CN111690545A - Method for isolating plant pathogenic fungi - Google Patents
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Abstract
The invention discloses a method for separating plant pathogenic fungi. The method comprises the following steps: (1) immersing a plant sample by using an ethanol solution with the volume fraction of 70-75%, and blowing the ethanol solution for 6-12 seconds by adopting an external force; drying the plant sample treated by the ethanol solution to obtain a pretreated plant sample; (2) placing the pretreated plant sample on a first separation culture medium, and culturing for 2-7 days at the temperature of 20-28 ℃; transferring hyphae at the edges of the colonies obtained by the first separation culture medium into a second separation culture medium, and culturing for 2-7 days at the temperature of 20-28 ℃; (3) and (4) purifying the colony obtained by culturing the second separation culture medium by adopting a single spore isolation method or a tip purification method. The method has high separation rate of pathogenic fungi and low contamination rate.
Description
Technical Field
The invention relates to a method for separating plant pathogenic fungi.
Background
The separation of plant pathogenic fungi means that the pathogenic fungi are separated from the infected plant tissues through artificial culture, and then the separated pathogenic fungi are purified in a proper indoor environment to separate the pathogenic fungi from other mixed fungi. The separation of plant pathogenic fungi is one of the most basic operation techniques of plant pathology experiments, and is a research means which is frequently used in the aspects of disease identification, pathogenic bacteria morphology observation, plant disease inoculum culture and the like.
CN104250616A discloses a separation method of endophytes, which comprises the following steps: (1) pretreatment of plant tissues: washing plant tissues with tap water, then washing with sterile water, airing surface water, cutting, placing the materials in ethanol with the volume concentration of 70-75% for disinfection for 3-5 min, washing with sterile water, soaking with mercuric chloride with the volume concentration of 0.1-0.3% for 1-3 min or sodium hypochlorite with the mass concentration of 4-6% for 1-5 min, then washing with sterile water, airing the surface water for later use; (2) plant tissue culture; (3) and (4) separating endophytes. The method has the advantages of complex operation of the plant tissue pretreatment process, long time consumption, large damage degree to sample materials and need of using chemical agents mercury bichloride or sodium hypochlorite which pollute the environment.
CN103667080B discloses a method for isolating plant pathogenic fungi. Wiping the part of the plant sample to be separated infected by pathogenic bacteria with alcohol with the volume ratio of 70-75%, removing soil on the surface, and drying the alcohol in the sun; placing the treated plant sample to be separated on a separation culture medium, and culturing for 1-2 days at 25-28 ℃; transferring hyphae at the edges of the bacterial colonies obtained by the culture of the separation culture medium into a purification culture medium for culture at 25-28 ℃ for 3-4 days, repeating the operation for 2-4 times, and transferring the hyphae at the edges of the bacterial colonies obtained by the previous culture into a new purification culture medium for culture at 25-28 ℃ for 3-4 days each time to obtain the purified pathogenic fungi. The method only adopts alcohol to wipe, and the contamination rate of pathogenic fungi is high.
Disclosure of Invention
In view of the above, the present invention is directed to provide a method for separating plant pathogenic fungi, which is simple and efficient, does not require the use of chemical reagents such as sodium hypochlorite or mercuric chloride, which may cause environmental pollution, and has a high separation rate of pathogenic fungi and a low contamination rate.
The invention provides a method for separating plant pathogenic fungi, which comprises the following steps:
(1) immersing a plant sample by using an ethanol solution with the volume fraction of 70-75%, and blowing the ethanol solution for 6-12 seconds by adopting an external force; drying the plant sample treated by the ethanol solution to obtain a pretreated plant sample;
(2) placing the pretreated plant sample on a first separation culture medium, and culturing for 2-7 days at the temperature of 20-28 ℃; transferring hyphae at the edges of the colonies obtained by the first separation culture medium into a second separation culture medium, and culturing for 2-7 days at the temperature of 20-28 ℃;
(3) and (4) purifying the colony obtained by culturing the second separation culture medium by adopting a single spore isolation method or a tip purification method.
According to the method for isolating plant pathogenic fungi, the plant sample is preferably selected from healthy tissues or plant seeds with fungi at the position 0.5-3 mm around the disease part.
According to the method for isolating plant pathogenic fungi, the disease part is vascular bundle tissue, and the plant sample is sheet vascular bundle tissue with the thickness of 0.5-3 mm.
According to the method for isolating plant pathogenic fungi, the disease part is preferably non-vascular bundle tissue, and the plant sample is a block of (2-5) mm x (2-5) mm.
According to the method for isolating a plant pathogenic fungus of the present invention, preferably, the first isolation medium is a water agar medium containing kanamycin to a final concentration of 25 to 100mg/mL, and the second isolation medium is a potato dextrose agar medium.
The method for isolating a plant pathogenic fungus according to the invention preferably comprises the following steps:
preparing spore suspension containing 3 × 10/ml of mycelia in colony obtained by culturing in second separation culture medium3～5×103Spores; transferring 40-60 mu L of spore suspension to a first purification culture medium to form a single colony; transferring the single colony to a second purification culture medium, and culturing at 20-28 ℃.
According to the method for isolating a plant pathogenic fungus of the present invention, preferably, the first purification medium is selected from the group consisting of water agar medium or potato dextrose agar medium, and the first purification medium contains kanamycin at a final concentration of 25 to 100 mg/mL; the second purification culture medium is a potato dextrose agar culture medium.
According to the method for isolating a plant pathogenic fungus of the present invention, preferably, the tip purification method comprises the steps of:
transferring the culture medium with hyphae at the edge of the colony obtained by culturing the second separation culture medium into a tip purification culture medium, and culturing at 20-28 ℃ until the colony is formed;
repeating the operation for 2-3 times, transferring the culture medium with the hyphae at the edge of the colony obtained by the last culture into another tip purification culture medium for each repetition, and culturing at 20-28 ℃ until the colony is formed.
According to the method for isolating a plant pathogenic fungus of the present invention, preferably, the tip purification medium is a potato dextrose agar medium.
According to the method for separating the plant pathogenic fungi, the plant sample is preferably from Astragalus membranaceus adsurges pall.
By adopting the method for separating the plant pathogenic fungi, the pretreatment of the plant sample can be finished in only 6-12 s, so that the separation efficiency is improved; the ethanol solution is blown by external force, so that the ethanol solution can be promoted to rapidly infiltrate the surface of the plant sample and enter somatic cells to achieve the aim of sterilization, and the ethanol can wash the surface of the plant sample to play a role in removing surface impurities or other microorganisms; the use of mercury bichloride or sodium hypochlorite is avoided, and the damage to the fresh plant sample with larger water content is reduced.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
The method for isolating pathogenic fungi of the present invention comprises the steps of: (1) pretreating a plant sample; (2) separating pathogenic fungi; (3) and (5) purifying the pathogenic fungi. As described in detail below.
< pretreatment of plant specimen >
And immersing the plant sample by using an ethanol solution, blowing the ethanol solution by adopting an external force, and airing the plant sample treated by the ethanol solution to obtain a pretreated plant sample.
The plant sample of the invention may be derived from a diseased organ or tissue of the plant, for example: leaves, branches, roots, stems, fruits, seeds, etc. Preferably, the plant is derived from a newly diseased plant. This can reduce saprophytic bacteria contamination.
The plant sample can be a healthy tissue at a position of 0.5-3 mm around a diseased part. Preferably, the plant sample is healthy tissue 1-2 mm around the diseased part. This can prevent the interior or surface of the necrotic area from being contaminated with saprophytic microorganisms.
The plant sample of the present invention may also be a granular plant seed with fungi. For example, tomato, eggplant, pepper, sargassum, adzuki, alfalfa, and other plant seeds. Shabaowang is also known as Astragalus orthodoxus (Astragalus adsurgensPall.).
The fungus of the present invention may be a fungus capable of forming a granular or sheet-like dormant structure, such as a fungus capable of forming a granular or sheet-like dormant structure, for example, a fungus having a large sclerotium, a small sclerotium, a rhizoctonia solani, and the like.
When the diseased part is vascular bundle tissue, the plant sample is sheet vascular bundle tissue with the thickness of 0.5-3 mm. Preferably, the plant sample is a sheet vascular bundle tissue with the size of (3-5) mm x (3-5) mm and the thickness of 1-2 mm. This is advantageous for the ethanol solution to sterilize and clean the surface.
When the diseased part is non-vascular bundle tissue, the plant sample is a block with (2-5) mm x (2-5) mm. Preferably, the plant sample is a block of (3-5) mm x (3-5) mm. This is advantageous for the ethanol solution to sterilize and clean the surface.
According to one embodiment of the present invention, the plant sample is a stem of Astragalus membranaceus, which is a new symptom of verticillium wilt, and a sheet vascular bundle tissue with a thickness of 1-2 mm is included in a healthy tissue with a thickness of 1-2 mm around the diseased part. Thus, the separation rate can be improved and the contamination rate can be reduced.
In the invention, the step of blowing the ethanol solution by using an external force refers to promoting the contact of the ethanol solution and the plant sample under the pushing of a slight external force, so that the ethanol solution quickly infiltrates the surface of the plant sample and washes the surface of the plant sample. By "slight external force" is meant that the ethanol solution is able to be brought into contact with the plant sample without causing damage to the plant sample. According to one embodiment of the invention, the ethanol solution is blown by a pipette gun, so that the ethanol solution quickly infiltrates and washes the surface of the plant sample.
The volume fraction of the ethanol solution is 70-75%; preferably 73 to 75%. Thus, better sterilization and washing effects can be achieved.
In the invention, the time for blowing the ethanol solution by external force is 6-12 s; preferably 7-11 s; more preferably 8 to 10 seconds. Therefore, the surface of the plant sample can be quickly infiltrated by the ethanol solution and enters the thallus cells to achieve the aim of sterilization, and the surface of the plant sample can be washed by the ethanol to play a role in removing surface impurities or other microorganisms; the short blow-beating time has unobvious disinfection effect, the too long blow-beating time increases the damage to pathogenic fungi, and reduces the separation rate.
< isolation of pathogenic fungi >
Placing the pretreated plant sample on a first separation culture medium, culturing for 2-7 days at 20-28 ℃, transferring hyphae at the edge of a colony obtained from the first separation culture medium into a second separation culture medium, and culturing for 2-7 days at 20-28 ℃.
The first isolation medium is an aqueous agar medium containing kanamycin to a final concentration of 25-100 mg/mL. Preferably, the first isolation medium is an aqueous agar medium containing kanamycin to a final concentration of 50 mg/mL. The culture temperature of the first separation culture medium is 20-28 ℃; preferably 22-26 ℃; more preferably 23 to 25 ℃. The culture time of the first separation culture medium is 2-6 days; preferably 3-5 days; more preferably 3 to 4 days.
The second separation medium may be potato dextrose agar medium. The culture temperature of the second separation culture medium is 20-28 ℃; preferably 22-26 ℃; more preferably 23 to 25 ℃. The culture time of the second separation culture medium is 2-7 days; preferably 3-6 days; more preferably 3 to 5 days.
< purification of pathogenic fungi >
And (4) purifying the spore-forming isolate by adopting a single spore isolation method. The method can be directly used for separating, purifying and culturing fungi capable of producing mildew or powder.
The purification method specifically comprises the following steps: and preparing a hypha block in the bacterial colony obtained by culturing the second separation culture medium into a spore suspension, taking the spore suspension, transferring the spore suspension to the first purification culture medium to form a single bacterial colony, and transferring the single bacterial colony to the second purification culture medium for culturing.
In the present invention, the spore suspension contains 3 × 10 per ml3～5×103Spores, preferably 3.5 × 103～4.5×103More preferably 3.9 × 103～4.1×103And (4) spores. This facilitates the obtaining of single colonies to obtain a pure culture. Pure culture refers to biological culture performed in the presence of only a single species.
And (3) coating 40-60 mu L of spore suspension on the first purification culture medium to form a single colony. Preferably, 45-55 μ L of spore suspension is spread onto the first purification medium to form a single colony. This facilitates the obtaining of single colonies to obtain a pure culture.
The first purification medium may be a water agar medium or a potato dextrose agar medium containing kanamycin to a final concentration of 25 to 100 mg/mL. Preferably, the first purification medium is water agar medium or potato dextrose agar medium containing kanamycin to a final concentration of 50 mg/mL. According to one embodiment of the invention, the first purification medium is an aqueous agar medium containing kanamycin to a final concentration of 50 mg/mL.
The culture temperature of the first purification culture medium can be 20-28 ℃; preferably 22-26 ℃; more preferably 23 to 25 ℃.
The second purification medium may be potato dextrose agar medium. The culture temperature of the second purification culture medium is 20-28 ℃; preferably 22-26 ℃; more preferably 23 to 25 ℃.
Non-spore-forming isolates (e.g., large sclerotium, small sclerotium, rhizoctonia solani, etc.) are purified by a top-end purification method. The purification method specifically comprises the following steps: transferring the culture medium with hyphae at the edge of the colony obtained by culturing the second separation culture medium into a tip purification culture medium for culturing until the colony is formed; repeating the operation for 2-3 times, and transferring the culture medium with the hyphae at the edge of the colony obtained by the last culture into another tip purification culture medium for culture every time, so as to form the colony. This allows the formation of colonies with typical morphology and without undesired bacteria.
The tip purification medium may be potato dextrose agar medium. The culture temperature can be 20-28 ℃; preferably 22-26 ℃; more preferably 23 to 25 ℃.
The test method is described below:
after the pretreated plant sample is cultured on the first separation culture medium for 4 days, observing and counting the number of plant sample materials which can grow out the target pathogen colony on the first separation culture medium, summing to obtain the separation times of the target pathogen, and according to a formula: the separation rate (the number of times of separation of the target pathogenic substance/total number of samples supplied) x 100%, and the separation rate of the target pathogenic substance was calculated; meanwhile, observing and counting the number of sample materials breeding bacteria or saprophytic fungi on the first separation culture medium according to a formula: the contamination rate (number of samples in which bacteria or saprophytic fungi are grown/total number of samples to be tested) is multiplied by 100%, and the contamination rate of the separated sample material is calculated.
The materials and reagents used in the examples and comparative examples are as follows:
water agar medium containing antibiotics: kanamycin was added to a water agar medium at a temperature of about 50 ℃ in a super clean bench by aseptic technique to a final concentration of 50mg/L, and then poured into 90mm disposable petri dishes at about 20mL per dish to form a plate 2 to 3mm thick.
Water agar medium: 15g of agar powder (NO.141614, Qingdao Haibo), 1L of distilled water, and sterilizing at 121 ℃ for 30 min.
Potato dextrose agar medium: 200g of potato, 20g of glucose (NO.14431-43-7, Dalochi chemical reagent factory, Tianjin, China), 15g of agar powder (NO.141614, Qingdao Haibo, China), 1L of tap water, and high-temperature sterilization at 121 ℃ for 30 min.
Examples 1 to 3
(1) Pre-treating a plant sample:
selecting upright astragalus stems with new symptoms of verticillium wilt, washing the stems with tap water, taking healthy tissues 2mm around the diseased parts, removing epidermis, cortex and medulla tissues of the stems with a sterile scalpel, and making vascular bundle tissues into slices 2mm thick to obtain plant samples.
The plant sample was placed in a sterile centrifuge tube, and then 75% by volume ethanol solution was added to the sterile centrifuge tube until the plant sample was submerged. And blowing the ethanol solution for 6-12 seconds by using a liquid transfer gun to promote the ethanol solution to quickly infiltrate the surface of the plant sample, washing the surface of the plant sample, then extracting the ethanol in the sterile centrifuge tube by using the liquid transfer gun, transferring the plant sample in the sterile centrifuge tube to sterile filter paper, and airing to obtain the pretreated plant sample.
(2) Separation of pathogenic fungi:
uniformly placing the pretreated plant samples on a water agar culture medium plate containing antibiotics by using sterilization tweezers, placing 7-10 plates in each plate, placing the culture plates in a constant-temperature incubator at 24 ℃, culturing for 4 days, picking typical bacterial colonies without sundry bacteria growing around the plant samples, picking the culture medium with hyphae at the edges of the bacterial colonies by using an inoculating needle, transferring the culture medium into a potato glucose agar culture medium, and culturing for 7 days in the constant-temperature incubator at 24 ℃.
(3) And (3) pathogenic fungi purification:
selecting potato glucose agar culture medium, culturing to obtain hypha blocks in bacterial colony, making into spore suspension, and regulating spore suspension concentration to 4 × 10 in each milliliter of spore suspension by using blood counting plate3And (3) coating 50 mu L of spore suspension on an antibiotic-containing water agar medium plate, and culturing at 24 ℃ until a single colony is formed on the antibiotic-containing water agar medium plate. Transferring the single colony to a potato glucose agar culture medium plate, and culturing in a thermostat at 24 ℃ to obtain the separated pathogenic fungi.
TABLE 1
| Serial number | Time(s) for blowing and beating the ethanol solution | Separation Rate (%) | Bacterial contamination ratio (%) |
| Example 1 | 6 | 78.67 | 8.67 |
| Example 2 | 9 | 95.33 | 1.33 |
| Example 3 | 12 | 74.67 | 0.67 |
Comparative example 1
The same astragalus membranaceus stem as in example 1 was used to isolate the pathogenic fungi. Example 1 was followed, except that:
the plant sample was placed in a sterile centrifuge tube, and then 75% by volume ethanol solution was added to the sterile centrifuge tube until the plant sample was submerged. Soaking the plant sample in an ethanol solution for 9s, then using a liquid transfer gun to extract ethanol in the sterile centrifuge tube, transferring the plant sample in the sterile centrifuge tube to sterile filter paper, and airing to obtain a pretreated plant sample.
The separation rate of pathogenic fungi is 27.25 percent, and the contamination rate is 55.75 percent.
Comparative examples 2 to 3
The same astragalus membranaceus stem as in example 1 was used to isolate the pathogenic fungi. The procedure of example 1 was repeated except that the time for blowing the ethanol solution was changed, and the details are shown in Table 2.
TABLE 2
| Serial number | Time(s) for blowing and beating the ethanol solution | Separation Rate (%) | Bacterial contamination ratio (%) |
| Comparative example 2 | 3 | 40.00 | 51.33 |
| Comparative example 3 | 15 | 31.33 | 0.00 |
Comparative example 4
(1) Pre-treating a plant sample:
using the same Astragalus membranaceus stem as in example 1, the stem was washed with tap water, and healthy tissue was collected 2mm around the diseased part, and then the epidermis, cortex and medulla of the stem was removed with a sterile scalpel, and vascular bundle tissue was made into 2mm thick sheets to obtain a plant sample.
And (3) placing the plant sample in a dish with a volume fraction of 75% for soaking for 30s, then carrying out surface disinfection for 5min by using 3% by mass of sodium hypochlorite, washing for 3 times by using sterile water, transferring to sterile filter paper, and airing to obtain the pretreated plant sample.
(2) Separation of pathogenic fungi:
uniformly placing the pretreated plant samples on a water agar culture medium plate containing antibiotics by using sterilization tweezers, placing 7-10 plates in each plate, placing the culture plates in a constant-temperature incubator at 24 ℃, culturing for 4 days, picking typical bacterial colonies without sundry bacteria growing around the plant samples, picking the culture medium with hyphae at the edges of the bacterial colonies by using an inoculating needle, transferring the culture medium into a potato glucose agar culture medium, and culturing for 7 days in the constant-temperature incubator at 24 ℃.
(3) And (3) pathogenic fungi purification:
selecting potato glucose agar culture medium, culturing to obtain hypha blocks in bacterial colony, making into spore suspension, and regulating spore suspension concentration to 4 × 10 in each milliliter of spore suspension by using blood counting plate3And (3) coating 50 mu L of spore suspension on an antibiotic-containing water agar medium plate, and culturing at 24 ℃ until a single colony is formed on the antibiotic-containing water agar medium plate. Transferring the single colony to a potato glucose agar culture medium plate, and culturing in a thermostat at 24 ℃ to obtain the separated pathogenic fungi.
The separation rate of pathogenic fungi is 84.00 percent, and the contamination rate is 9.33 percent.
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.
Claims (10)
1. A method for isolating a plant pathogenic fungus comprising the steps of:
(1) immersing a plant sample by using an ethanol solution with the volume fraction of 70-75%, and blowing the ethanol solution for 6-12 seconds by adopting an external force; drying the plant sample treated by the ethanol solution to obtain a pretreated plant sample;
(2) placing the pretreated plant sample on a first separation culture medium, and culturing for 2-7 days at the temperature of 20-28 ℃; transferring hyphae at the edges of the colonies obtained by the first separation culture medium into a second separation culture medium, and culturing for 2-7 days at the temperature of 20-28 ℃;
(3) and (4) purifying the colony obtained by culturing the second separation culture medium by adopting a single spore isolation method or a tip purification method.
2. The method for isolating phytopathogenic fungi according to claim 1, wherein the plant specimen is selected from healthy tissue at 0.5 to 3mm around the locus of attack or plant seeds in the form of granules with fungi.
3. The method for isolating a plant pathogenic fungus according to claim 2, wherein the diseased site is vascular bundle tissue, and the plant sample is sheet vascular bundle tissue 0.5 to 3mm thick.
4. The method for isolating a plant pathogenic fungus according to claim 2, wherein the diseased site is a non vascular bundle tissue and the plant specimen is a block of (2-5) mm x (2-5) mm.
5. The method for isolating a plant pathogenic fungus according to claim 1, wherein the first isolation medium is a water agar medium containing kanamycin to a final concentration of 25 to 100mg/mL, and the second isolation medium is a potato dextrose agar medium.
6. The method for isolating phytopathogenic fungi according to claim 1, characterized in that said monospore isolation comprises the following steps:
preparing spore suspension containing 3 × 10/ml of mycelia in colony obtained by culturing in second separation culture medium3～5×103Spores; transferring 40-60 mu L of spore suspension to a first purification culture medium to form a single colony; transferring the single colony to a second purification culture medium, and culturing at 20-28 ℃.
7. The method for isolating a plant pathogenic fungus according to claim 6, wherein the first purification medium is selected from the group consisting of water agar medium or potato dextrose agar medium, and the first purification medium contains kanamycin at a final concentration of 25 to 100 mg/mL; the second purification culture medium is a potato dextrose agar culture medium.
8. The method for isolating phytopathogenic fungi according to claim 1, characterized in that said tip purification process comprises the following steps:
transferring the culture medium with hyphae at the edge of the colony obtained by culturing the second separation culture medium into a tip purification culture medium, and culturing at 20-28 ℃ until the colony is formed;
repeating the operation for 2-3 times, transferring the culture medium with the hyphae at the edge of the colony obtained by the last culture into another tip purification culture medium for culture at the temperature of 20-28 ℃ every time, and waiting for the formation of the colony.
9. The method for isolating phytopathogenic fungi according to claim 8, characterized in that the tip purification medium is potato dextrose agar medium.
10. The method of claim 1, wherein the plant sample is from Astragalus Astragalus adsurgins pall.
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| LUISA PISTELLI等: "Antimicrobial and antifungal activity of crude extracts and isolated saponins Astragalus verrucosus", 《FITOTERAPIA》 * |
| 焉兆萍等: "藏红花内生真菌的分离和代谢产物的初步研究", 《上海师范大学学报(自然科学版)》 * |
| 韩凤英等: "胡萝卜根腐病病原菌的分离鉴定", 《北方农业学报》 * |
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