CN103045529A - Method of inducing phytophthora nicotianae breda de haan bacteria to generate sporangiums and release zoospores - Google Patents
Method of inducing phytophthora nicotianae breda de haan bacteria to generate sporangiums and release zoospores Download PDFInfo
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- CN103045529A CN103045529A CN2012105934297A CN201210593429A CN103045529A CN 103045529 A CN103045529 A CN 103045529A CN 2012105934297 A CN2012105934297 A CN 2012105934297A CN 201210593429 A CN201210593429 A CN 201210593429A CN 103045529 A CN103045529 A CN 103045529A
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Abstract
The invention belongs to the technical field of plant protection, and discloses a method of inducing phytophthora nicotianae breda de haan bacteria to generate sporangiums and release zoospores. The method comprises the following steps: activating the phytophthora nicotianae breda de haan bacteria, preparing the induction liquid generating sporangiums and inducing to generate sporangiums and release zoospores. According to the invention, the used induction liquid is the Bache wild vegetable composite fruit and vegetable juice with the volume concentration of 15-25 percent and the compound fertilizer solution with the mass concentration of 0.05-0.20 percent, and when the phytophthora nicotianae breda de haan is induced to generate the sporangiums, the continuous illumination culture is performed under the white fluorescent lamp. The method provided by the invention is simple, convenient, rapid and pollution-free, and can be used for the researches on virulence determination of the phytophthora nicotianae breda de haan bacteria, the biological activity assay of the phytophthora nicotianae breda de haan bacteria by the sterilizing agent, the evaluation of disease resistance of an epidemic disease by the luffa varieties and the like.
Description
Technical field
The invention belongs to the plant protection technology field, relate to the method that a kind of evoking tobacco phytophthora produces sporocyst and discharges zoospore.
Background technology
Phytophthora nicotianae (Phytophthora nicotianae Breda de Haan) is the pathogenic bacteria that causes the sponge gourd epidemic disease, and it is serious to cause harm in sponge gourd production.Under the condition of benign climate, the epidemic disease short-term gets final product outbreak of epidemic, causes very large financial loss.Therefore, often need to obtain sporocyst or the zoospore of a large amount of free of contamination Phytophthora nicotianae bacterium, be used for monitoring various places Phytophthora nicotianae bacterium virulence, measure various sterilant to the biologic activity of its sporocyst and zoospore, and the resistance level etc. of measuring variety of luffa.At present, the method for indoor acquisition a large amount of Phytophthora nicotianae bacterium sporocyst and zoospore mainly contains tradition and cultivates 2 kinds of revulsion and fresh host plant material revulsions.
Tradition is cultivated revulsion: mycelia is placed oat medium or rye substratum or V8 juice nutrient solution, activation culture is after longer for some time, add again soil extract or Pi Shi liquid, induce the generation sporocyst under the illumination condition, after subzero treatment, produce zoospore.The method induces phytophthora to produce sporocyst to be needed more than 10 days, and the time is long.
Fresh host plant material revulsion: will activate good phytophthora and be inoculated in the cotyledon or fruit of fresh host plant, and can induce the generation sporocyst after cultivation for some time, and after subzero treatment, produce zoospore.The needed time of the method is shorter, but suffers easily the pollution of bacterium, and the difficult acquisition of vegetable material.
Summary of the invention
The object of the invention is to overcome the shortcoming of prior art with not enough, provide a kind of easy, rapid induction Phytophthora nicotianae bacterium to produce a large amount of free of contamination sporocysts and discharge the method for zoospore, be used for Phytophthora nicotianae bacterium Pathogenic Tests, sterilant to the researchs such as Disease Resistance Identification to epidemic disease of the biological activity determination of Phytophthora nicotianae bacterium and variety of luffa.
Purpose of the present invention is achieved through the following technical solutions: a kind of evoking tobacco phytophthora produces sporocyst and discharges the method for zoospore, comprises the steps:
(1) the Phytophthora nicotianae bacterium activates: the Phytophthora nicotianae bacterium is inoculated in carries out activation culture on the PDA substratum.
(2) preparation produces sporangial induced liquid: the strange edible wild herbs composite vegetable juice of the shellfish beverage preparation that water is produced strange (Fujian) Food Co., Ltd of the shellfish of buying on the market becomes the strange edible wild herbs composite vegetable of the shellfish juice of volumetric concentration 15%～25%, and is for subsequent use after the sterilization; Preparation quality concentration is composite fertilizer's solution of 0.05%～0.2%, and is for subsequent use after the sterilization.
(3) induce the generation sporocyst: get in the strange edible wild herbs composite vegetable of the shellfish juice of volumetric concentration 15%～25% that Phytophthora nicotianae bacterium that step (1) activated is inoculated into step (2) preparation 25 ℃～30 ℃ dark culturing 2d～4d; Then use up the strange composite vegetable juice of shellfish, the mass concentration of changing step (2) preparation is composite fertilizer's solution of 0.05%～0.20%, cultivate 2d～4d in lower 25 ℃～30 ℃ continuous illuminations of the white fluorescent lamp of intensity of illumination 500Lux～700Lux, can induce the generation sporocyst.
(4) induce the release zoospore: with Phytophthora nicotianae bacterium 4 ℃～10 ℃ placement 20min～40min of step (3) through inducing, then place 25 ℃～30 ℃, can discharge a large amount of zoospores behind the 30min.
Activation culture described in the step (1) is 25 ℃～30 ℃ dark culturing 2d～4d; The temperature of preferred described activation culture is 27 ℃.
Water described in the step (2) is preferably distilled water.
The volumetric concentration of the strange edible wild herbs composite vegetable juice of shellfish described in the step (2) is preferably 20%.
The described composite fertilizer of step (2) is preferably potassium sulfate type 15-15-15 composite fertilizer, and the mass concentration of described composite fertilizer solution is preferably 0.1%.
The condition optimization of the sterilization described in the step (2) is 121 ℃ of moist heat sterilization 25min.
Inducing described in the step (3) produces sporangial method and is preferably: get the Phytophthora nicotianae bacterium edge mycelia piece that step (1) activated and place on the aseptic culture dish, the strange edible wild herbs composite vegetable of the shellfish juice that adds the volumetric concentration 20% of step (2) preparation, make fruit juice liquid just soak the mycelia piece, 27 ℃ of dark culturing 2d; Then confide all the strange edible wild herbs composite vegetable of shellfish juice, change composite fertilizer's solution of step (2) preparation, make composite fertilizer's solution also just soak the mycelia piece, place 27 ℃ of continuous illuminations of white fluorescent lamp illumination box of intensity of illumination 600Lux to cultivate 3d, can induce to produce a large amount of sporocysts.
The method that discharges zoospore of inducing described in the step (4) is preferably: with the Phytophthora nicotianae bacterium 4 ℃ placement 30min of step (3) through inducing, then place 27 ℃, can discharge a large amount of zoospores behind the 30min.
The present invention has following advantage and effect with respect to prior art:
(1) quick: method of the present invention can obtain sporocyst and the zoospore of a large amount of Phytophthora nicotianae bacterium in 5d, and tradition cultivation induction method is compared, and induction duration can shorten half; Suitable with the induction duration of fresh host plant material revulsion.
(2) pollution-free: induced liquid process sterilising treatment of the present invention, the also adding in pollution-free source in operating process, the sporocyst that induces and zoospore are all less than polluting.
(3) induced liquid is can prolonged preservation for subsequent use: induced liquid of the present invention can place 4 ℃ of refrigerators to save backup through sterilising treatment, can take out use if needed.
Description of drawings
Fig. 1 is that the evoking tobacco phytophthora produces sporocyst and discharges the zoospore schema.
Fig. 2 is Phytophthora nicotianae bacterium reference culture (numbering: GIM3.567) produce sporangial as a result figure.
Fig. 3 is Phytophthora nicotianae bacterium reference culture (numbering: GIM3.567) the as a result figure of release zoospore.
Fig. 4 is the growing state figure of Phytophthora nicotianae bacterium in induced liquid that the sick sample of fresh sponge gourd epidemic disease separates.
Fig. 5 is that the Phytophthora nicotianae bacterium of inducing the sick sample of fresh sponge gourd epidemic disease to separate produces sporangial as a result figure.
Fig. 6 is the as a result figure that induces the Phytophthora nicotianae release zoospore of the sick sample separation of fresh sponge gourd epidemic disease.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Specifications of raw materials
The strange edible wild herbs composite vegetable juice of shellfish beverage: the commercially available drink of strange (Fujian) Food Co., Ltd of shellfish.
The strange edible wild herbs composite vegetable of 20% shellfish juice: use by volume 4:1 mixing of distilled water and the strange edible wild herbs composite vegetable juice of shellfish beverage, for subsequent use behind 121 ℃ of moist heat sterilization 25min.
Composite fertilizer: potassium sulfate type 15-15-15 compound manure, the commercially available fertilizer of Norway Hydro company limited.
0.1% composite fertilizer's solution: take by weighing the composite fertilizer of 1.0g, be dissolved in the 999mL distilled water, for subsequent use behind 121 ℃ of moist heat sterilization 25min.
The needed culture dish of other experimental implementation, PDA substratum, pipettor, Autoclave, refrigerator, distilled water, illumination box etc. are the conventional instrument in laboratory or material.
Embodiment 1 Phytophthora nicotianae bacterium reference culture produces sporocyst and zoospore
(1) on Bechtop, Phytophthora nicotianae bacterium (strain number: GIM3.567, available from microbial strains preservation center, Guangdong Province) is inoculated on the PDA substratum 27 ℃ of dark culturing 2d.
(2) on Bechtop, with diameter be the punch tool of 0.5cm in the punching of colony edge place, getting 1 ferfas silk piece, to place diameter be the sterile petri dish of 6cm, adds volumetric concentration and be 20% the strange edible wild herbs composite vegetable of shellfish juice 5mL, 27 ℃ of dark culturing 2d; Then use up fruit juice liquid, changing aseptic mass concentration is 0.1% composite fertilizer solution 5mL, (the safe grand medicine equipment of Shaoguan City of Guangdong Province company limited product, model: LRH-250-G), 3d are cultivated in 27 ℃ of continuous illuminations to place the illumination box of the white fluorescent lamp of intensity of illumination 600Lux.
(3) will through the Phytophthora nicotianae bacterium of above-mentioned inducing culture in 4 ℃ of refrigerators, place 30min; Then be placed in 27 ℃ of incubators, collect liquid behind the 30min.
(4) with range be 2.5 μ L liquid-transfering guns draw 0.2 μ L liquid as on the slide glass, drag into band, under 4 times of mirror visuals field, calculate the quantity of zoospore.Take a sample 10 times, calculate the mean number of zoospore in the 0.2 μ L liquid, then be converted into the concentration of every milliliter of zoospore suspension.
The sporocyst that produces and the zoospore of release are as shown in Figures 2 and 3; The zoospore number that discharges after measured reaches 5.92 * 10
5Individual spore/mL, release rate is more than 80%.
Embodiment 2 induces the Phytophthora nicotianae bacteria strain that separates in the sick sample of fresh sponge gourd epidemic disease to produce sporocyst and zoospore
(1) gets the sick sample sample of sponge gourd epidemic disease of fresh morbidity, be good for the tissue block that intersection clip size is about 2mm * 5mm with scissors in disease.
(2) on Bechtop, place 75% alcohol to soak 30s tissue block, then in 5% chlorine bleach liquor, soak rinsing 1min; Thereafter change clothes 3 times with aqua sterilisa.After sterilization filter paper blots water, it is transferred on the PDA culture medium flat plate 27 ℃ of dark culturing 4d.
(3) after growing bacterium colony, the picking colony color is the edge mycelia piece of white on Bechtop, and it is carried out the mycelia purifying, can obtain the Phytophthora nicotianae bacterium.
(4) on Bechtop, it is 20% the strange edible wild herbs composite vegetable of shellfish juice that the bacterial strain behind the purifying is placed volumetric concentration, makes fruit juice liquid just soak the mycelia piece, 27 ℃ of dark culturing 2d; Then confide all liquid, the mass concentration that changes to the bacterium of going out is 0.1% composite fertilizer's solution, make composite fertilizer's solution also just soak the mycelia piece, place white fluorescent lamp (the Shaoguan City of Guangdong Province Thailand grand medicine equipment company limited product of intensity of illumination 600Lux, model: in illumination box LRH-250-G), 3d is cultivated in 27 ℃ of continuous illuminations.
(5) above-mentioned bacterial strain through inducing culture is placed 4 ℃ of refrigerator 30min, then be positioned in 27 ℃ of incubators, collect liquid behind the 30min.Draw the liquid of 0.2 μ L with liquid-transfering gun as on the slide glass, under 4 times of mirror visuals field, calculate the quantity of zoospore.Take a sample 10 times, calculate the mean number of zoospore in the 0.2 μ L liquid, then be converted into the concentration of zoospore suspension.
From the Phytophthora nicotianae bacterium of fresh sponge gourd epidemic disease sample separation and purification at the growing state of induced liquid as shown in Figure 4, the sporocyst of generation and the zoospore of release are as shown in Figure 5 and Figure 6.After measured, the zoospore of release several 1.91 * 10
5Individual spore/mL, release rate is more than 80%.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. an evoking tobacco phytophthora produces sporocyst and discharges the method for zoospore, it is characterized in that comprising the steps:
(1) the Phytophthora nicotianae bacterium activates: the Phytophthora nicotianae bacterium is inoculated in carries out activation culture on the PDA substratum;
(2) preparation produces sporangial induced liquid: the strange edible wild herbs composite vegetable juice of the shellfish beverage preparation that water is produced strange (Fujian) Food Co., Ltd of the shellfish of buying on the market becomes the strange edible wild herbs composite vegetable of the shellfish juice of volumetric concentration 15%～25%, and is for subsequent use after the sterilization; Preparation quality concentration is composite fertilizer's solution of 0.05%～0.2%, and is for subsequent use after the sterilization;
(3) induce the generation sporocyst: get in the strange edible wild herbs composite vegetable of the shellfish juice of volumetric concentration 15%～25% that Phytophthora nicotianae bacterium that step (1) activated is inoculated into step (2) preparation 25 ℃～30 ℃ dark culturing 2d～4d; Then confide all the strange composite vegetable juice of shellfish, the mass concentration of changing step (2) preparation is 0.05%～0.20% composite fertilizer's solution, cultivate 2d～4d in lower 25 ℃～30 ℃ continuous illuminations of the white fluorescent lamp of intensity of illumination 500Lux～700Lux, can induce to produce a large amount of sporocysts;
(4) induce the release zoospore: with Phytophthora nicotianae bacterium 4 ℃～10 ℃ placement 20min～40min of step (3) through inducing, then place 25 ℃～30 ℃, can discharge a large amount of zoospores behind the 30min.
2. evoking tobacco phytophthora according to claim 1 produces sporocyst and discharges the method for zoospore, and it is characterized in that: the activation culture described in the step (1) is 25 ℃～30 ℃ dark culturing 2d～4d.
3. evoking tobacco phytophthora according to claim 2 produces sporocyst and discharges the method for zoospore, and it is characterized in that: the temperature of described activation culture is 27 ℃.
4. evoking tobacco phytophthora according to claim 1 produces sporocyst and discharges the method for zoospore, it is characterized in that:
Water described in the step (2) is distilled water;
The volumetric concentration of the strange edible wild herbs composite vegetable juice of shellfish described in the step (2) is 20%;
The described composite fertilizer of step (2) is preferably potassium sulfate type 15-15-15 composite fertilizer, and the mass concentration of described composite fertilizer solution is 0.1%.
5. evoking tobacco phytophthora according to claim 1 produces sporocyst and discharges the method for zoospore, and it is characterized in that: the condition of the sterilization described in the step (2) is 121 ℃ of moist heat sterilization 25min.
6. evoking tobacco phytophthora according to claim 1 produces sporocyst and discharges the method for zoospore, it is characterized in that:
Inducing described in the step (3) produces sporangial method: get the Phytophthora nicotianae bacterium edge mycelia piece that step (1) activated and place on the aseptic culture dish, the strange edible wild herbs composite vegetable of the shellfish juice that adds the volumetric concentration 20% of step (2) preparation, make fruit juice liquid just soak the mycelia piece, 27 ℃ of dark culturing 2d; Then confide all composite vegetable juice, the mass concentration of changing step (2) preparation is 0.1% composite fertilizer's solution, make composite fertilizer's solution also just soak the mycelia piece, place 27 ℃ of continuous illuminations of white fluorescent lamp illumination box of intensity of illumination 600Lux to cultivate 3d, can induce the generation sporocyst.
7. evoking tobacco phytophthora according to claim 1 produces sporocyst and discharges the method for zoospore, it is characterized in that:
The method that discharges zoospore of inducing described in the step (4) is: with the Phytophthora nicotianae bacterium 4 ℃ placement 30min of step (3) through inducing, then place 27 ℃, can discharge a large amount of zoospores behind the 30min.
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| 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 |
| CN105766573A (en) * | 2016-03-08 | 2016-07-20 | 云南省烟草农业科学研究院 | Method for inoculating wound tobacco root system with phytophthora nicotianae |
| CN106967648A (en) * | 2017-05-08 | 2017-07-21 | 广东省农业科学院植物保护研究所 | Long-term store method in a kind of cabbage heart anthrax bacteria |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
| CN110819583A (en) * | 2018-08-10 | 2020-02-21 | 云南农业大学 | Spore-producing culture method of phytophthora sojae |
| CN113373064A (en) * | 2021-07-14 | 2021-09-10 | 海南大学 | Method for inducing phytophthora litchi sporangium to release zoospores |
| CN115044528A (en) * | 2022-06-10 | 2022-09-13 | 华南农业大学 | Method for inducing oomycetes saprophyta to generate zoospores |
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| CN104726349A (en) * | 2015-04-15 | 2015-06-24 | 云南省烟草农业科学研究院 | Tobacco phytophthora culture medium and preparation method thereof |
| CN104726349B (en) * | 2015-04-15 | 2018-05-25 | 云南省烟草农业科学研究院 | A kind of Phytophthora nicotianae culture medium and preparation method thereof |
| 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 |
| 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 |
| CN105766573A (en) * | 2016-03-08 | 2016-07-20 | 云南省烟草农业科学研究院 | Method for inoculating wound tobacco root system with phytophthora nicotianae |
| CN105766573B (en) * | 2016-03-08 | 2018-11-20 | 云南省烟草农业科学研究院 | A kind of method of Phytophthora nicotianae inoculation wound tobacco root system |
| CN106967648A (en) * | 2017-05-08 | 2017-07-21 | 广东省农业科学院植物保护研究所 | Long-term store method in a kind of cabbage heart anthrax bacteria |
| CN106967648B (en) * | 2017-05-08 | 2020-06-09 | 广东省农业科学院植物保护研究所 | Medium-and long-term preservation method for colletotrichum gloeosporioides |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
| 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 |
| CN110819583A (en) * | 2018-08-10 | 2020-02-21 | 云南农业大学 | Spore-producing culture method of phytophthora sojae |
| CN110819583B (en) * | 2018-08-10 | 2021-07-06 | 云南农业大学 | Spore-producing culture method of phytophthora sojae |
| CN113373064A (en) * | 2021-07-14 | 2021-09-10 | 海南大学 | Method for inducing phytophthora litchi sporangium to release zoospores |
| CN115044528A (en) * | 2022-06-10 | 2022-09-13 | 华南农业大学 | Method for inducing oomycetes saprophyta to generate 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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