CN115044528B - Method for inducing oomycetes saphenous pythium to generate zoospores - Google Patents
Method for inducing oomycetes saphenous pythium to generate zoospores Download PDFInfo
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- CN115044528B CN115044528B CN202210651561.2A CN202210651561A CN115044528B CN 115044528 B CN115044528 B CN 115044528B CN 202210651561 A CN202210651561 A CN 202210651561A CN 115044528 B CN115044528 B CN 115044528B
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- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Abstract
The invention belongs to the technical field of inducing saphenous procyanidins to produce spores, and discloses a method for inducing saphenous procyanidins to produce zoospores. The study is based on juice extracted from induced liquid and herbaceous plants, and has obvious effect of inducing zoospores after the juice is combined with Pythium insidiosum. Because the oomycete saphenous procyanidins are difficult to generate zoospores, the invention provides a method capable of improving the spore yield compared with the existing spore production induction technology, and the method can induce the generation of zoospores more efficiently and simplify the collection of zoospores.
Description
Technical Field
The invention relates to the technical field of zoospores induced by oomycetes, in particular to a method for inducing saphenous procyanidins to generate zoospores.
Background
Pythium insidiosum Pythium insidiosum is a fungus-like aquatic oomycete, the only pathogen in mammals that causes Pythium insidiosum. The ecological advantages are achieved in swamp areas, and are common in tropical, subtropical and temperate areas. Pythium cryptogami belongs to the biological kingdom of Protozoa (Stranipila) (the same synonym as the algae kingdom), oomycota, peronosporales, pythicaceae. Pythium is an ecologically versatile microorganism found in almost all soil and humid environments. They are one of the most damaging plant pathogens, causing economic losses to a variety of crops. Members of the genus Pythium are unique both ecologically and physiologically. They often inhabit the soil and waters around the world. However, of all microorganisms described in this genus, only one, pythium insidioum, is considered to be the only pathogen causing disease in mammals.
The organism generally occurs in two forms, one is hyphae with right-angle branches or broad filaments, the other is zoospores with double flagella, and is an infectious propagule that is only present in the aquatic environment. The sexual reproduction form produced by pathogenic oomycetes is a spherical, thick-walled structure that can withstand adverse external conditions. Oospores can survive in the soil for long periods of time (months to years), which perpetuates the presence of pathogens in the environment, increasing the chances of infecting the host. Zoospores are the infectious unit of P.insidiosum, which can swim to invade host tissues as germinated hyphae. Hyphae are common in laboratory cultures, whereas zoospores are rare in laboratory cultures due to the harsh conditions under which they are produced.
In order to better identify the strain and study the biological characteristics of saphenous mould, induction of saphenous mould to produce zoospores is an indispensable step in the study. However, the methods for inducing saphenous mould to produce spores are still few, so that the method is low in spore yield and difficult to separate, and a new method for inducing saphenous mould to produce zoospores is needed.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art, and provide a method for inducing saphenous procyanidins to generate zoospores.
The invention aims at realizing the following technical scheme:
a compound inducing liquid comprises Pythium insidiosum spore inducing liquid and herbal juice;
the saphenous pythium aphanidermatum spore induction liquid comprises the following components:
and (3) solution A: k (K) 2 HPO 4 ·3H 2 O 11.4g；KH 2 PO 4 6.8g,(NH 4 ) 2 HPO 4 6.6g; 50ml of ultrapure water;
and (2) liquid B: mgCI (MgCI) 2 ·6H 2 O 2.54g,CaCl 2 ·2H 2 0.84 g; 25ml of ultrapure water;
the mixing volume ratio of the solution A to the solution B is 5:1-1:1. The herbal plants in the herbal plant juice are the branches and leaves of the acetogenins.
As a preferable technical scheme, the mixing volume ratio of the annona sinensis branches and leaves to the water is 1:1-1:5.
As a preferable technical scheme, the solution A and the solution B are mixed and then added into 1L of water to obtain the saphenous procymidone spore induction solution.
As a preferable technical scheme, the mixing volume ratio of the saphenous procymidone spore induction liquid and the herb juice is 7-1: 1.
the application simultaneously protects the application of the composite induction liquid in the induction of saphenous procymidone to generate zoospores.
Compared with the prior spore production technology, the invention has the following beneficial effects:
the invention provides a method for inducing saphenous procyanidins to generate zoospores, which can remarkably increase the yield of saphenous procyanidins zoospores compared with the existing spore induction technology, can induce zoospore production more efficiently, and simplify spore production and zoospore collection.
Drawings
FIG. 1 is a photograph of a microscope taken red blood cell count plate counting zoospores.
Figure 2 is a photograph taken with a microscope of zoospore production.
FIG. 3 is a view of the zoospore induced by the leaf edge under the microscope under the condition of comparative example 1, and FIGS. 3a and 3b represent views at different angles of view, respectively.
FIG. 4 is a graph comparing plant leaf juice to inducer ratio versus number of zoospores produced.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The induction liquid and the herb juice are prepared for self-preparation without commercial purchase.
Example 1 method of inducing Pythium insidiosum to produce zoospores
1. Experimental materials:
(1) Induced fluid, herb leaves (sweetsop leaves), optical microscope, etc.
(2) Test medium: 2% glucose agar (SDA) from Shandong Staran Biotechnology Co., ltd.), brain heart extract agar medium from Guangdong Crypton, and sterile defibrinated sheep blood (purchased from Guangdong Staran Biotechnology Co., ltd.).
(3) Strains: isolate of Pythium insidiosum (stored in the laboratory, commercially available).
2. Preparation work before test:
(1) Preparing spore inducing liquid
Preparing spore induction liquid;
and (3) solution A: k (K) 2 HPO 4 ·3H 2 O 11.4g；KH 2 PO 4 6.8g,(NH 4 ) 2 HPO 4 6.6g; 50ml of ultrapure water;
and (2) liquid B: mgCI (MgCI) 2 ·6H 2 O 2.54g,CaCl 2 ·2H 2 0.84 g; 25ml of ultrapure water;
0.5ml of A solution and 0.1ml of B solution are added with 1L of dd water, and the pH is 7.0-7.1, stored at 4 ℃ and kept for use.
(2) Cleaning herb (sweetsop leaves) with sterile water, soaking with 70% alcohol for several minutes for disinfection, obtaining herb juice: 100g of the mixture is weighed, 500ml of sterile water is added, and juice is obtained by squeezing. Sterilizing and storing plant juice at 121deg.C.
(3) Pythium insidiosum is cultured in 90mm dish to the size of 40-60mm in mycelium growth diameter.
3. A method for inducing saphenous procyanidins to produce zoospores, comprising the steps of:
(1) Picking agar plugs with hyphae from the edge of sheep blood platelets of fresh brain heart leaching liquid, transferring to 2% Sa glucose agar (SDA) plate, and culturing in a 37 ℃ incubator;
(2) Taking agar plugs with mycelia on 2% of Saccharum sinensis Roxb (SDA) plates, placing in a centrifuge tube of 2% of Saccharum sinensis Roxb broth (SDB), and culturing at 180rpm in a shaker at 37deg.C for 4 hr;
(3) Taking out the centrifuge tube cultured in the step (2), removing broth in the centrifuge tube, adding the same amount of ultrapure water, and culturing at 37 ℃ and 180rpm for 1h;
(4) Taking out the centrifuge tube cultured in the step (3), removing ultrapure water in the centrifuge tube, adding the volume ratio of the induction liquid to the leaf juice to be (1:1), and culturing at 37 ℃ for 10-24 hours at 180 rpm;
(5) And (3) dripping the bacterial liquid cultured in the step (4) on a cell counting plate, counting the number of spores induced under an optical microscope, and carrying out statistical analysis after the experimental results are all subjected to three biological repetition.
As shown in FIG. 1, the number of zoospores produced can be counted with a red blood cell count plate under an optical microscope (10X).
As shown in FIG. 2, the zoospores produced were clearly observed under an optical microscope (10X).
Comparative example 1
1. Experimental materials:
(1) Induced fluid, herb leaves (sweetsop leaves), optical microscope, etc.
(2) Test medium: 2% glucose agar (SDA) from Shandong Staran Biotechnology Co., ltd.), brain heart extract agar medium from Guangdong Crypton, and sterile defibrinated sheep blood (purchased from Guangdong Staran Biotechnology Co., ltd.).
(3) Strains: isolate of Pythium insidiosum (laboratory preservation).
2. Preparation work before test:
(1) Preparing spore inducing liquid
Preparing spore induction liquid;
and (3) solution A: k (K) 2 HPO 4 ,87.09g；KH 2 PO 4 ,68.05g；(NH 4 ) 2 HPO 4 66.04g; ultrapure water, 500ml.
And (2) liquid B: caCl (CaCl) 2 ·2H 2 0,18.38g；MgCl 2 ·6H 2 O25.42 g; 250ml of ultrapure water.
0.5ml of A solution and 0.1ml of B solution are added with 1L of dd water, and the mixture is stored at a pH of 7.1 and stored at a temperature of 4 ℃ for use.
(2) Cutting leaf of herb (sweetsop leaf) into small pieces of 25mm, and sterilizing in sterilized water at 121deg.C.
(3) Pythium insidiosum is cultured in 90mm dish to the size of 40-60mm in mycelium growth diameter.
3. A method for inducing saphenous procyanidins to produce zoospores, comprising the steps of:
(1) The hyphal agar plugs were picked from sheep blood platelets from fresh brain heart extract and transferred to 2% glucose agar (SDA) plates.
(2) 2% agarose plugs with hyphae from a glucose agar (SDA) plate were removed and transferred to a water agar plate at pH 6.9,2%.
(3) Simultaneously, the sterilized sweetsop leaves are cut into small blocks with the size of 1-4mm, covered on each agar plug, and placed in a 37 ℃ incubator for incubation for 1d, and hyphae can be parasitic on the edges of the leaves in the process.
(4) The parasitized leaf is transferred to a culture dish containing 30ml of induction solution and incubated for 12-24h at 37 ℃.
(5) And taking down the grass leaves on the agar blocks, observing the quantity of induced spores under an optical microscope, and carrying out statistical analysis after three biological repetition of experimental results.
As shown in fig. 3, zoospores generated at the edge of the leaf can be observed under an optical microscope (40×).
Because zoospores are adsorbed on the edges of the blades, the zoospores cannot be well separated from the induced liquid between hyphae, the blades influence the observation field of view, so that the counting is difficult, and the spore production quantity cannot be recorded specifically.
The results of example 1 and comparative example 1 are shown in Table 1, the spore yield of the method for inducing Pythium insidioum to generate zoospores of the invention is greatly improved compared with that of comparative example 1, the method of the invention can obviously improve the saphenous pythium the number of zoospores is produced and the spore production step is simplified.
TABLE 1 method for inducing Pythium insidiosum to produce zoospores
Example 2 method of inducing Pythium insidiosum to produce zoospores
Referring to the procedure of example 1, the ratio of plant to inducer liquid was varied as follows:
fresh plant leaves were picked, weighed 100g, added with 500ml of sterile water and squeezed into juice. Plant leaf juice and induction liquid are mixed according to the proportion of 1:1,1: 4. 1:7, adding fresh mycelium agar blocks, culturing at 37 ℃ and 180rpm for 12-24 hours, and counting the number of induced spores. The results are shown in table 2 and fig. 3, and the plant leaf juice was found to be the same as the induction liquid 1: the spore yield is best in proportion 1.
TABLE 2
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (2)
1. A method of inducing saphenous pythium to produce zoospores, the method comprising the steps of:
(1) Picking an agar plug with hyphae from the edge of a sheep blood plate of a fresh brain heart leaching liquid, transferring the agar plug to a 2% Sa glucose agar plate, and culturing in a 37 ℃ incubator;
(2) Taking an agar plug with hyphae of a 2% glucose agar plate, placing the agar plug into a centrifuge tube of 2% glucose broth, and placing the centrifuge tube into a shaking table at 37 ℃ for culturing for 4 hours at 180 rpm;
(3) Taking out the centrifuge tube cultured in the step (2), removing broth in the centrifuge tube, adding the same amount of ultrapure water, and culturing at 37 ℃ and 180rpm for 1h;
(4) Taking out the centrifuge tube cultured in the step (3), removing ultrapure water in the centrifuge tube, adding the induction liquid and the herbal plant juice, wherein the volume ratio of the induction liquid to the herbal plant juice is 1:1, then culturing at 37 ℃ and 180rpm for 10-24 hours;
the induction liquid comprises liquid A and liquid B;
and (3) solution A: k (K) 2 HPO 4 ·3H 2 O 11.4 g；KH 2 PO 4 6.8 g, (NH 4 ) 2 HPO 4 6.6g; ultrapure water 50ml;
and (2) liquid B: mgCl 2 ·6H 2 O 2.54 g，CaCl 2 ·2H 2 O1.84 g; 25ml of ultrapure water;
0.5ml of solution A and 0.1ml of solution B were added to 1L of ddH 2 O, pH 7.0-7.1, storing at 4deg.C to obtain induced solution;
the herbal juice is obtained by cleaning the sweetsop leaves with sterile water, soaking with 70% alcohol for several minutes for disinfection, and obtaining the herbal juice: 100g of the mixture is weighed, 500ml of sterile water is added, juice is obtained by squeezing, and plant juice is sterilized at 121 ℃.
2. Use of the method of claim 1 for inducing the production of zoospores by saphenous pythium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045529A (en) * | 2012-12-31 | 2013-04-17 | 广东省农业科学院植物保护研究所 | Method of inducing phytophthora nicotianae breda de haan bacteria to generate sporangiums and release zoospores |
| 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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| US6833136B2 (en) * | 1997-07-17 | 2004-12-21 | Board Of Trustees Of Michigan State University | Vaccine for preventing pythiosis in humans and animals |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045529A (en) * | 2012-12-31 | 2013-04-17 | 广东省农业科学院植物保护研究所 | Method of inducing phytophthora nicotianae breda de haan bacteria to generate sporangiums and release zoospores |
| 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 |
Non-Patent Citations (5)
| Title |
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| "Pythium insidiosum Keratitis Clinical Profile and Role of DNA Sequencing and Zoospore Formation in Diagnosis";Savitri Sharma et al.;Cornea;第34卷(第4期);第438-442页 * |
| "柬埔寨常用传统药用植物探讨";姚霞等;中国现代中药(第02期);第142-146页 * |
| ColinK.Campbell著.《病原真菌鉴定》.上海：上海科学技术出版社,2019,第226-227页. * |
| Leonel Mendiza et al.."A method to obtain rapid zoosporogenesis of Pythium insidiosum".Mycopathologia.1998,第104卷(第1期),第59-62页. * |
| 苟亚峰等."圆滑番荔枝叶乙醇提取物中抑菌活性成分的初步分离".植物保护.2016,(第1期),第134-137页. * |
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