CN113337404B - Fungus strain for promoting germination of fescue seeds, germination promoter and application - Google Patents
Fungus strain for promoting germination of fescue seeds, germination promoter and application Download PDFInfo
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- CN113337404B CN113337404B CN202110616161.3A CN202110616161A CN113337404B CN 113337404 B CN113337404 B CN 113337404B CN 202110616161 A CN202110616161 A CN 202110616161A CN 113337404 B CN113337404 B CN 113337404B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention relates to the technical field of microorganism application, and particularly discloses a fungus strain for promoting germination of fescue seeds, wherein the strain is number 65, the preservation date is 2021, 03 and 29 days, the preservation number is CGMCC 21937, and the strain is classified and named as Tulasnella sp. The strain No. 65 obtained by the invention has a promoting effect on the germination of the fescue seeds, so that the germination rate of the fescue seeds reaches 85.24%.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to a fungus strain for promoting germination of fescue seeds, a germination promoter and application.
Background
The herba Caprae Seu Ovis (Liparis campylostalix) is whole herb with root of herba Caprae Seu Ovis of Orchidaceae, and is distributed in northeast, northwest, anhui, hubei, sichuan, guizhou, yunnan, etc. Has effects of promoting blood circulation, stopping bleeding, and relieving swelling and pain. It is commonly used for metrorrhagia, puerperal abdominal pain, leukorrhagia, tonsillitis, traumatic injury, and burn.
The festuca sativa has higher medicinal value, the part to be used for taking the medicine is whole grass with roots, the market demand is large, however, the festuca sativa is mainly acquired and obtained in the field at present, no report of artificial cultivation and planting exists, and the festuca sativa is slow to grow, has higher requirements on the growing environment condition, and causes serious damage to wild plant resources of the festuca sativa.
The seed quantity of the festuca sativa is large, but due to the tiny seed and lack of endosperm, the germination is very difficult under natural conditions, the germination rate is extremely low and is less than 0.01%, and proper mycorrhizal fungi symbiosis is needed for germination, so that the seeds are greatly influenced by environmental factors. Therefore, it is extremely important to obtain a method for promoting the germination of the fescue garlic, and no research on the promotion of the germination of the fescue garlic by using fungi is available in the prior art.
Disclosure of Invention
In order to solve the technical problems, the invention provides a fungus strain for promoting the germination of the fescue garlic seeds, a germination promoter and application thereof, wherein the fungus strain has a promoting effect on the germination of the fescue garlic seeds, so that the germination rate of the fescue garlic seeds reaches 85.24%.
The first object of the invention is to provide a fungus strain for promoting the germination of the seeds of the garlicus caprae seu ovis, wherein the preservation date of the fungus strain is 2021, 03 and 29, the preservation number is CGMCC 21937, and the strain is named as Tulasnella sp.
The second object of the present invention is to provide the method for culturing the fungus strain for promoting the germination of the seeds of the garlicus caprae seu ovis, wherein the fungus strain is cultured by adopting a PDA culture medium, and the culture temperature is 23-27 ℃.
The third object of the invention is to provide the application of the fungus strain No. 65 in promoting the germination of the seeds of the fescue.
Further, the fungus strain No. 65 promotes germination of the seeds of the garlics caprae seu ovis by co-culturing with the seeds of the garlics caprae seu ovis.
Further, the culture medium used in the co-culture process is a symbiotic culture medium.
Further, the symbiotic culture medium has a mass concentration of 5 g.L -1 Oat medium of (a) and (b) a plant.
The invention also provides a germination promoter of the tremella melitensis seeds, which is prepared from the fungus strain No. 65 according to claim 1.
Further, oat medium is added to the promoter.
Further, the oat medium is prepared according to the following steps: taking 5g of oat, adding 500-600ml of distilled water, boiling for 1h, filtering by 8 layers of gauze, taking filtrate to fix the volume to 1L, adding 15g of agar powder, uniformly stirring, and sterilizing at the high temperature of 121 ℃ for 20 minutes.
Compared with the prior art, the invention has the beneficial effects that:
1. the fungus strain obtained by separation can promote germination of the tremella melitensis seeds, the germination rate is as high as 85.24%, and the germination rate of a control group is 0 under the same time.
2. The fungus strain 65 obtained in the invention can specifically symbiotic with the tremella melitensis seeds and promote the germination of the tremella melitensis seeds.
3. The invention obtains the fungus for specifically promoting the germination of the tremella and garlic seeds for the first time, and provides a new way for efficiently cultivating seedlings by utilizing the symbiotic germination of the tremella and garlic seeds and the fungus.
Description of biological Material preservation information
No. 65, referred to herein as the fungus strain, has been deposited at China general microbiological culture Collection center, CGMCC 21937, at about 2021, 03, 29, with the deposit unit address being the national institute of microbiology, national institute of sciences, national center for sciences, 1, beijing, kogyo, postal code: 100101, a strain of the genus Tulasnella sp.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a microscopic image of strain 65 according to the present invention on four different concentrations of oat medium;
wherein FIG. 1a shows 2.5g.L of comparative example 1 of the present invention -1 Hypha map of oat culture medium;
FIG. 1b shows 5 g.L in example 1 of the present invention -1 Hypha map of oat culture medium;
FIG. 1c shows 7.5 g.L in comparative example 2 according to the invention -1 Hypha map of oat culture medium;
FIG. 1d shows 10 g.L in comparative example 3 according to the invention -1 Hypha map of oat culture medium;
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified.
Specific examples are as follows:
example 1
1. Test materials and methods
1. Test material
Sheep ear garlic plants: wild fescue plants from Shenyang Tong Ling park, shenyang, liaoning province are obtained.
Fungus strain: obtained by separation and screening.
2. Isolation of strains
Taking roots of the fescue plants, cleaning and carrying out surface disinfection, cutting the root sections into small sections with the length of about 3mm, placing the small sections in a PDA culture medium for culture at 25 ℃, observing colony growth conditions around the root sections after one week, picking up edge hyphae, transferring the edge hyphae into a new PDA culture medium for single purification culture at the temperature of 25 ℃ and then extracting DNA to identify fungus types after a culture dish with the length of 90mm is full.
3. Germination rate experiment
Experimental group (28 days): the method comprises the steps of performing three repetition, namely, respectively numbering a, b and c, respectively selecting 5 seed pods, sequentially soaking the complete seed pods in distilled water, 75% alcohol, 100% alcohol, 75% alcohol and sterile water for one minute in an ultra-clean workbench, and airing for later use.
Peeling off seed pod with sterilized toothpick, spreading seeds, mixing different seed pods, and sowing seeds in 5g.L with small medicine spoon -1 The oat medium was inoculated with the isolated and screened strain No. 65 of the present invention simultaneously in the center of the dish. The sealed culture dish is placed in a light incubator with 12h light and 12h dark for culturing at 25 ℃.
Control group (ck, 28 days): the same three repetitions are carried out, the numbers are d, e and f, and the seeds after disinfection are respectively sown in 5 g.L directly -1 The oat culture medium is not inoculated with bacteria, and other conditions are the same as those of the experimental group.
The symbiotic culture medium has the mass concentration of 5 g.L -1 Oat medium of (a): taking 5g of oat, adding 550ml of distilled water, boiling for 1h, filtering by 8 layers of gauze, taking filtrate to reach a constant volume of 1L, adding 15g of agar powder, and sterilizing at high temperature and high pressure for 20min at 121 ℃ for later use.
Nutrient rich medium (28 days) set: three repetitions were made, numbered h1, i1, j1, and the sterilized seeds were directly sown on nutrient rich medium, without inoculation, with the other conditions being the same as in example 1;
nutrient rich medium (65 days) group: three repetitions were made, numbered h2, i2, j2, and the sterilized seeds were directly sown on nutrient rich medium, without inoculation, with the other conditions being the same as in example 1;
the formula of the nutrient-rich culture medium is as follows: 1/2MS+100ml/L coconut juice.
2. Experimental results
TABLE 1 Effect of different conditions on seed germination of Caprae Seu Ovis
The seeds of the festuca sativa are tiny, the sowing quantity can not be controlled during sowing, so the seeds can only be counted after sowing, the total number of the seeds in each group in the control group and the experimental group is not equal, and the germination rate comparison is calculated.
As can be seen from the table, the germination rate of the fescue garlic seeds is up to 85.24% when the fungus strain No. 65 obtained by the invention and the fescue garlic seeds are subjected to symbiotic germination, and the germination rate of the non-inoculation bacteria is 0 under the same time;
it can also be seen from Table 1 that the seed germination rate was 0 when the seeds were given rich nutrition (nutrient rich medium), inoculated with no bacteria, and cultured for 28 days (the same days as in example 1);
the seeds are rich in nutrition (nutrient rich culture medium) and are not inoculated, and the seeds germinate when being cultured for 65 days, but the germination rate of the seeds is only 9.13%, and compared with the embodiment 1 of the invention, the germination rate is less than 1/9 as long as more than 2 times. Therefore, the fungus strain No. 65 disclosed by the invention can be used for effectively promoting the germination of the seeds of the fescue.
Comparative example 1
Comparative example 1 substantially identical to example 1 in experimental procedure, except that:
the mass concentration of the symbiotic culture medium is 2.5 g.L -1 Oat medium of (a) and (b) a plant.
The 2.5 g.L -1 The formula of the oat culture medium is as follows: 2.5g of oat is taken, 550ml of distilled water is added for boiling for 1h,8 layers of gauze is used for filtering, the filtrate is taken to be constant in volume to 1L, and 15g of agar powder is addedStirring uniformly, and sterilizing at high temperature and high pressure of 121 ℃ for 20min for later use.
Comparative example 2
Comparative example 2 substantially identical to the experimental procedure of example 1, except that:
the symbiotic culture medium is used with the mass concentration of 7.5 g.L -1 Oat medium of (a) and (b) a plant.
The 7.5 g.L -1 The formula of the oat culture medium is as follows: taking 7.5g of oat, adding 550ml of distilled water, boiling for 1h, filtering by 8 layers of gauze, taking filtrate to fix the volume to 1L, adding 15g of agar powder, uniformly stirring, and sterilizing at the high temperature of 121 ℃ for 20min for later use.
Comparative example 3
Comparative example 3 substantially identical to the experimental procedure of example 1, except that:
the mass concentration of the symbiotic culture medium is 10 g.L -1 Oat medium of (a) and (b) a plant.
The 10 g.L -1 The formula of the oat culture medium is as follows: taking 10g of oat, adding 550ml of distilled water, boiling for 1h, filtering by 8 layers of gauze, taking filtrate to reach a constant volume of 1L, adding 15g of agar powder, uniformly stirring, and sterilizing at high temperature and high pressure for 20min at 121 ℃ for later use.
Comparing the mycelium growth conditions of example 1 of the present invention with those of comparative examples 1 to 3 in the germination process of the sheep ear garlic seeds, the mycelium growth conditions of comparative example 1 are shown in fig. 1a, the mycelium growth conditions of comparative example 2 are shown in fig. 1c, the mycelium growth conditions of comparative example 3 are shown in fig. 1d, the mycelium growth conditions of example 1 are shown in fig. 1b, and the mycelium growth conditions are shown in fig. 1:
as shown in figure 1, the fungus strain 65 has regular edges, the front and back sides are grey white, hyphae are closely attached to an oat culture medium for growth, the hyphae are straight, transparent and separated, the diameter is 4.24+/-0.74 mu m, the diaphragm is obvious, the diaphragm is formed near right-angle branches, and spores are not seen; the hyphae basically overflowed and contracted, but the deformation is not obvious.
In FIG. 1a (comparative example 1), the mycelium growth rate is the slowest, 3.46mm/d,13 days full of culture dishes, the branches are the least, the mycelium density is the least, the mycelium coverage rate is about 35%, the probability of meeting mycelium with the seeds of the sheep ear garlic is smaller due to the low coverage rate, the infection rate is lower, and the seed germination rate is lower;
in FIG. 1c (comparative example 2), the hypha growth rate was moderate, 5mm/d,9 days of growth on the petri dish, more hypha branches, and about 70% hypha coverage, and higher hypha coverage wrapped the seeds, making the seeds lack oxygen and hinder germination.
In FIG. 1d (comparative example 3), the growth rate of hypha is the fastest, 6.43mm/d, the culture dish is full for 7 days, the hypha coverage rate is about 85%, the seeds are covered by the excessive hypha coverage rate, the seeds lack oxygen to prevent germination, and the pollution rate of the culture medium is high;
in FIG. 1b (example 1), the hyphal branches are clearly ordered, the hyphal coverage is about 55%, the coverage is moderate, and the hyphal growth rate is 5mm/d. Therefore, the symbiotic culture medium has the mass concentration of 5 g.L -1 The oat culture medium can ensure that the hypha of the fungus No. 65 obtained by the invention has clear and ordered branches and moderate hypha coverage rate, and the hypha can fully infect the seeds of the fescue, and can not completely wrap the seeds to cause hypoxia, so that the germination of the seeds of the fescue can be promoted.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (8)
1. The fungus strain for promoting the germination of the fescue seeds is characterized by being 65-numbered, and the preservation date of the strain is 2021, 03 and 29 days, and the preservation number of the strain is CGMCC 21937.
2. The method of claim 1, wherein the fungus strain is cultured in PDA medium at 23-27 ℃.
3. Use of fungus strain No. 65 according to claim 1 for promoting germination of seeds of fescue garlic.
4. Use of fungus strain No. 65 according to claim 3 for promoting germination of seeds of fescue garlic, wherein fungus strain No. 65 promotes germination of seeds of fescue garlic by co-cultivation with seeds of fescue garlic.
5. The use of fungus strain 65 according to claim 4 for promoting germination of seeds of fescue, wherein the medium used in the co-cultivation process is symbiotic medium.
6. The use of fungus strain 65 according to claim 5 for promoting germination of seeds of Boyle-Prinsepia utilis, wherein the symbiotic medium has a mass concentration of 5 g.L -1 Oat medium of (a) and (b) a plant.
7. A germination promoter for fescue seeds, wherein the germination promoter is prepared from the fungus strain No. 65 of claim 1.
8. The germination promoter of sheep ear garlic seeds of claim 7, wherein oat medium is added to the promoter.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048334A (en) * | 2017-12-22 | 2018-05-18 | 中国林业科学研究院林业研究所 | It is a kind of to promote the blue screening of Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system method for building up |
| CN109536391A (en) * | 2018-12-20 | 2019-03-29 | 沈阳农业大学 | A kind of fungi and its application for promoting iris protocorm Stem nematode |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048334A (en) * | 2017-12-22 | 2018-05-18 | 中国林业科学研究院林业研究所 | It is a kind of to promote the blue screening of Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system method for building up |
| CN109536391A (en) * | 2018-12-20 | 2019-03-29 | 沈阳农业大学 | A kind of fungi and its application for promoting iris protocorm Stem nematode |
Non-Patent Citations (4)
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| Chie Tsutsumi等.In vitro Seed Propagation and Conservation of the Rediscovered Rare Liparis hostifolia (Orchidaceae).Bull. Natl. Mus. Nat. Sci..2020,第46卷(第3期),111-118. * |
| Ding R等.Identity and specificity of Rhizoctonia-like fungi from different populations of Liparis japonica (Orchidaceae) in Northeast China.PLoS One.2014,第9卷(第8期),e105573:1-8. * |
| 丁锐 ; 李萌凯 ; 任光宇 ; 金宇琦 ; 张浩琪 ; 陈旭辉 ; .羊耳蒜内生真菌的分离鉴定与抑菌活性筛选.北方园艺.2017,(第01期),第126-130页. * |
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