CN116179361A - Achillea-aculeatum and application thereof in promoting growth of dendrobium candidum - Google Patents
Achillea-aculeatum and application thereof in promoting growth of dendrobium candidum Download PDFInfo
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- 229910052799 carbon Inorganic materials 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 10
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- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 description 1
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Abstract
The invention discloses a carbon horn fungus and application thereof in promoting growth of dendrobium candidum. The classification is named as the Xylobacter pinnatifidus Xylaria apiculata, the classification is that the ascomycetes are the Xylomycetes of the Ascomycota, the strain number is JNLSHCZ-3, and the Xylobacter pinnatifidus is preserved in the China general microbiological culture collection center with the preservation number of CGMCC No.18561 and the preservation date of 2019, 09 and 02 days. The strain JNLSHCZ-3 of the invention has obvious growth promoting effect on dendrobium candidum seedlings; meanwhile, the artificial culture can be realized, the propagation speed is high, the large-scale production is convenient, and the development and application prospect is good.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a carbon horn fungus and application thereof in promoting the growth of dendrobium candidum.
Background
Dendrobium officinale is perennial epiphyte of Dendrobium genus of Orchidaceae, and is mainly distributed in Zhejiang, anhui, fujian, etc. in the same river basin, and has effects of nourishing yin, moistening lung, and nourishing intestines and stomach, etc., and is a rare Chinese medicinal material recorded in Chinese pharmacopoeia. As the seeds have no endosperm, are difficult to germinate, and can complete the whole life history only by symbiotic with fungi under natural conditions. The wild dendrobium candidum resources are endangered day by day due to the long-term uncontrolled mining and ecological environment damage.
At present, although the tissue rapid propagation technology realizes the large-scale propagation of dendrobium candidum seedlings and reduces the collection pressure of wild dendrobium, the tissue culture seedlings have the problems of low transplanting survival rate, slow growth, low content of effective components and the like. Mycorrhizal fungi of dendrobium candidum can be symbiotic with dendrobium candidum in seeds, protocorms and nutrient roots, so that the germination of the seeds can be promoted, the survival rate and the growth speed of transplanting tissue culture seedlings can be improved, the stress resistance of the seedlings can be increased, mineral nutrition can be provided for plants, vitamins, hormone substances and the like can be produced, and the growth and development of the plants can be promoted.
In recent years, a great deal of researches are carried out by a plurality of scholars on the mechanism and related actions of the mycorrhizal fungi of the dendrobium plants on the growth mechanism of the dendrobium, and how to obtain mycorrhizal fungi with remarkable effects, so that the growth of the dendrobium candidum is further effectively promoted, and the development of the dendrobium candidum industry is in a direction of continuous research.
Disclosure of Invention
The invention aims to: aiming at the defects and shortcomings of the prior art, one of the purposes of the invention is to provide a mycorrhizal fungi-carbonium gracile Xylaria apiculata strain JNLSHCZ-3 with remarkable growth promoting effect in dendrobium candidum cultivation. Another object of the invention is to provide the use of the above fungus strain for promoting the growth of dendrobium candidum.
The technical scheme is as follows: the invention relates to a kind of Xylobacter oxydans, its classification is named as Xylobacter oxydans Xylaria apiculata, its classification status is ascomycetes, chaetocerales, xylodaceae, strain number is JNLSHCZ-3, it has been preserved in China general microbiological culture Collection center, preservation number is CGMCCNO.18561, and preservation date is 2019, 09, 02. The preservation address is the postal code 100101 of the institute of microbiology of national academy of sciences, national institute of sciences, 1 st, 3 rd, north chen west way, the morning of the Beijing city.
The main biological characteristics of the strain are as follows: the colonies were grown on PDA medium for 7d, 4.3cm in diameter and slower to grow. The bacterial colony is flat, the central hypha of the bacterial colony is compact, gray and black are alternately colored, the edge is irregular and is serrated, and the bacterial colony is not easy to produce spores under the artificial culture condition.
The ITS region complete sequence was amplified and sequenced, and the PCR amplified 16sRNA complete sequence (SEQ ID NO. 1) TTTAGAAATTAGGGGGTTTTACGGCAAGGGACCGGCTCATCTATAGGCGAGATAAAGTTTACTACGTCTAGAGTGTGAAACCGACTCCGCCACTAACTTTAAGGAACTACAGAGGGTATCTGTAGGCTCCCAACGCTAAGCAACAGGGCTTAAGGGTTGAAATGACGCTCGAACAGGCATGCCCACTAGAATACTAATGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTAACTTATTTAGTTATATTATTCAGAATGACATAGTAAACAGAGTTTAGTAGACCACCGGCAGGTTATGCCCGCAACTACCGGGTAAGGTACCTACAGGGTAGGTCCCCTCAGGGTAAGTTGCAGACCTGCCGAGGCAACAAAGGTAGGTTTCACATGGGTTTAGGAGTTGTTTTTAACTCTTTAATGATCCCTCCGCAGGTTCACCTACGG.
The invention discloses application of a carbon horn fungus on promotion of growth of dendrobium candidum.
The application comprises the steps of preparing the carbon horn fungus on the tip into a liquid microbial inoculum, adding the liquid microbial inoculum into a dendrobium candidum culture medium, and maintaining the relative water content of the medium at 60% -80% for 5-6 days so as to ensure the survival of fungi in the medium.
When the liquid microbial inoculum is applied, the inoculation amount of the liquid microbial inoculum for each cube of dendrobium candidum culture medium is 30-50 g.
When the liquid microbial inoculum is prepared, wheat bran is used as a culture medium, ammonium sulfate is used as an exogenous nitrogen source, and glucose is used as an exogenous carbon source to prepare a liquid culture medium; inoculating the Xylopsis pinicola after sterilization, performing liquid shake culture, and breaking the fermentation liquor containing mycelium pellet into uniform slurry bacterial agent after the culture is finished.
The liquid shaking culture conditions are 25-30 ℃, 10-12 h of illumination/12-14 h of darkness, pH of 5.5-6.5, 100-150 r/min shaking culture for 15-25 d.
The mass concentration of ammonium sulfate and glucose in the liquid culture medium is respectively 4.0 g/L-7.0 g/L and 10.0 g/L-20.0 g/L.
The liquid culture medium is prepared by adding wheat bran in the form of wheat bran juice, adding 300ml distilled water into 30-50 g wheat bran, boiling for 30min, and filtering to obtain wheat bran juice
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: through detection, the strain JSLSHCZ-3 of the invention has obvious growth promoting effect on dendrobium candidum seedlings, and can remarkably improve the chlorophyll content of the dendrobium candidum seedlings after application and promote the growth of the dendrobium candidum seedlings. Meanwhile, the strain can be artificially cultured, has high propagation speed, simple culture conditions, convenient application, easy preservation and easy mass production, and has good development and application prospects.
Drawings
FIG. 1 is a front morphology of a colony cultivated by a strain of the present invention;
FIG. 2 is a rear morphology of colonies cultivated by the strain of the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and the specific embodiments.
Example 1: isolation and identification of strains
The strain JNLSHCZ-3 is obtained by separating root systems of dendrobium moniliforme in dendrobium cultivation areas in Jiangsu nongbo garden in period and city of Jiangsu province in 2017.
The separation method comprises the following steps: taking fresh dendrobium moniliforme root, washing the substrate on the surface of the root section with flowing tap water, sucking the water on the surface with filter paper in an ultra-clean workbench, immersing in 75% alcohol 45s for several times, and immersing in 0.1% secondary mercury solution for disinfection for 5min. After the disinfection is completed, the disinfection is thoroughly removed by washing 3-4 times with sterilized deionized water, residual water drops on the root surface are sucked by sterilized filter paper, and then the root section is cut into tissue sections with the thickness of about 0.5-1 mm by a sterilized scalpel. The tissue sections were transferred to PDA medium plates with streptomycin sulfate (50-100. Mu.g/m L), each plate was inoculated with 5 tissue sections, and each medium was inoculated with 15 plates. The endophytic fungi obtained through separation are subjected to tieback test to obtain the strain, the strain is named as JNLSHCZ-3, the strain can obviously promote the growth of dendrobium seedlings, and the survival rate of the seedlings is improved.
The colony characteristics of strain JSNLSHCZ-3 are as follows: the colonies were grown on PDA medium for 7d, 4.3cm in diameter and slower to grow. The bacterial colony is flat, the central hypha of the bacterial colony is compact, gray and black are alternately colored, the edge is irregular and is serrated, and the bacterial colony is not easy to produce spores under the artificial culture condition.
The ITS region complete sequence was amplified and sequenced, and the PCR amplified 16s RNA complete sequence was as follows. The complete sequence obtained by PCR amplification (SEQ ID NO. 1):
TTTAGAAATTAGGGGGTTTTACGGCAAGGGACCGGCTCATCTATAGGCGAGATAAA
GTTTACTACGTCTAGAGTGTGAAACCGACTCCGCCACTAACTTTAAGGAACTACAG
AGGGTATCTGTAGGCTCCCAACGCTAAGCAACAGGGCTTAAGGGTTGAAATGACG
CTCGAACAGGCATGCCCACTAGAATACTAATGGGCGCAATGTGCGTTCAAAGATTC
GATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCAT
CGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTAACTTATTTAGTTATATTAT
TCAGAATGACATAGTAAACAGAGTTTAGTAGACCACCGGCAGGTTATGCCCGCAAC
TACCGGGTAAGGTACCTACAGGGTAGGTCCCCTCAGGGTAAGTTGCAGACCTGCC
GAGGCAACAAAGGTAGGTTTCACATGGGTTTAGGAGTTGTTTTTAACTCTTTAATGATCCCTCCGCAGGTTCACCTACGG。
morphological and molecular biological identification shows that the strain is a Xylobacter oxydans (Xylaria apiculata), the separation position of the strain is Xylobacter oxydans genus of Xylomycetes order Ascomycota, the strain number is JNLSHCZ-3, the strain is preserved in China general microbiological culture Collection center (CGMCC) No.18561, and the preservation date is 2019, 09 and 02. The preservation address is the postal code 100101 of the institute of microbiology of national academy of sciences, national institute of sciences, 1 st, 3 rd, north chen west way, the morning of the Beijing city.
Example 2: analysis of growth promoting effect of Tamarindus (Xylaria apiculata) JNLSHCZ-3 on dendrobium candidum
1) The liquid culture medium is prepared by taking wheat bran as a culture medium, ammonium sulfate as an exogenous carbon source and glucose as an exogenous carbon source. Adding 40g of wheat bran into 300ml of distilled water, boiling for 30min, filtering to obtain wheat bran juice, adding 5g of ammonium sulfate and 15g of glucose into distilled water, fixing the volume to 1L, adjusting the pH to 5.8, sterilizing for 20 min under the high pressure condition of 121 ℃ and 101.3 kilopascals, cooling a culture medium, inoculating the mycorrhizal fungi JSLSHCZ-3 (Xylaria apiculata) under the sterile condition, carrying out shaking culture for 20d under the dark condition of 28 ℃, 120h/min and 12h light, breaking a fermentation liquor containing mycelium pellets into a uniform slurry microbial inoculum after the culture is finished, mixing the microbial inoculum into a dendrobium candidum culture medium, and maintaining the relative water content of the medium to be above 60% -80% for 5 days so as to ensure the survival of the fungi in the medium. The inoculation amount of the liquid microbial inoculum inoculated to each dendrobium candidum culture medium is 30-50 g.
2) Selecting dendrobium candidum tissue culture seedlings as a cultivation material, cleaning and airing the dendrobium candidum seedlings until the roots turn white, weighing, planting the dendrobium candidum seedlings in the inoculation cultivation substrate according to the planting density of 5 plants/cluster and 30 clusters/square meter, and setting an un-inoculated substrate as an un-control (ck).
3) And (3) carrying out normal water and fertilizer management, after 60d cultivation, taking out dendrobium candidum seedlings by adopting multipoint random sampling, and carrying out the analysis of the fruits of the dendrobium candidum.
The pro-effect analysis includes:
1. and (3) heavy increment measurement: cleaning the culture medium on the root and tuber, air-drying under natural condition, weighing fresh weight of dendrobium candidum seedling with analytical balance (0.0001 g), subtracting the weight of dendrobium candidum seedling before transplanting to obtain net weight increment.
2. Chlorophyll a, chlorophyll b and chlorophyll a+b content: weighing about 0.5g of healthy dendrobium candidum leaves, cutting, adding a mixed leaching solution of 25m L absolute ethyl alcohol and acetone (volume ratio is 1:1), and leaching for 24 hours in a dark place. After 24h, the extract was centrifuged and the absorbance A645 and A663 of each supernatant was measured at 645nm and 663nm wavelengths using an ultraviolet-visible spectrophotometer.
The measured data were used to calculate chlorophyll a, chlorophyll b and total chlorophyll content, respectively, according to the following formula:
Ca(mg/g)＝(12.71A663－2.59A645)*V/1000m；
Cb(mg/g)＝(22.88A645－4.67A663)*V/1000m；
Ca+b(mg/g)＝(8.04A663+20.29A645)*V/1000m；
in the above formula: v represents the final volume (mL) of the mixed leaching solution of the absolute ethyl alcohol and the acetone in the volume ratio of 1:1, and m represents the fresh weight (g) of the dendrobium candidum leaves.
The analysis of the dendrobium candidum tissue culture seedling promoter effect by the Acremonium acutum (Xylaria apiculata) JNLSHCZ-3 is as follows:
TABLE 1 influence of JNLSHCZ-3 on Dendrobium officinale growth
Through detection, the strain JSLSHCZ-3 of the invention has obvious growth promoting effect on dendrobium candidum seedlings, and can remarkably improve the chlorophyll content of the dendrobium candidum seedlings after application and promote the growth of the dendrobium candidum seedlings. Meanwhile, the strain can be artificially cultured, has high propagation speed, simple culture conditions, convenient application, easy preservation and easy mass production, and has good development and application prospects.
Claims (8)
1. The Xylaria pinnatifida is classified and named as Xylaria pinnatifida Xylaria apiculata, has a strain number of JNLSHCZ-3 and is preserved in China general microbiological culture Collection center (CGMCC) No.18561, and has a preservation date of 2019, 09 and 02.
2. The use of the carbon horn fungus of claim 1 in promoting the growth of dendrobium candidum.
3. The use according to claim 2, characterized in that: the preparation method comprises the steps of preparing the carbon horn bacteria on the tip into a liquid microbial inoculum, adding the liquid microbial inoculum into a dendrobium candidum culture medium, and maintaining the relative water content of the medium at 60% -80% for 5-6 days so as to ensure the survival of fungi in the medium.
4. A use according to claim 3, characterized in that: the inoculation amount of the dendrobium candidum culture medium inoculated liquid microbial inoculum per cube is 30 g-50 g.
5. The use according to claim 2, characterized in that: when the liquid microbial inoculum is prepared, wheat bran is used as a culture medium, ammonium sulfate is used as an exogenous nitrogen source, and glucose is used as an exogenous carbon source to prepare a liquid culture medium; inoculating the Xylopsis pinicola after sterilization, performing liquid shake culture, and breaking the fermentation liquor containing mycelium pellet into uniform slurry bacterial agent after the culture is finished.
6. The use according to claim 5, characterized in that: the liquid shaking culture conditions are 25-30 ℃, 10-12 h of illumination/12-14 h of darkness, pH of 5.5-6.5, 100-150 r/min shaking culture for 15-25 d.
7. The use according to claim 5, characterized in that: the mass concentration of ammonium sulfate and glucose in the liquid culture medium is respectively 4.0 g/L-7.0 g/L and 10.0 g/L-20.0 g/L.
8. The use according to claim 7, characterized in that: the wheat bran is added into the liquid culture medium in the form of wheat bran juice, 30 g-50 g of wheat bran is taken when the wheat bran juice is prepared, 300ml of distilled water is added, and the wheat bran juice is obtained after boiling for 30 min.
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| CN202211488709.1A CN116179361A (en) | 2022-11-25 | 2022-11-25 | Achillea-aculeatum and application thereof in promoting growth of dendrobium candidum |
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| CN202211488709.1A CN116179361A (en) | 2022-11-25 | 2022-11-25 | Achillea-aculeatum and application thereof in promoting growth of dendrobium candidum |
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