CN106967648B - Medium-and long-term preservation method for colletotrichum gloeosporioides - Google Patents
Medium-and long-term preservation method for colletotrichum gloeosporioides Download PDFInfo
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Abstract
The invention belongs to the technical field of plant protection and discloses a medium-long term storage method of colletotrichum gloeosporioides. The method comprises the following steps: separating and culturing colletotrichum gloeosporioides; preparing an SNA slant culture medium, namely adding the molten SNA culture medium into a freezing storage tube, obliquely placing the frozen storage tube, and solidifying the frozen storage tube to obtain the SNA slant culture medium; inoculating a colony edge hypha block of colletotrichum gloeosporioides to a cryopreservation tube SNA slant culture medium, screwing a sealing cover opening, sealing a tube opening with a sealing film, putting the tube opening into a cryopreservation tube box, and culturing and storing at 4-15 ℃. The method is simple and convenient to operate, and expensive equipment is not needed; a large space is not needed; the preservation time is longer: the colletotrichum gloeosporioides can survive for more than 4 years in the environment of low-temperature storage on the SNA slant culture medium, and has strong pathogenicity. The method is simple, convenient and practical, and is used for long-term monitoring of the pathogenicity difference and the drug resistance change of anthracnose of flowering cabbage, evaluating the disease resistance level of flowering cabbage varieties, screening high-activity bactericides and the like.
Description
Technical Field
The invention belongs to the technical field of plant protection, and particularly relates to a medium-and-long-term preservation method for colletotrichum gloeosporioides.
Background
The flowering cabbage is an important special vegetable in Guangdong province and even south China, has large cultivation area and high multiple cropping index, and can be produced and supplied all year round. Anthracnose of cabbage heart is one of the most important diseases in the production of cabbage heart, and the quality and the yield of the cabbage heart are seriously influenced. Researches on measuring pathogenicity difference of Colletotrichum brassicae (also called Colletotrichum higginsanium) in different areas, monitoring drug resistance change of the germs, evaluating disease resistance level of the Colletotrichum brassicae varieties, screening bactericides with activity on the germs and the like all need a large amount of Colletotrichum brassicae strains which are stored for a medium-long period and have stable biological characteristics.
At present, the main preservation methods of plant pathogenic fungi comprise 3. (1) Bevel low-temperature preservation method: the strain is inoculated in a glass test tube filled with a proper solid slant culture medium, a tube opening is bundled by a cotton plug and kraft paper, and the strain is transferred to a refrigerator at 4-10 ℃ for storage after the strain fully grows. The preservation method is simple to operate and convenient to use, special instruments and equipment are not needed, but the slant culture medium is easy to dry, and the preservation time is usually not more than one year; in addition, the storage space required for storing the strains is large, the storage of vessels is difficult, the probability of contaminating sundry bacteria is high, and the strains are not suitable for large-batch long-time storage. (2) Freezing vacuum drying preservation method: suspending spore suspension of the strain to be preserved in a protective agent (such as skimmed milk and glycerol), rapidly freezing at low temperature (-45 deg.C), sublimating ice under vacuum to remove most of water, sealing ampoule, and storing in refrigerator at 4 deg.C. The method has long strain preservation time and high survival rate, but needs expensive professional equipment such as freeze drying and the like, has high preservation cost, and is not suitable for common laboratories. (3) Liquid nitrogen ultra-low temperature preservation method: suspending the strain hypha or spore in liquid culture medium containing protectant (such as 20% glycerol), subpackaging in low temperature resistant ampoule bottle or plastic centrifuge tube, pre-cooling, and transferring to liquid nitrogen tank (196 deg.C below zero) for storage. The method has long strain preservation time, is suitable for preserving various microorganisms, but needs special equipment such as a liquid nitrogen tank and the like, and has the defects of frequent addition of volatile liquid nitrogen, high management cost, inconvenient use and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for preserving colletotrichum gloeosporioides for a medium and a long term. The method is simple, convenient and practical, and is used for long-term monitoring of the pathogenicity difference and the drug resistance change of anthracnose of flowering cabbage, evaluating the disease resistance level of flowering cabbage varieties, screening high-activity bactericides and the like.
The purpose of the invention is realized by the following technical scheme:
a method for preserving colletotrichum gloeosporioides for a medium and long term comprises the following steps:
(1) separating and culturing colletotrichum gloeosporioides;
(2) preparing an SNA slant culture medium: adding the melted SNA culture medium into a freezing storage tube, obliquely placing, and preparing an SNA slant culture medium after the SNA culture medium is solidified;
(3) preservation of colletotrichum gloeosporioides: inoculating the colony edge hypha blocks of the colletotrichum gloeosporioides purified in the step (1) to the SNA slant culture medium of the cryopreservation tube in the step (2), screwing a sealing cover opening, sealing a tube opening with a sealing film, and storing at 4-15 ℃ after culturing.
In order to better implement the invention, the method further comprises the following steps:
(4) activation of the preserved strain: taking out the freezing tube filled with colletotrichum gloeosporioides from a refrigerator at 4 ℃, picking out the culture medium blocks with bacteria, and placing the culture medium blocks on a PDA (Potato dextrose agar) culture medium for activated culture, so that hyphae grow out for later use.
The separation and culture of colletotrichum gloeosporioides in the step (1) comprises the following steps:
collecting anthracnose samples of cabbage hearts, selecting tissues with typical disease spots, washing the tissues with clear water, and shearing tissue blocks at the junction of disease centers; sequentially sterilizing in 75% alcohol and 1% sodium hypochlorite solution, and rinsing in sterile water for 3 times; taking out, absorbing excessive water with sterilized filter paper, blow-drying on a superclean bench, and culturing on a PDA plate; after hyphae grow out, picking hyphae blocks at the edges of the colonies for re-culture; the separated bacterial strain is purified by adopting a single hypha picking method or a single spore separation method, and the colletotrichum gloeosporioides is obtained after the verification of the Koch's law.
The tissue block is 0.3cm multiplied by 0.5 cm.
The time for sterilization is 15s to 45s, preferably 30 s.
The temperature of the culture is 25-30 ℃, and preferably 27 ℃.
The SNA (synthetic low nutrient agar medium) culture medium in the step (2): 1g/L KH2PO4，1g/L KNO3，0.5g/L MgSO4·7H2O, 0.5g/L KCl, 0.2g/L glucose, 0.2g/L sucrose and 15g/L agar powder; 120 deg.C pesticideAnd (5) sterilizing for 20min for later use.
The culture conditions in step (3) are 25 to 30 ℃ for 2 to 5 days, preferably 27 ℃ for 2 days.
The preservation time in the step (3) is at least 4 years.
The activation culture in step (4) is carried out under conditions of 25 to 30 ℃ for 3 to 7 days, preferably 27 ℃ for 5 days.
Compared with the prior art, the invention has the following advantages and effects:
(1) the operation is simple and convenient: the strain is stored in a clean and common refrigerator without expensive equipment.
(2) Does not need a large space: the volume of the 2mL freezing storage tube is small, and a square plastic box with the length of 12cm can be put into an 81 tube, which is equivalent to the capacity of storing 81 strains.
(3) The preservation time is longer: the colletotrichum gloeosporioides can survive for more than 4 years in the environment of low-temperature storage on the SNA slant culture medium, and has strong pathogenicity.
Drawings
FIG. 1 is a flow chart of the procedure for preserving Colletotrichum album.
FIG. 2 is a colony morphology of Colletotrichum album cultured on PDA at 27 ℃ for 5 days.
FIG. 3 is a diagram of a freezing tube and a freezing tube box for colletotrichum gloeosporioides preservation.
FIG. 4 is a diagram showing the colony growth morphology of Colletotrichum album L.var.carinatum activated at 27 ℃ for 5 days after being stored for 4 years.
FIG. 5 is a diagram showing the pathogenic effects of Colletotrichum album after 4 years of storage.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Raw material specification:
the SNA (synthetic low nutrient agar medium) culture medium: 1g/L KH2PO4，1g/L KNO3，0.5g/LMgSO4·7H2O, 0.5g/L KCl, 0.2g/L glucose, 0.2g/L sucrose and 15g/L agar powder; 1Sterilizing at 20 deg.C for 20 min.
The materials of the formula components of the SNA culture medium are all analytical pure chemical reagents.
The 2mL cryopreservation tube, PDA culture medium, distilled water, culture dish, pipettor, sterilizer, refrigerator, clean bench and the like required by other experimental operations are all conventional materials or tools in the laboratory.
The operational flow chart for preserving colletotrichum gloeosporioides is shown in fig. 1.
Example 1 isolation and preservation of pathogens from field-derived anthracnose samples of cabbage
(1) Selecting a typical fresh disease sample of anthracnose of cabbage heart in the field, cleaning the sample with clear water, shearing tissue blocks (about 0.3cm multiplied by 0.5cm) at the boundary of the disease and the health, respectively disinfecting the sample on a super clean workbench with 75% alcohol and 1% sodium hypochlorite solution for 30s, flushing the sample with sterile water for three times, drying the sample in the air, transferring the sample to a PDA (personal digital assistant) plate, culturing the sample at 27 ℃ for 5d, selecting hypha blocks at the edges of colonies, and performing purification culture to obtain the isolate bacteria.
(2) Inserting sterile sun-dried cowpea tissue blocks (with seeds removed) into the edges of bacterial colonies of the bacteria to be separated, culturing at 27 ℃ for 5d, and performing microscopic examination on the cowpea tissue blocks, wherein if a large number of oblong conidia can be microscopically detected on the epidermis of the cowpea tissue blocks, the cowpea tissue blocks can be primarily determined as pathogenic bacteria of anthracnose cabbage (Seggins anthracnose bacteria). Inoculating the obtained conidium into a cabbage heart, and confirming that the obtained conidium causes diseases to the cabbage heart and causes typical anthracnose symptoms; then single spore separation and purification culture are carried out, thus obtaining the colletotrichum gloeosporioides. The colony morphology of Colletotrichum brassicae is shown in FIG. 2.
(3) Placing purified colletotrichum gloeosporioides on a PDA (potato dextrose agar) culture medium for culturing for 5d at 27 ℃, then picking out colony edge mycelium blocks by using aseptic toothpicks to inoculate the colony edge mycelium blocks on an SNA slant culture medium in a 2mL freezing tube, screwing a sealing cover opening, sealing a tube opening by using a sealing film, placing the tube into a freezing tube box, culturing for 2d at 27 ℃, and then placing the tube box in a refrigerator at 4 ℃ for storage. The cryopreservation tube and the cryopreservation tube box for colletotrichum gloeosporioides preservation are shown in FIG. 3.
(4) A small piece of culture medium with hyphae is picked from colletotrichum gloeosporioides stored for 1 year, 2 years and 3 years and is put on a PDA plate, and cultured for 3 days at 27 ℃ to observe whether hyphae grow out. New hyphae grow out, namely survival is judged; no new hyphae were grown, and the test was judged to be dead. The strains in 20 stored frozen tubes were randomly picked and activated on PDA plates, and the proportion of the tubes surviving was examined and counted. The test results show that all the randomly selected strains in the tube stored for 1 year, 2 years and 3 years survive, and the survival rate is 100%.
Example 2 detection of preservation Effect of Alternaria caerulea in Medium-and Long-term preservation
(1) Detection of survival rate of preserved colletotrichum gloeosporioides
A small piece of the culture medium with hyphae was randomly picked up from a 2mL freezing tube stored for 4 years in example 1 and plated on a PDA plate, and cultured at 27 ℃ for 3 days to observe the presence or absence of hyphae growth. New hyphae grow out, namely survival is judged; no new hyphae were grown, and the test was judged to be dead. The bacteria in 50 frozen tubes stored for 4 years are randomly picked to be activated on a PDA plate, and the proportion of the survival tubes is detected and counted. The results of the tests showed that all randomly selected tube-borne bacteria stored for 4 years were viable with a survival rate of 100% (fig. 4).
(2) Determination of pathogenicity of preserved colletotrichum gloeosporioides
Randomly picking a small piece of culture medium with hyphae from a 2mL freezing storage tube stored for 4 years in example 1, culturing at 27 deg.C on a PDA plate, inserting sterile sun-dried cowpea pod on the plate after hyphae grow, culturing in dark for 7d, washing conidia with clear water, measuring the concentration of the spore suspension with a blood counting plate, and diluting to 1 × 105spores/mL. Inoculating the spore suspension to 3-4 leaf slices of cabbage heart with a hand-held sprayer, spreading small water drops on the leaf surfaces without dropping, keeping the water in the dark for 12h, placing the leaf slices in a greenhouse at 25 +/-2 ℃, and watering the leaf slices with a shower head in the morning and at the evening. The incidence of the leaves was investigated 5 days after inoculation. The pathogenicity of 3 strains of different origin was thus tested at random. The test result shows that 3 strains of colletotrichum gloeosporioides stored for 4 years can cause the leaf of the colletotrichum gloeosporioides to suffer from diseases, the area of the suffered leaves accounts for more than 50% of the area of the whole leaves, and the colletotrichum gloeosporioides stored for 4 years still has strong pathogenicity (figure 5).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. A medium-long term preservation method of colletotrichum gloeosporioides is characterized by comprising the following steps:
(1) separating and culturing colletotrichum gloeosporioides; the colletotrichum gloeosporioides is the colletotrichum schizanioides;
(2) preparing an SNA slant culture medium: adding the melted SNA culture medium into a freezing storage tube, obliquely placing, and preparing an SNA slant culture medium after the SNA culture medium is solidified; the SNA culture medium: 1g/L KH2PO4，1 g/L KNO3，0.5 g/LMgSO4·7H2O, 0.5g/L KCl, 0.2g/L glucose, 0.2g/L sucrose and 15g/L agar powder; sterilizing at 120 deg.C for 20 min;
(3) preservation of colletotrichum gloeosporioides: inoculating the colony edge hypha blocks of the purified colletotrichum gloeosporioides in the step (1) to the SNA slant culture medium of the cryopreservation tube in the step (2), screwing a sealing cover opening, sealing a tube opening with a sealing film, and storing at 4-15 ℃ after culturing;
the culture condition in the step (3) is culture for 2d to 5d at 25 ℃ to 30 ℃.
2. The method for the medium-and long-term preservation of colletotrichum gloeosporioides according to claim 1, further comprising:
activation of the preserved strain: taking out the freezing tube filled with colletotrichum gloeosporioides from a refrigerator at 4 ℃, picking out the culture medium blocks with bacteria, and placing the culture medium blocks on a PDA (PDA) culture medium for activated culture, wherein hyphae grow out for later use.
3. The method for the medium-and long-term preservation of colletotrichum gloeosporioides according to claim 1 or 2, characterized in that:
the separation and culture of colletotrichum gloeosporioides in the step (1) comprises the following steps:
collecting anthracnose samples of cabbage hearts, selecting tissues with typical disease spots, washing the tissues with clear water, and shearing tissue blocks at the junction of disease centers; sequentially sterilizing in 75% alcohol and 1% sodium hypochlorite solution, and rinsing in sterile water for 3 times; taking out, absorbing excessive water with sterilized filter paper, blow-drying on a superclean bench, placing on a PDA plate, and culturing; after hyphae grow out, picking hyphae blocks at the edges of the colonies for re-culture; the separated bacterial strain is purified by adopting a single hypha picking method or a single spore separation method, and the colletotrichum gloeosporioides is obtained after the verification of the Koch's law.
4. The method for the medium-and long-term preservation of colletotrichum gloeosporioides according to claim 3, wherein:
the disinfection time is 15-45 s.
5. The method for the medium-and long-term preservation of colletotrichum gloeosporioides according to claim 3, wherein:
the culture temperature is 25-30 ℃.
6. The method for the medium-and long-term preservation of colletotrichum gloeosporioides according to claim 2, wherein:
the activation culture condition is activation culture for 3d to 7d at the temperature of 25 ℃ to 30 ℃.
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| CN102399696A (en) * | 2010-09-09 | 2012-04-04 | 上海海帝园艺有限公司 | Method for preserving microbial strains for long time |
| 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 |
| CN106191197A (en) * | 2016-09-14 | 2016-12-07 | 江苏丘陵地区镇江农业科学研究所 | A kind of anthrax bacteria method for quick drug-fast to various medicaments |
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| JP2006296419A (en) * | 2005-03-04 | 2006-11-02 | Hiroshima Univ | Method for producing antibiotic-producing microorganism by gene disruption and the resultant antibiotic-producing microorganism, and method for producing antibiotic metabolic intermediate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102399696A (en) * | 2010-09-09 | 2012-04-04 | 上海海帝园艺有限公司 | Method for preserving microbial strains for long time |
| 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 |
| CN106191197A (en) * | 2016-09-14 | 2016-12-07 | 江苏丘陵地区镇江农业科学研究所 | A kind of anthrax bacteria method for quick drug-fast to various medicaments |
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| Title |
|---|
| 草莓炭疽病菌鉴定技术规程;张丽勍等;《上海农业科技》;20160131;第145-146页 * |
| 菜心炭疽病菌菌丝生长 、产孢和孢子萌发的影响因素;周而勋等;《南京农业大学学报》;20021231;第25卷(第2期);第47-51页 * |
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