CN111676144B - Fusarium fungus, cultivation method and application thereof, and cultivation method of achyranthes bidentata - Google Patents
Fusarium fungus, cultivation method and application thereof, and cultivation method of achyranthes bidentata Download PDFInfo
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Abstract
The invention provides Fusarium fungus, which is classified and named Fusarium sp.C 53 (Fusarium sp.) and is preserved in China general microbiological culture collection center (CGMCC) in 1 month and 13 days in 2020 year with the preservation number of CGMCC No.19364. The invention also provides application of the fusarium fungus in achyranthes cultivation. The cultivation method of achyranthes bidentata comprises the following steps: taking achyranthes bidentata Blume seedlings 25-30 days after sowing, respectively applying 2.0-3.5g of fungus material cultivated by the cultivation method of the fusarium fungus for improving the yield of achyranthes bidentata Blume and the content of polysaccharide components to the roots of each achyranthes bidentata Blume seedling, covering the fungus material with soil, and planting the fungus material in a greenhouse or a field for 3-5 months to harvest. After 3-5 months of symbiotic cultivation with achyranthes bidentata seedling, it can increase the yield of achyranthes bidentata and the content of polysaccharides.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to fusarium fungi, a cultivation method and application thereof, and a cultivation method of achyranthes bidentata.
Background
Achyranthes bidentata (Achyranthus bidentata Blume.) is a deep-root herbaceous plant of Achyranthes in Amaranthaceae (Achyranthus), grows in hillside forest with an altitude of 200-1750 m, is mainly produced in Wu \38495inthe Jordan province, qinyang, mengzhou, boai, xiu and Wen county (the district of ancient Huaqing Fu), has one of famous local medicinal materials in China, and is named because the stem of the Achyranthes bidentata is in a section and similar to the shape of the Achyranthes bidentata. Achyranthes bidentata has a long history of use, and the earliest pharmaceutical work in China listed as the top grade achyranthes bidentata in Shen nong Ben Cao Jing, which can lighten the body and prolong the life after long-term use. The Chinese pharmacopoeia records that the characters are bitter, sweet, sour and neutral; the functions of 'removing blood stasis and stimulating the menstrual flow, nourishing liver and kidney, strengthening bones and muscles, inducing diuresis and treating stranguria, and leading blood to descend'; can be used for treating amenorrhea, dysmenorrhea, soreness of waist and knees, myasthenia of bones and muscles, stranguria, edema, headache, and vertigo. Radix Achyranthis bidentatae contains various components such as sterone, saponin, polysaccharide, flavone, alkaloid, etc., wherein the polysaccharide has the effects of resisting aging, resisting tumor, improving immunity, etc.
The yield of achyranthes bidentata and the content of polysaccharide components in achyranthes bidentata are important value indexes for measuring achyranthes bidentata, but the yield of achyranthes bidentata and the content of polysaccharide components in achyranthes bidentata are low by adopting the prior art.
Disclosure of Invention
The invention aims to provide fusarium fungus, a cultivation method and application thereof and a cultivation method of achyranthes bidentata.
The technical scheme adopted by the invention is as follows:
on one hand, the disclosure provides Fusarium fungus which is classified and named as Fusarium sp, the strain name is C53 (Fusarium sp.), the Fusarium sp is preserved in China general microbiological culture collection center of China general microbiological culture Collection management Committee in 1 month and 13 days in 2020, the address is No. 3 North road No.1 of West Chen of the sunward area in Beijing, and the preservation number is CGMCC No.19364; .
A cultivation method of the fusarium fungus is characterized in that the fusarium fungus is added into a solid culture medium for cultivation; the components of the solid culture medium comprise 1 part of wheat bran, 4 parts of buckwheat hull and 0.05-0.1 part of corn flour in parts by volume, and the solution A is added and stirred uniformly until water can seep out by holding a hand, so as to obtain a fungus material; the preparation method of the solution A comprises the following steps: adding water to 10 parts by mass of sucrose and 10 parts by mass of gypsum to a volume of 1 part by volume to obtain a solution A, wherein 1 part by mass to 1 part by volume =1g to 1L.
The invention also provides application of the fusarium fungus in achyranthes cultivation.
On the other hand, the invention also provides a cultivation method of achyranthes bidentata, which comprises the following steps: taking Achyranthis radix seedling 25-30 days after sowing, applying 2.0-3.5g of fungus material cultured by the above Fusarium fungus culture method to the root of each Achyranthis radix seedling, covering with soil, and planting in greenhouse or field for 3-5 months.
The invention has the beneficial effects that:
the strain C53 of the fusarium fungus is an endophytic fungus of wild achyranthes bidentata, and can improve the yield of achyranthes bidentata medicinal materials and the content of polysaccharide components compared with the achyranthes bidentata which is not inoculated with bacteria under the same cultivation conditions after 3-5 months of symbiotic cultivation with seedlings of achyranthes bidentata.
Detailed Description
The present invention is further illustrated below with reference to specific examples.
The first embodiment is as follows:
the embodiment provides Fusarium fungus, which is classified and named Fusarium sp, has a strain name of C53, is preserved in the common microorganism center of the china committee for culture collection and management of microorganisms in 1 month and 13 months in 2020, and has a preservation number of CGMCC No.19364.
The separation method of the strain C53 comprises the following steps:
adopting a tissue mass separation method: randomly selecting 80 tissue blocks from the root of wild Huai cattle knee collected from Qinyang in Henan, washing the tissue blocks under running water, drying the tissue blocks, cutting the tissue blocks into tissue blocks with the size of 0.5cm, respectively washing the tissue blocks in 75% ethanol for 30-60 s under aseptic condition, then soaking the tissue blocks in 3% hypochlorous acid solution for 8-12 min, washing the tissue blocks in aseptic water for 4 times, and finally sucking the water by aseptic filter paper. The surface sterilized samples were then cut into tissue pieces 0.2cm x 0.2cm in size under sterile conditions for isolation of wild achyranthes bidentata endophytic fungi. Tissue blocks were placed on PDA medium and menglar red medium, and 4 tissue blocks were inoculated per plate. Culturing the sample at a constant temperature of 25 ℃ for two months, periodically observing the colony formation condition of endophytic fungi, when the diameter of a filamentous fungi colony is about 1cm, selecting the edge part of the colony, inoculating the colony to a new PDA (personal digital assistant) plate, and repeatedly inoculating to form a single colony; according to the colony characteristics of the strains, merging the strains to obtain fungal strains with different colony characteristics, numbering, counting and describing the fungal strains, and identifying the fungal strains by using morphological and molecular biological methods. The bacterial strain is preserved in a PDA test tube inclined plane and properly preserved in a 10% glycerol tube; the PDA test tube is stored in a refrigerator at 4 deg.C, and 10% glycerol tube is stored in an ultra-low temperature refrigerator at-80 deg.C.
Wherein, the separation culture medium is as follows:
potato medium (PDA) formula (1L): 200g of peeled potatoes, 20g of glucose, 12g of agar and deionized water with the volume of 1000mL, and the pH is natural. Sterilizing at 122 deg.C for 22min.
The formula (1L) of a Bengal culture medium comprises: 35g of Bengal red, adding deionized water to 1000mL, and naturally adjusting the pH value. Sterilizing at 122 deg.C for 22min.
The result of conservative sequence sequencing (SEQ ID No. 1) of the strain C53 is as follows:
ttggcctgcggagggatcattaccgagtttacaactcccaaacccctgtgaacataccaattgttgcctcggcggatcagcccgctcccggtaaaacgggacggcccgccagaggacccctaaactctgtttctatatgtaacttctgagtaaaaccataaataaatcaaaactttcaacaacggatctcttggttctggcatcgatgaagaacgcagcaaaatgcgataagtaatgtgaattgcagaattcagtgaatcatcgaatctttgaacgcacattgcgcccgccagtattctggcgggcatgcctgttcgagcgtcatttcaaccctcaagcccagcttggtgttgggactcgcgagtcaaatcgcgttccccaaattgattggcggtcacgtcgagcttccatagcgtagtagtaaaaccctcgttactggtaatcgtcgcggccacgccgttaaaccccaacttctgaatgttgacctcggatcaggtaggaatacccgctgaacttaagcatatcaaaagcccggaggaaaa
example two:
a method for culturing fusarium fungus comprises adding the fusarium fungus of the embodiment I to a solid culture medium for culturing; the components of the solid culture medium comprise 1 part of wheat bran, 4 parts of buckwheat hull and 0.05-0.1 part of corn flour by volume part, and the solution A is added and stirred uniformly until water can seep out by holding with hands, so as to obtain the fungus material; the preparation method of the solution A comprises the following steps: adding water to 10 parts by mass of sucrose and 10 parts by mass of gypsum to a volume of 1 part by volume to obtain a solution A, wherein 1 part by mass to 1 part by volume =1g to 1L. As a specific mode, the components of the culture medium of the solid culture comprise the following components in volume: wheat bran-buckwheat husk culture medium: wheat bran: buckwheat husk = 1: 4 (V: V), fine corn flour 0.05V-0.1V, and solution A is stirred uniformly, and water just seeps out when holding by hand. Solution a formulation (1L): sucrose 10g, gypsum Fibrosum (CaSO) 4 ·2H 2 O) 10g, adding water to a constant volume of 1L. Weighing the above materials, stirring, packaging, sterilizing at 122 deg.C for 180min, and cooling. By diameter 0A8 cm puncher takes a C53 (Fusarium sp.) fungus slice cultured on the PDA culture medium, the fungus slice is placed in the middle of the solid culture medium, and the fungus is cultured in the dark at 25 ℃ until the C53 (Fusarium sp.) fungus grows to 2/3 of the culture medium in a bottle for use.
Example three:
the invention also provides an application of the fusarium fungus in achyranthes cultivation.
Example four:
the invention also provides a cultivation method of achyranthes bidentata, which comprises the following steps: taking achyranthes bidentata seedlings 25-30 days after sowing, respectively applying 2.0-3.5g of fungus material cultivated by the cultivation method of the fusarium fungus in the embodiment to the roots of each achyranthes bidentata seedling, covering the achyranthes bidentata seedling with soil, and planting the achyranthes bidentata seedling in a greenhouse or a field for 3-5 months to harvest. The method comprises the following specific steps:
(1) Solid culture of C5 (Fusarium sp.) 3 fungi: the components of the culture medium for solid culture are as follows according to the volume: wheat bran-buckwheat husk culture medium: wheat bran: buckwheat husk = 1: 4 (V: V), fine corn flour 0.05V-0.1V, and solution A is stirred uniformly, and water just seeps out when holding by hand. Solution a formulation (1L): sucrose (10 g), gypsum Fibrosum (CaSO) 4 ·2H 2 O) 10g, adding water to a constant volume of 1L. Weighing the above materials, stirring, packaging, sterilizing at 122 deg.C for 180min, and cooling. A C53 (Fusarium sp.) fungal plate cultured on PDA medium was taken out with a 0.8cm diameter punch, placed in the middle of the solid medium, and cultured in the dark at 25 ℃ until the C53 (Fusarium sp.) fungal grows to 2/3 of the medium in the flask for use.
(2) And (3) symbiotic cultivation of the bacterins: sowing seeds of achyranthes bidentata in 6 months, taking achyranthes bidentata seedlings 25-30 days after germination, respectively applying 2.5-3.0g of C53 (Fusarium sp.) fungus material for solid culture to the roots of each achyranthes bidentata seedling, covering with soil, planting in a greenhouse or a field, and performing planting management by a conventional method.
(3) The strain C53 (Fusarium sp.) is subjected to symbiotic cultivation with achyranthes bidentata seedlings for 3-4 months, and the dry weight and polysaccharide content of achyranthes bidentata are improved to different degrees compared with those of achyranthes bidentata which are not inoculated under the same cultivation conditions.
The disclosure is further illustrated below with specific tests:
test one:
the cultivation method of achyranthes bidentata comprises the following steps:
c53 (Fusarium sp.) solid culture of fungi: wheat bran-buckwheat husk culture medium: wheat bran: buckwheat husk = 1: 4 (V: V), fine corn flour 0.05V is added, the solution A is stirred uniformly, and most preferably, water just seeps out when the solution A is held by hand. Solution a formulation (1L): sucrose (10 g), gypsum Fibrosum (CaSO) 4 ·2H 2 O) 10g, adding water to a constant volume of 1L. Weighing the above materials, stirring, packaging, sterilizing at 122 deg.C for 180min, and cooling. A C53 (Fusarium sp.) fungal plate cultured on a PDA culture medium is taken by a puncher with the diameter of 0.8cm, placed in the middle of a solid culture medium, and cultured in the dark at 25 ℃ until the C53 fungal grows to 2/3 of the culture medium in a bottle for use.
And (3) symbiotic cultivation of the bacterins: sowing achyranthes bidentata seeds in 6 months by using a greenhouse potting method, and taking achyranthes bidentata seedlings 25-30 days after germination; the achyranthes bidentata Blume seedling is divided into two groups, one group is given with 2.5g of solid culture C53 (Fusarium sp.) fungal material at the root of each achyranthes bidentata seedling, the other group is given with no solid culture C53 (Fusarium sp.) fungal material, each pot is provided with 1 strain, each group has 6 pots, the repeat is arranged for 6 times, the achyranthes bidentata Blume seedlings are covered with soil and planted in a greenhouse, the cultivation management is carried out by a conventional method, and the achyranthes bidentata Blume seedlings are harvested after 3 months.
The dry weight was weighed and the polysaccharide content was determined. Achyranthes bidentata cultivated symbiotically with C53 (Fusarium sp.) fungus, the dry weight of each root was 12.6g on average, and the polysaccharide content of the root was 11.00% on average. The non-inoculated achyranthes bidentata has an average dry weight of 8.2g per root and an average root polysaccharide content of 8.60%.
And (2) testing II:
the cultivation method of achyranthes bidentata comprises the following steps:
c53 (Fusarium sp.) solid culture of fungus, testa Tritici-testa Fagopyri Esculenti culture medium, testa Tritici: testa Fagopyri Esculenti = 1: 4 (V: V), adding fine grain semen Maydis powder 0.75V, stirring with solution A, and holding with hand to allow water to just seep out. Solution a formulation (1L): sucrose (10 g), gypsum Fibrosum (CaSO) 4 ·2H 2 O) 10g, adding water to a constant volume of 1L. Weighing the above materials, stirring, packaging, sterilizing at 122 deg.C for 180 deg.Cmin, cooling for use. A C53 (Fusarium sp.) fungal plate cultured on PDA medium was taken out with a 0.8cm diameter punch, placed in the middle of the solid medium, and cultured in the dark at 25 ℃ until the C53 (Fusarium sp.) fungal grows to 2/3 of the medium in the flask for use.
And (3) symbiotic cultivation of the bacterins: the method comprises the steps of planting achyranthes bidentata through a greenhouse, sowing achyranthes bidentata seeds in 6 months, taking achyranthes bidentata seedlings 25-30 days after germination, dividing the achyranthes bidentata seedlings into two groups, applying C53 (Fusarium sp.) fungus material for solid culture to the roots of the achyranthes bidentata seedlings in one group for 2.75g, applying no C53 (Fusarium sp.) fungus material to the roots of the achyranthes bidentata seedlings in the other group for 1 plant in each pot, setting 6 pots in each group, repeating the steps for 6 times, covering the pots with soil, planting the seedlings in the greenhouse, carrying out cultivation management through a conventional method, and harvesting the seedlings after 4 months.
The dry weight was weighed and the polysaccharide content was determined. Achyranthes bidentata cultivated symbiotically with C53 (Fusarium sp.) fungus, the average dry weight of each root is 15.2g, and the average polysaccharide content of the root is 13.00%. The average dry weight of each root of the non-inoculated achyranthes bidentata is 10.0g, and the average polysaccharide content of the roots is 9.00%.
And (3) test III:
the cultivation method of achyranthes bidentata comprises the following steps:
c53 (Fusarium sp.) solid culture of fungus, testa Tritici-testa Fagopyri Esculenti culture medium, testa Tritici: testa Fagopyri Esculenti = 1: 4 (V: V), adding fine corn flour 0.1V, stirring with solution A, and holding with hand to allow water to permeate out. Solution a formulation (1L): sucrose 10g, gypsum Fibrosum (CaSO) 4 ·2H 2 O) 10g, adding water to a constant volume of 1L. Weighing the above materials, stirring, packaging, sterilizing at 122 deg.C for 180min, and cooling. A C53 (Fusarium sp.) fungal plate cultured on PDA medium was taken out with a 0.8cm diameter punch, placed in the middle of the solid medium, and cultured in the dark at 25 ℃ until the C53 (Fusarium sp.) fungal grows to 2/3 of the medium in the flask for use.
And (3) symbiotic cultivation of the bacterins: the method comprises the steps of planting achyranthes bidentata seeds in a greenhouse in 6 months, taking achyranthes bidentata seedlings 25-30 days after germination, dividing the achyranthes bidentata seedlings into two groups, applying 3.0g of C53 (Fusarium sp.) fungus material for solid culture to the roots of the achyranthes bidentata seedlings in one group, applying no C53 (Fusarium sp.) fungus material in the other group, applying 1 strain to each pot, setting 6 pots in each group, repeating the steps for 6 times, covering the pots with soil, planting the pots in the greenhouse, performing cultivation management by a conventional method, and harvesting the achyranthes bidentata seedlings after 5 months.
The dry weight was weighed and the polysaccharide content was determined. Achyranthes bidentata cultivated symbiotically with C53 (Fusarium sp.) fungus, the dry weight of each root averages 16.8g, and the polysaccharide content of the root averages 15.00%. The average dry weight of each root of the non-inoculated achyranthes bidentata is 11.0g, and the average polysaccharide content of the roots is 10.00%.
In conclusion, after the achyranthes bidentata are symbiotically cultivated with the achyranthes bidentata seedlings for 3-5 months, the yield of the achyranthes bidentata medicinal material and the content of polysaccharide components can be obviously improved compared with the achyranthes bidentata which is not inoculated with bacteria under the same cultivation condition.
The present invention is not limited to the above alternative embodiments, and other various forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the claims, and which the description is intended to be interpreted accordingly.
Sequence listing
<110> school of schchang
<120> fusarium fungus, cultivation method, application and cultivation method of achyranthes bidentata
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<211> 542
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ttggcctgcg gagggatcat taccgagttt acaactccca aacccctgtg aacataccaa 60
ttgttgcctc ggcggatcag cccgctcccg gtaaaacggg acggcccgcc agaggacccc 120
taaactctgt ttctatatgt aacttctgag taaaaccata aataaatcaa aactttcaac 180
aacggatctc ttggttctgg catcgatgaa gaacgcagca aaatgcgata agtaatgtga 240
attgcagaat tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc cagtattctg 300
gcgggcatgc ctgttcgagc gtcatttcaa ccctcaagcc cagcttggtg ttgggactcg 360
cgagtcaaat cgcgttcccc aaattgattg gcggtcacgt cgagcttcca tagcgtagta 420
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ttgacctcgg atcaggtagg aatacccgct gaacttaagc atatcaaaag cccggaggaa 540
aa 542
Claims (4)
1. A fusarium fungus characterized by: classified and named as Fusarium (Fusarium sp.) The strain is named as C53 and is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 13 months in 2020, and the preservation number is CGMCC No.19364.
2. A method of cultivating the fungus of the genus Fusarium of claim 1, wherein the fungus of the genus Fusarium is cultured by adding it to a solid medium; the components of the solid culture medium consist of 1 part of wheat bran, 4 parts of buckwheat hull and 0.05-0.1 part of corn flour in parts by volume, and the solution A is added and stirred uniformly until water can seep out by holding the hand, so as to obtain fungus material; the preparation method of the solution A comprises the following steps: adding water into 10 parts by mass of sucrose and 10 parts by mass of gypsum to a constant volume of 1 part by volume to obtain a solution A, wherein 1 part by mass: 1 part by volume =1g:1L of the compound.
3. Use of the fusarium fungus of claim 1 in achyranthes bidentata cultivation.
4. A cultivation method of achyranthes bidentata is characterized by comprising the following steps: taking achyranthes bidentata Blume seedlings 25-30 days after sowing, respectively applying 2.0-3.5g of fungus material cultivated by the cultivation method containing the fusarium fungus according to claim 2 to roots of each achyranthes bidentata Blume seedling, covering with soil, and planting in a greenhouse or a field for 3-5 months before harvesting.
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| 不同诱导子对怀牛膝细胞生长及多糖含量的影响;李萍等;《植物学报》;20171231;第52卷(第05期);第615-621页 * |
| 不同连作年限怀牛膝根际土壤理化性质及微生物多样性;王娟英等;《生态学报》;20170908;第37卷(第17期);第5621-5629页 * |
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