CN104686196B - Method for preserving and separating toadstool strain through sporocarp dried in shade - Google Patents

Abstract

The invention belongs to the technical field of microbes, and in particular relates to a method for preserving and isolating hickory chick strains by drying fruiting bodies in the shade. The technical problem to be solved by the present invention is to provide that the existing morel strain preservation method is limited by seasons, and the operation is complex, easy to pollute and high in cost. The technical solution of the present invention is a method for preserving and isolating hickory chick strains by drying fruiting bodies in the shade, comprising the following steps: a. preservation of strains; b. separation of strains. The method of the invention is low in cost, simple in operation, free from seasonal restrictions, and can effectively preserve the vitality of bacteria, and is convenient for transportation and preservation.

Description

A method for preserving and isolating hickory chick strains by drying fruiting bodies in the shade

technical field

The invention belongs to the technical field of microbes, and in particular relates to a method for preserving and isolating hickory chick strains by drying fruiting bodies in the shade.

Background technique

Morchella is a rare and rare edible fungus. Although it can be cultivated artificially in the field, its output is extremely unstable, which leads to its high price. The acquisition of strains is the first step in field production of morels, and it is also a key step. At present, the separation of morel strains mainly uses fresh fruiting bodies as the separation material, and there are two specific technical routes: in the morel fruiting body output season (every March-April), take fresh fruiting bodies and hang them upside down above the culture medium , the ascospores fall naturally on the culture medium (spore separation method, commonly used); or take fresh fruit body tissue, add sterile water to grind, prepare the bacterial suspension and spread the culture medium (tissue separation method, not commonly used) . After the bacteria are grown by the above method, the pure and pollution-free strains are obtained through repeated purification several times, stored at 4°C, and used in production. Since fresh fruit bodies are perishable and cannot be preserved for a long time, the acquisition of morel strains has a strong seasonality. If the production season is missed, the strains cannot be obtained. Moreover, using this method to preserve the strains requires a test tube to be stored in a 4°C freezer or refrigerator, which is costly; to preserve the strains in a test tube, it needs to be subcultured once in a certain period of time to prevent the depletion of nutrients in the medium, the degradation of the vitality of the strains, and complicated operations. ; Fresh fruit bodies carry nematodes and other pests, as well as a large number of miscellaneous bacteria, and the pollution is serious when the strains are isolated.

Contents of the invention

The technical problem to be solved by the present invention is that the existing hickory chick strain preservation method is limited by seasons, and the operation is complicated, the cost is high, and the separation is easy to be polluted.

Technical scheme of the present invention is a kind of method for preserving and isolating hickory chick bacterial classification by drying fruiting body in the shade, comprises the following steps:

a. Preservation of strains: In 3-4 months, select morels that are mature, robust, free from diseases and insect pests, with full caps, fully expanded ribs and pits on the caps, milky white stipe, normal shape, and moderate volume. Well-ventilated, temperature 10-15°C, dark, dry place, spread out and turn over from time to time until the fruiting bodies are completely dry; packed in paper bags, stored in a dry, dark and cool place indoors;

b. Strain separation: When strains need to be used, under aseptic conditions, take 0.5-1cm2 shade ^- dried fruiting body cap tissues, soak them in sterile water, and put them into sterilized research equipment after softening completely. Bowl, add 1 ml of sterile water, grind to prepare a tissue suspension, take the tissue suspension and inoculate it in the center of the wheat grain medium; incubate at 15-25°C in the dark for 10-20 days until the aerial hyphae or sclerotia of Morchella grow out, and then use an inoculation needle to pick aerial hyphae or sclerotia, inoculate them on PDA medium, and culture again, and then carry out repeated purification and culture on PDA medium until the bacteria without pollution are obtained.

Preferably, in step a, check the number of ascospores remaining in the shade-dried fruiting body before storage, the method is as follows: take a 0.5-1cm ² cap tissue block, soak it in water, soften it completely, put it into a mortar, add a small amount of water , Grind to prepare tissue suspension, examine under a microscope, discard fruiting bodies with no ascospores or too few ascospores, and select fruiting bodies with many ascospores for preservation.

Preferably, the culture temperature in step b is 20°C.

Preferably, the culture time in step b is 15 days.

Preferably, in step b, the inoculum used for the second purification is the sclerotium obtained during the first isolation.

During the preservation of the strains in step a, if there is poor ventilation, high temperature or humid environment, the fruiting body is prone to rot, and light will cause the fruiting body to eject a large number of ascospores, so low temperature, ventilation, dryness and dark environment are required. Since some ascospores may be ejected before fruiting bodies are harvested and during the drying process, the number of remaining ascospores can be checked before storage. Measures should be taken to prevent pests and rodents during storage.

In the step b strain separation process, the shade-dried fruit body amount is too little, the spore quantity that can germinate is too little, it is not easy to grow aerial hyphae, and the amount is too much, although there are many spores germinated, there are more miscellaneous bacteria, 0.5- ^-1cm2 is more suitable; if the amount of sterile water is too small, it is not easy to grind into a bacterial suspension; if the amount of sterile water is too large, a water layer will form at the bottom of the wheat grains, which will accelerate the spread of bacteria. 1ml is more suitable. Because the shade-dried fruiting body cap tissue is very thin and very light, the area commonly used in this field represents the size of the shade-dried fruiting body tissue.

Production of wheat grain medium in step b: select wheat grains that are free from diseases and insect pests, plump, relatively fresh, shelled and skinned, soaked in water for 24 hours, put into a pot, add water and boil until the grains have no white heart and no broken skin, remove Drain the water, put it into a plate, sterilize at 121°C for 30 minutes, cool down and set aside.

Production of PDA medium in step b: cut 200 grams of potatoes into small pieces, boil the juice and filter, add 20 grams of glucose, 7 grams of agar powder to the filtrate, dilute water to 1000 ml, sterilize at 121 °C for 30 minutes, pour out the bacteria The plate was cooled and solidified for later use.

In the present invention, according to previous knowledge, after the fruit body is dried, the ascospores therein will also dry out and lose the ability to germinate mycelium. And the present invention has chosen a lot of dry in the shade, the fruit body that contains a large amount of ascospores to carry out spore germination experiment, finds, although a lot of spores have lost germination ability, still have about 40-70% spores to absorb water and expand, germinate hyphae, form bacterium colony, This result is the technical basis of the present invention. However, the high-temperature dried fruiting body is not easy to germinate mycelium again because the spores in it are damaged by high temperature and lose their activity.

In the present invention, the culture medium of agar+nutrient+water is commonly used in previous studies to carry out the isolation of hickory chick strains, mainly conventional PDA culture medium. Since morels are harvested from the field, they carry a lot of germs, including bacteria and fungi. When growing on PDA, miscellaneous bacteria, especially bacteria, often grow into a thick layer of moss, covering morel hyphae, making it difficult to grow morel hyphae, even if morel grows aerial hyphae, When the aerial hyphae were picked and cultivated on a new PDA, the growth of bacteria carried by the aerial hyphae would often overwhelm the morel hyphae, and so repeatedly, resulting in separation failure. This problem cannot be solved when using the spore separation method to isolate the strains of fresh hickory chicks. When using the tissue separation method, although the method of respectively carrying out plate medium coating after the bacterial suspension is diluted step by step can alleviate the pollution situation, but this This method is suitable for fresh fruiting bodies, not for dry fruiting bodies. Because the spore germination rate of fresh fruiting bodies is almost 100%, after a suitable dilution level is obtained through experiments, the dilution level can be applied to the isolation of different fruiting bodies; while the spore germination rate of dry fruiting bodies is different among different individuals Relatively large, a series of dilution experiments are required for each fruiting body to obtain a dilution level suitable for the individual, and the workload is very large. However, the research of the present invention has found that when the tissue suspension made of dry tissue is inoculated on the wheat grain medium, the Morchella can grow a large amount of aerial hyphae, which are strong and dense, and even develop sclerotium tissue, which is much better than PDA medium. This is because the volume of bacteria is much smaller than that of hickory hyphae, and they need a continuous medium, such as agar medium, to expand on the medium, while the grain medium has a large gap between grains, which greatly hinders the speed of bacterial expansion, and It cannot form a lawn, but morel mycelium is slender, the gap has almost no hindrance to it, and the air permeability is good for its growth, so the growth and diffusion speed is faster than that of bacteria, although there is mold pollution, but the inhibitory ability to morel It is not as strong as bacteria, so on wheat medium, morel mycelia grow better and more robustly than on PDA, and when these aerial mycelia are taken to PDA for purification and culture, although there are also miscellaneous bacteria pollution, but The morel mycelium grows better than miscellaneous bacteria, which speeds up the process of strain purification. In addition, since the bacterial contamination rate of wheat grain medium is lower than that of PDA, as long as the amount of fruiting body tissue and the amount of sterile water are controlled within a rough ratio range, robust aerial hyphae can grow without Need to do too complex dilution level experiments, very suitable for dry fruiting bodies with inconsistent spore germination rate. The above is the technical basis of the present invention.

Beneficial effects of the present invention:

1, adopt the method of the present invention, all can obtain bacterial classification from dry fruiting body isolation at any time. The dry ascospores in the dry fruiting body can maintain germination vigor in at least 48 months (due to not carrying out a longer preservation experiment, it is possible that the ascospore vigor can be maintained for a longer period of time) to obtain strains. This breaks through the seasonal restrictions on the production of morel strains; in addition, the excellent fruiting bodies obtained in the field can be preserved for a long time by drying in the shade;

2. The test-tube strains obtained from fresh fruiting bodies must be stored in a refrigerator at 4°C. Dried fruiting bodies can be stored in a refrigerator freezer at 4°C, or in a dark, dry and cool place at room temperature, with low cost;

3. The test tube strains isolated from fresh fruiting bodies need to be transferred to new test tubes regularly to prevent the depletion of nutrients and the degradation of the strains. The dry seed entity can be used to isolate strains, no need to transfer test tubes, and the operation is simple;

4. The volume of dried fruiting bodies is much smaller than that of test tubes, and it can be crushed into small pieces for storage. Even if many varieties are stored, it does not require much space;

5. The test-tube strains obtained by fresh fruiting bodies are prone to mutations during preservation, and the excellent species degenerate. The mutation probability of dry ascospores in dry fruiting bodies is very low, and can maintain excellent seed quality for a long time;

6. If the species is introduced from other places, the fresh fruiting bodies are perishable during long-distance transportation, the test tube species are easily polluted, and the packaging is complicated. If it is in the high temperature season, the strains will die. Dried fruit bodies can be simply packaged during long-distance transportation, and the activity is not limited by temperature and will not rot;

7. Compared with strains isolated from fresh fruiting bodies, shade-dried fruiting bodies can reduce pollution to a certain extent. Nematodes and other micro-pests mainly come from the soil where fruiting bodies grow. They need to eat fresh and tender fruiting bodies to survive. When separating strains, these pests move freely in the medium and spread miscellaneous bacteria, greatly increasing the chance of contamination . The dry fruiting bodies become hard, which is not conducive to the pests to eat and parasitize them. They will die, escape or dormant. Although the dormant worms may survive again when they are separated later, the number has been greatly reduced compared with the fresh fruiting bodies. Pollution probability is reduced; the growth of mycelia of miscellaneous bacteria requires a humid environment. When the environment becomes dry, the mycelium of miscellaneous fungi also loses water and shrinks, and loses their growth activity. , but the number has been reduced, and the chance of contamination is less than that of fresh fruiting bodies;

8. Wheat culture medium was used instead of conventional PDA medium for the initial separation. Because the gaps between wheat grains are large, the expansion of bacteria is greatly hindered, so it is beneficial to reduce pollution, and it is breathable, which is conducive to the growth of Morchella mycelium and accelerates the growth of bacteria. Strain purification process.

detailed description

The dry fruiting body isolate strain of embodiment 1 different storage time compares with the fresh fruiting body isolate strain

From March to April, select morels that are mature, robust, free from diseases and insect pests, with full caps, fully expanded ribs and pits on the caps, milky white stipe, normal shape, and moderate volume, and make 2 parts on average, 1 part Immediately isolate the strains, and place the other part in a well-ventilated, low temperature (10-15°C), dark, and dry place. Spread it out and turn it from time to time until the fruiting body is completely dry. Take a small amount of cap tissue, After soaking and softening in clean water, grind it in a mortar to make a tissue suspension. After microscopic examination, select the dry fruiting body with many ascospores, put it in an envelope, and store it in a dry, dark and cool place indoors, and take measures to prevent pests and rodents.

Due to microscopic examination of spore germination, PDA medium was used for isolation here. Production of PDA medium: cut 200 grams of potatoes into small pieces, boil the juice and filter, add 20 grams of glucose and 7 grams of agar powder to the filtrate, dilute water to 1000 ml, sterilize at 121 ° C for 30 minutes, pour out the sterilized plate, and cool it for backup use.

Fresh sporocarp spore isolation method (spore separation method): On the ultra-clean workbench, put the fresh sporocarp with the cap downward and the stipe upward, and hang it upside down above the culture medium. Pay attention that the sporocarp does not touch the medium. The light illuminates, prompting the fruiting body to eject the ascospores. After about 12 hours, take a plate, incubate at 20°C in the dark, and regularly check under a microscope to record the spore germination. After the colony grows, the inoculation needle picks up a little hyphae at the edge of the colony, inoculates it in the center of another PDA medium, and cultures it again. Purification and cultivation are repeated in this way until the contamination-free strains are obtained.

Method for isolating strains from dry fruiting bodies: the dry fruiting bodies that have been preserved for 12, 24, 36, and 48 months by the above-mentioned method of drying in the shade, respectively take 0.5-1cm ² cap tissue pieces, and use sterile water on the ultra-clean workbench After soaking in water and completely softening, put it into a sterilized mortar, add 1ml of sterilized water, grind to prepare tissue suspension, add it to the PDA medium with a sterilized dropper, and use an L-shaped glass rod to coat it for use. The tissue suspension is evenly distributed on the surface of the culture medium. Cultivate at 20°C in the dark, check regularly under the microscope, and record the spore germination. After the colony grows, the inoculation needle picks up a little mycelium at the edge of the colony, inoculates it in the center of another PDA medium, and cultures it again. Purification and cultivation are repeated in this way until the contamination-free strains are obtained.

Inoculate the PDA with the purified bacterial strain obtained above, 3 dishes/treatment, and record the mycelial growth situation.

Table 1 Comparison of the growth of strains obtained from dry fruiting bodies and fresh fruiting bodies at different storage times

Note: "+" in the above table is a quantitative expression of the indicators. The more "+", the better the growth, and vice versa. The same below.

It can be seen from the results in Table 1 that although the dry spores germinated slower than the fresh spores due to the longer imbibition process, the growth of mycelium and sclerotia was not different from that of the fresh spores. The growth of mycelium and sclerotia of the dried spores with different storage times is not much different, and it can be seen that the dried spores can maintain their vitality in at least 48 months.

The influence of embodiment 2 separation medium on dry spore isolation strain

In view of the previous experiments, it was found that conventional PDA was rich in nutrients, and other miscellaneous bacteria, especially bacteria, also grew in large numbers at the same time as the morel mycelium grew, and the pollution was serious. Therefore, the following experiments were performed and cultured with pure agar without any nutrients Base, wheat grain medium and conventional PDA medium were compared. Make the following 3 media:

Medium 1: conventional PDA medium, the composition and preparation method are the same as in Example 1.

Medium 2: pure agar medium, compared with conventional PDA medium, does not contain potato juice and glucose, and the rest of the ingredients and production methods are the same.

Medium 3: Wheat grain medium, select wheat grains that are free from diseases and insect pests, plump, relatively fresh, shelled and skinned, soaked in water for 24 hours, add water to the pot and cook until the grains have no white heart and no broken skin, then remove Drain, pour into a plate, sterilize at 121°C for 30 minutes, cool down and set aside.

Isolate strains with the dry fruiting bodies preserved for 12 months, the method is the same as in Example 1, inoculate 3 kinds of culture media respectively, 3 dish/treatment, when wherein inoculating culture media 1 and 2, be that the bacterial suspension is evenly coated on the culture medium When inoculating medium 3 on the base surface, the bacterial suspension was inoculated in the center of the medium. After inoculation, culture at 20°C in the dark, and regularly check the germination of spores on medium 1 and 2 under a microscope. After colony growth, record the growth of aerial hyphae.

Table 2 Comparison of growth of Morchella mycelium on different isolation media

From the above results, it can be seen that if there is no nutrient in the medium, although there are few bacteria, it is beneficial for purification, and morel spores can also germinate, but after the germination reaches a certain level, the nutrients carried by the spores are exhausted, and the hyphae stop growing. , cannot further grow into colonies. However, with nutrient-rich PDA, bacterial contamination is very serious. Although aerial hyphae can grow, the growth is weak and unfavorable for purification. Although the growth time of the aerial hyphae in the wheat grain medium is obviously longer than that of PDA, the mycelium is strong and grows well, and can even develop into sclerotia, and the pollution is small. Therefore, the wheat grain medium is still required for the initial isolation of strains.

Embodiment 3 The influence of culture temperature on the growth of dry spore isolate strain.

Isolate strains with dry fruiting bodies stored for 12 months, the method is the same as in Example 1, inoculate the center of the wheat grain medium, culture them in the dark at 15°C, 20°C, and 25°C respectively, 3 dishes/treatment, and record the mycelial sclerotia growing situation.

Table 3 Effects of different culture temperatures on the growth of strains isolated from dry spores

Although the hyphae grow faster at 25°C, their growth vigor is not as good as at 20°C, and there are fewer sclerotia. Because Morchella is a low-temperature fungus, too high a temperature can easily lead to premature aging of the hyphae. Although spores can germinate at 15°C, and the growth of colonies and sclerotias is also good, but due to the slow growth of mycelium, it is easy to be contaminated by miscellaneous bacteria. Therefore, 20°C is the optimal growth temperature.

The comparison of different inoculums to hickory chick mycelium growth during the second purification of embodiment 4

In Example 2, because wheat grains are suitable for the growth of Morchella mycelium, Morchella can develop sclerotium, this study takes the Morchella sclerotia and mycelium grown on the wheat grain medium in Example 2 respectively. 1. Wheat grains (1-2 grains/dish) with Morchella hyphae were inoculated in PDA medium, and the purification effect was compared. 3 dishes/treatment. After culturing at 20°C for 15 days in the dark, observe the growth of mycelia.

Table 4 The effect of the inoculum during the second purification on the growth of hickory chick mycelium

As can be seen from Table 4, sclerotia is the best inoculum, so when separating for the second time, if sclerotia can develop on the wheat grain medium, it is best to inoculate with sclerotia, the purification is fast, the obtained strain is excellent, and the hybrid Less bacterial contamination. If there is no sclerotia, wheat grains can be used for inoculation. First observe the pollution of the wheat grains, and try to inoculate the wheat grains with strong growth of morel mycelium and few miscellaneous bacteria.

Claims (4)

1. a kind of by sporophore preservation and the method for separating Morchella esculenta (L.) Perss strain of drying in the shade, it is characterised in that：Comprise the steps：

A, fungi preservation：In the 3-4 months, choose that maturation, stalwartness, no disease and pests harm, cap be full, rib and pit are opened up completely on cap Open, stem is milky, the Morchella esculenta (L.) Perss that form is normal, volume is moderate, in well-ventilated, 10～15 DEG C of temperature, dark, drying Place, spreads out and puts, stir frequently, be completely dried until drying in the air to sporophore, take 0.5-1cm²The cap piece of tissue of size, clear water leaching Bubble, after softening completely, is put into mortar, plus a small amount of clear water, and grinding prepares tissue suspension, under the microscope microscopy, no ascus spore Son or ascospore sporophore very little is discarded, and selects the sporophore more than ascospore to preserve, with paper bag packing, does indoors Dry dark shady place is preserved；

B, strain separating：When needing using strain, under aseptic condition, 0.5-1cm is taken²The sporophore cap group of drying in the shade of size Knit, soaked with sterilized water, after softening completely, be put into sterilized mortar, add 1 milliliter of sterilized water, grinding prepares tissue suspension, Take tissue suspension and be inoculated in wheat grain culture medium central；In 15～25 DEG C of lucifuge cultures 10-20 days to Morchella esculenta (L.) Perss aerial hyphae or bacterium Core grows, and then with Inoculating needle picking aerial hyphae or sclerotium, is inoculated in PDA culture medium, cultivates again, hereafter trains in PDA Purification culture repeatedly is carried out on foster base, till obtaining free of contamination strain.

2. the method for claim 1, it is characterised in that：In step b, cultivation temperature is 20 DEG C.

3. the method for claim 1, it is characterised in that：In step b, incubation time is 15 days.

4. the method for claim 1, it is characterised in that in step b, inoculum used by second purification are first separation When the sclerotium that obtains.