | 2021-07-14 | 2023-04-14 | 海南大学 | Method for inducing phytophthora litchi sporocyst to release zoospores |
| CN115044528B (en) * | 2022-06-10 | 2023-06-02 | 华南农业大学 | Method for inducing oomycetes saphenous pythium to generate zoospores |
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