CN103045529B - 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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- CN103045529B CN103045529B CN201210593429.7A CN201210593429A CN103045529B CN 103045529 B CN103045529 B CN 103045529B CN 201210593429 A CN201210593429 A CN 201210593429A CN 103045529 B CN103045529 B CN 103045529B
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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 a kind of method that 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, in sponge gourd production, causes harm serious.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, for the virulence of monitoring various places Phytophthora nicotianae bacterium, measure the biologic activity of various sterilant to 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 in to 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 illumination condition, produce zoospore after subzero treatment.The method induces phytophthora to produce sporocyst to be needed more than 10 days, and the time is long.
Fresh host plant material revulsion: the phytophthora activated is inoculated in the cotyledon or fruit of fresh host plant, after cultivation for some time, can induces the generation sporocyst, produce zoospore after subzero treatment.The needed time of the method is shorter, but easily suffers the pollution of bacterium, and the more 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, the researchs such as Disease Resistance Identification to epidemic disease to the biological activity determination of Phytophthora nicotianae bacterium and variety of luffa for Phytophthora nicotianae bacterium Pathogenic Tests, sterilant.
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) Phytophthora nicotianae bacterium activation: the Phytophthora nicotianae bacterium is inoculated on the PDA substratum and carries out activation culture.
(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 market becomes the strange edible wild herbs composite vegetable of the shellfish juice of volumetric concentration 15%～25%, standby after sterilizing; Composite fertilizer's solution that preparation quality concentration is 0.05%～0.2%, standby after sterilizing.
(3) induce the generation sporocyst: get in the strange edible wild herbs composite vegetable of the shellfish juice that Phytophthora nicotianae bacterium that step (1) activated is inoculated into volumetric concentration 15%～25% prepared by step (2) 25 ℃～30 ℃ dark culturing 2d～4d; Then use up the strange composite vegetable juice of shellfish, composite fertilizer's solution that to change mass concentration prepared by step (2) be 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: 4 ℃～10 ℃ placement 20min～40min of Phytophthora nicotianae bacterium by step (3) through inducing, then be placed in 25 ℃～30 ℃, can discharge a large amount of zoospores after 30min.
Activation culture described in step (1) is 25 ℃～30 ℃ dark culturing 2d～4d; The temperature of preferred described activation culture is 27 ℃.
Water described in step (2) is preferably distilled water.
The volumetric concentration of the strange edible wild herbs composite vegetable juice of shellfish described in 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 sterilizing described in step (2) is 121 ℃ of moist heat sterilization 25min.
Inducing described in step (3) produces sporangial method and is preferably: get the Phytophthora nicotianae bacterium edge mycelia piece that step (1) activated and be placed on aseptic culture dish, the strange edible wild herbs composite vegetable of the shellfish juice of the volumetric concentration 20% that adds step (2) to prepare, 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 prepared by step (2), make composite fertilizer's solution also just soak the mycelia piece, 3d is cultivated in the 27 ℃ of continuous illuminations of white fluorescent lamp illumination box that are placed in intensity of illumination 600Lux, can induce and produce a large amount of sporocysts.
The method that discharges zoospore of inducing described in step (4) is preferably: the 4 ℃ of placement 30min of Phytophthora nicotianae bacterium by step (3) through inducing, then be placed in 27 ℃, and can discharge a large amount of zoospores after 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 more traditional 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 of the present invention is through sterilising treatment, and also adding of pollution-free source in operating process, the sporocyst induced and zoospore be not pollution all.
(3) induced liquid is can prolonged preservation standby: induced liquid of the present invention, through sterilising treatment, can be placed in 4 ℃ of refrigerators and save backup, and can take out use if needed.
The accompanying drawing explanation
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 figure as a result.
Fig. 3 is Phytophthora nicotianae bacterium reference culture (numbering: the figure as a result that GIM3.567) discharges 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 figure as a result.
Fig. 6 is the figure as a result that induces the Phytophthora nicotianae release zoospore of the sick sample separation of fresh sponge gourd epidemic disease.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, 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: with distilled water and the strange edible wild herbs composite vegetable juice of shellfish beverage by volume 4:1 mix, standby after 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 the composite fertilizer of 1.0g, be dissolved in the 999mL distilled water, standby after 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, purchased from microbial strains preservation center, Guangdong Province) is inoculated on the PDA substratum to 27 ℃ of dark culturing 2d.
(2), on Bechtop, the punch tool that is 0.5cm with diameter, in the punching of colony edge place, is got 1 ferfas silk piece and is placed in the sterile petri dish that diameter is 6cm, the strange edible wild herbs composite vegetable of the shellfish that adds volumetric concentration to be 20% 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 be placed in 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 after 30min.
(4) with range be 2.5 μ L liquid-transfering guns draw 0.2 μ L liquid as on slide glass, drag into band, calculate the quantity of zoospore under 4 times of mirror visuals field.Sample 10 times, calculate the mean number of zoospore in 0.2 μ L liquid, then be converted into the concentration of every milliliter of zoospore suspension.
The sporocyst produced and the zoospore of release are as shown in Figures 2 and 3; The zoospore number discharged after measured reaches 5.92 * 10
5individual spore/mL, release rate is more than 80%.
Embodiment 2 induces the Phytophthora nicotianae bacteria strain separated in the sick sample of fresh sponge gourd epidemic disease to produce sporocyst and zoospore
(1) get the sick sample sample of sponge gourd epidemic disease of fresh morbidity, be about the tissue block of 2mm * 5mm with scissors in the strong intersection clip size of disease.
(2) on Bechtop, tissue block is placed in to 75% alcohol and soaks 30s, then in 5% chlorine bleach liquor, soak rinsing 1min; Thereafter with aqua sterilisa, change clothes 3 times.Blot water on sterilizing filter paper after, it is transferred on the PDA culture medium flat plate to 27 ℃ of dark culturing 4d.
(3), after growing bacterium colony, on Bechtop, the picking colony color is white edge mycelia piece, and it is carried out to the mycelia purifying, can obtain the Phytophthora nicotianae bacterium.
(4) on Bechtop, the bacterial strain after purifying is placed in to the strange edible wild herbs composite vegetable of the shellfish juice that volumetric concentration is 20%, make fruit juice liquid just soak the mycelia piece, 27 ℃ of dark culturing 2d; Then confide all liquid, changing to sterilized mass concentration is 0.1% composite fertilizer's solution, make composite fertilizer's solution also just soak the mycelia piece, be placed in 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) the above-mentioned bacterial strain through inducing culture is placed in to 4 ℃ of refrigerator 30min, then is positioned in 27 ℃ of incubators, collect liquid after 30min.Draw the liquid of 0.2 μ L with liquid-transfering gun as on slide glass, calculate the quantity of zoospore under 4 times of mirror visuals field.Sample 10 times, calculate the mean number of zoospore in 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 preferably 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 principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in 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) Phytophthora nicotianae bacterium activation: the Phytophthora nicotianae bacterium is inoculated on the PDA substratum and carries out activation culture;
(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 market becomes the strange edible wild herbs composite vegetable of the shellfish juice of volumetric concentration 15%～25%, standby after sterilizing; The potassium sulfate type 15-15-15 composite fertilizer solution that preparation quality concentration is 0.05%～0.2%, standby after sterilizing;
(3) induce the generation sporocyst: get in the strange edible wild herbs composite vegetable of the shellfish juice of volumetric concentration 15%～25% prepared to step (2) by Phytophthora nicotianae bacterium edge inoculated by hypha block that step (1) activated 25 ℃～30 ℃ dark culturing 2d～4d; Then confide all the strange composite vegetable juice of shellfish, changing mass concentration prepared by step (2) 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 and produce a large amount of sporocysts;
(4) induce the release zoospore: 4 ℃～10 ℃ placement 20min～40min of Phytophthora nicotianae bacterium by step (3) through inducing, then be placed in 25 ℃～30 ℃, can discharge a large amount of zoospores after 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 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 step (2) is distilled water;
The volumetric concentration of the strange edible wild herbs composite vegetable juice of shellfish described in step (2) is 20%;
The mass concentration of the described potassium sulfate type 15-15-15 of step (2) 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 sterilizing described in 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 step (3) produces sporangial method: get the Phytophthora nicotianae bacterium edge mycelia piece that step (1) activated and be placed on aseptic culture dish, the strange edible wild herbs composite vegetable of the shellfish juice of the volumetric concentration 20% that adds step (2) to prepare, make fruit juice liquid just soak the mycelia piece, 27 ℃ of dark culturing 2d; Then confide all composite vegetable juice, changing mass concentration prepared by step (2) is 0.1% potassium sulfate type 15-15-15 composite fertilizer solution, make composite fertilizer's solution also just soak the mycelia piece, 3d is cultivated in the 27 ℃ of continuous illuminations of white fluorescent lamp illumination box that are placed in intensity of illumination 600Lux, 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 step (4) is: the 4 ℃ of placement 30min of Phytophthora nicotianae bacterium by step (3) through inducing, then be placed in 27 ℃, and can discharge a large amount of zoospores after 30min.
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| 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 |
| 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 |
| 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 |
| 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 |
| 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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