CN116731866A - Pediopside parvulum for promoting dendrobium candidum growth and application thereof - Google Patents
Pediopside parvulum for promoting dendrobium candidum growth and application thereof Download PDFInfo
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- 241000026010 Dendrobium candidum Species 0.000 title claims abstract description 44
- 230000001737 promoting effect Effects 0.000 title claims abstract description 17
- 241001674041 Pestalotiopsis microspora Species 0.000 claims abstract description 12
- 241000223261 Trichoderma viride Species 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 230000000813 microbial effect Effects 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000011081 inoculation Methods 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 241001076416 Dendrobium tosaense Species 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 5
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- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
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- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
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- 241000233866 Fungi Species 0.000 description 4
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- NSMUHPMZFPKNMZ-VBYMZDBQSA-M chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 description 2
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- 241001523681 Dendrobium Species 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
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Abstract
The invention discloses a trichoderma viride for promoting dendrobium candidum to grow and application thereof. The classification is named as Pelaralospora microphylla Pestalotiopsis microspora, the classification is named as Pelaralospora microphylla of Pelaralosporales (family) of Pelaralosporales of Deuteromycotina, the strain number is JNLSHGS-13, and the strain is preserved in China general microbiological culture Collection center (CGMCC) No.18543, and the preservation date is 2019, 09 and 02. The trichoderma microsporidianum Pestalotiopsis microspora strain JNLSHGS-13 has obvious growth promoting effect on dendrobium candidum seedlings and has a certain effect on reducing the application of chemical fertilizers; meanwhile, the artificial culture can be realized, 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 trichoderma viride for promoting dendrobium candidum growth and application thereof.
Background
At present, the market demand of dendrobium candidum mainly depends on artificial cultivation, but the growth rate of the dendrobium candidum planted by the artificial cultivation is slow, the phenomenon of chemical and pesticide abuse is serious for pursuing the yield, so that the dendrobium candidum is low in quality, high in pesticide residue and easy to occur in food safety event, based on the dendrobium candidum, scientific researchers gradually shift the research direction of fertilizer application in the dendrobium candidum production and cultivation process to microbial resources, the dendrobium candidum can complete the whole life history only by symbiotic with fungi, and the dendrobium candidum is numerous in nature, and the mycorrhizal fungi resources are also rich, so that abundant microbial resources for research are provided for vast scientific researchers. How to screen out mycorrhizal fungus agents with remarkable effects and further promote the growth of dendrobium candidum becomes an important direction of research and development of the dendrobium candidum industry.
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 mycorrhizal fungi-trichoderma viride Pestalotiopsis microspora with remarkable growth promotion effect in dendrobium candidum cultivation, and the strain number is JSLSHGS-13. Another object of the invention is to provide the use of the strain described above for promoting the growth of dendrobium candidum.
The technical scheme is as follows: the invention relates to a pseudodisc-like hirsutella microsporum for promoting dendrobium candidum growth, which is classified as pseudodisc-like hirsutella microsporum Pestalotiopsis microspora, the classification status of which is the pseudodisc-like hirsutella genus of the order of the family of the Uighurales of the phylum Heterosporida, the strain number is JNLSHGS-13, which is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 18543 and a preservation date of 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.
The main biological characteristics of the strain are as follows: the colonies were grown on PDA medium for 7d, with a colony diameter of 5.4cm and slower growth. The colony is flat, the surface is white and flocculent, the back is provided with annular pigment sediment, the colony grows in a wheel shape, the edge is irregular, the mycelium is undeveloped, and the growth speed is low.
The ITS region complete sequence was amplified and sequenced, and the PCR amplified 16sRNA complete sequence (SEQ ID NO. 1) GTGACTTACCTTTTGTTGCCTCGGCAGAAGTTATAGGTCTTCTTATAGCTGCTGCCGGCGGACCATTAAACTCTTGTTATTTTATGTAATCTGAGCGTCTTATTTTAATAAGTCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCTTAAGCCTAGCTTAGTGTTGGGAATCTACTTCTCTTAGGAGTTGTAGTTCCTGAAATACAACGGCGGATTTGTAGTATCCTCTGAGCGTAGTAATTTTTTTCTCGCTTTTGTTAGGTGCTATAACTCCCAGCCGCTAAACCCCCAATTTTTTGTGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGA.
The invention relates to an application of trichoderma viride for promoting dendrobium candidum growth.
The microbial agent produced by the strain JNLSHGS-13.
The preparation method of the microbial agent produced by the strain JNLSHGS-13 comprises the following steps: comprising the following steps:
1) Taking corn flour as a culture medium, ammonium sulfate as an exogenous nitrogen source and glucose as an exogenous carbon source to prepare a solid culture medium; the pH is adjusted to 5.5-6.5, and the strain JNLSHGS-13 is inoculated after sterilization;
2) Culturing for 20-30 d under the conditions of 25-30 ℃ and 12h illumination/12 h dark culture;
3) And (3) crushing the solid microbial agent cultured in the step (2) into powder to obtain the microbial agent.
The mass ratio of corn flour to ammonium sulfate to glucose in the step 1) is 40-50:5:8-10; adding 45-55 g of purified water into each 100g of the mixture in the mass ratio, uniformly mixing and sterilizing to prepare a solid culture medium.
The sterilization condition is that the sterilization is carried out for 30min under the high pressure condition of 121 ℃ and 0.11 MP.
The invention discloses application of a microbial agent produced by a strain JNLSHGS-13 in dendrobium candidum cultivation.
The inoculation amount of the inoculation microbial inoculum of each cubic dendrobium candidum culture medium is 20 g-50 g.
The relative water content of the dendrobium candidum culture medium is maintained to be more than 10 days between 60 and 90 percent.
The dendrobium candidum tissue culture seedlings are selected as cultivation materials, the dendrobium candidum seedlings are cleaned, dried until the roots turn white, weighed, and planted in a inoculation cultivation matrix according to the planting density of 5 plants/cluster and 30 clusters/square meter.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the trichoderma microsporidianum Pestalotiopsis microspora strain JSLSHGS-13 has obvious growth promoting effect on dendrobium candidum seedlings, has a certain effect on reducing application of chemical fertilizers, can obviously improve chlorophyll content of the dendrobium candidum seedlings after application, and reduces enzyme activities of MDA and POD, thereby promoting growth of the dendrobium candidum seedlings. Meanwhile, the strain can be artificially cultured, has simple culture conditions, is convenient to apply and easy to store, is easy to produce in a large scale, 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:
and in 2017, 6 months, a bacterial strain JSLSHGS-13 which is obtained by separating and screening a strain capable of promoting the growth of dendrobium candidum seedlings from a dendrobium alpine root system with good growth state in Jiangsu nongboguan of sentence holding city in Jiangsu province is selected.
The colony characteristics of the strain JNLSHGS-13 are as follows: the colonies were grown on PDA medium for 7d, with a colony diameter of 5.4cm and slower growth. The colony is flat, the surface is white and flocculent, the back is provided with annular pigment sediment, the colony grows in a wheel shape, the edge is irregular, the mycelium is undeveloped, and the growth speed is low.
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):
GTGACTTACCTTTTGTTGCCTCGGCAGAAGTTATAGGTCTTCTTATAGCTGCTGCCG
GCGGACCATTAAACTCTTGTTATTTTATGTAATCTGAGCGTCTTATTTTAATAAGTCA
AAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAAT
GCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACA
TTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCTTAA
GCCTAGCTTAGTGTTGGGAATCTACTTCTCTTAGGAGTTGTAGTTCCTGAAATACAA
CGGCGGATTTGTAGTATCCTCTGAGCGTAGTAATTTTTTTCTCGCTTTTGTTAGGTG
CTATAACTCCCAGCCGCTAAACCCCCAATTTTTTGTGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGA。
morphological and molecular biological identification shows that the strain is Pelaralospora Microsporum (Pestalotiopsis microspora), the separation position of the strain is Pelaralospora Microsporum of Pelaralosporales (family) of Pelaralosporales of Desmodii, the strain number is JNLSHGS-13, the strain is preserved in China general microbiological culture Collection center (CGMCC) No.18543, 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:
a microbial agent produced by a strain JNLSHGS-13 of Pelargonium microsporum Pestalotiopsis microspora.
Taking corn flour as a culture medium, ammonium sulfate as an exogenous nitrogen source and glucose as an exogenous carbon source, respectively weighing 42g, 8g and 10g, uniformly mixing, adding 30g of water to prepare a solid culture medium, adjusting the pH to 5.5-6.5, sterilizing for 30min under the high pressure condition of 121 ℃ and 0.11MP, and inoculating a trichoderma microsporum (Pestalotiopsis microspora) strain JNLSHGS-13; culturing for 20-30 d under the dark culture condition of illumination for 12h at the temperature of 25-30 ℃. The cultured solid microbial inoculum is crushed into powder for standby.
Example 3:
the effect analysis of the microbial agent produced by the trichoderma microsporum (Pestalotiopsis microspora) strain JNLSHGS-13 on promoting the growth of dendrobium candidum is carried out.
1) The dendrobium candidum tissue culture seedlings are selected as cultivation materials, the dendrobium candidum seedlings are cleaned, dried until the roots turn white, weighed, planted in the inoculation cultivation substrate described in the example 2 according to the planting density of 5 plants/cluster and 30 clusters/square meter, and the non-inoculation substrate is set as a non-control (ck).
2) 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.
1. Average growth rate (%): cleaning the culture medium on the root and tuber, air-drying under natural condition, weighing fresh weight of Dendrobium officinale seedling with analytical balance (0.0001 g), subtracting the weight of Dendrobium officinale seedling before transplanting to obtain net weight increment (net weight increment/weight of Dendrobium officinale seedling before transplanting) ×100% to obtain average growth rate.
2. Chlorophyll a, chlorophyll b and chlorophyll a+b content: about 0.5g of healthy dendrobium candidum leaves are weighed, cut up, added with 25mL of mixed leaching solution of absolute ethyl alcohol and acetone (volume ratio is 1:1), and leached 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 calculated as 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.
3. Peroxidase (POD) activity assay: POD measurement was performed using a POD kit (available from Soviet biosciences Inc., st.) and 0.1g of the upper leaf of Dendrobium officinale planted with mycorrhizal fungi for growth promotion was weighed, and 1ml of the extract was added to the solution to perform ice bath homogenization. Centrifuge at 4 ℃ X12000 rpm for 10min. Taking the supernatant, and placing the supernatant on ice for testing. The spectrophotometer was tuned to 470nm and distilled water zeroed. Sample 40, reagent one, reagent two and reagent three are added into A1 mL cuvette in sequence, and are mixed uniformly, and the absorbance A1 is read immediately at 470nm, and the absorbance A2 is read after one minute. Data is recorded. And (3) injection: if DeltaA is less than 0.005, the reaction time can be prolonged to 5min.
POD(ΔOD 470 Per min/g fresh weight) =Δa ≡ (w×v1++v) ≡0.5+=50×Δa ≡w. Calculating enzyme activity definition: each gram of tissue is subjected to absorption value of 0.5 at 470nm in a reaction system as an enzyme activity unit.
The analysis result of the effect of the microbial agent produced by the trichoderma microsporum (Pestalotiopsis microspora) strain JNLSHGS-13 on promoting the growth of dendrobium candidum is shown in table 1.
TABLE 1 analysis of effect of microbial inoculant produced by strain JNLSHGS-13 on promotion of Dendrobium officinale growth
The trichoderma microsporidianum Pestalotiopsis microspora strain JSLSHGS-13 has obvious growth promoting effect on dendrobium candidum seedlings, has a certain effect on reducing application of chemical fertilizers, can obviously improve chlorophyll content of the dendrobium candidum seedlings after application, and reduces enzyme activities of MDA and POD, thereby promoting growth of the dendrobium candidum seedlings. Meanwhile, the strain can be artificially cultured, has simple culture conditions, is convenient to apply and easy to store, is easy to produce in a large scale, and has good development and application prospects.
Claims (9)
1. A kind of trichoderma viride for promoting dendrobium candidum growth is named as trichoderma viride Pestalotiopsis microspora, the strain number is JNLSHGS-13, and the trichoderma viride is preserved in China general microbiological culture Collection center (CGMCC) No.18543 and the preservation date is 2019, 09 and 02.
2. The use of a trichoderma viride for promoting the growth of dendrobium candidum of claim 1.
3. The microbial agent produced by the strain JNLSHGS-13 as claimed in claim 1.
4. The method for producing a microbial agent according to claim 3, wherein: comprising the following steps:
1) Taking corn flour as a culture medium, ammonium sulfate as an exogenous nitrogen source and glucose as an exogenous carbon source to prepare a solid culture medium; the pH is adjusted to 5.5-6.5, and the strain JNLSHGS-13 is inoculated after sterilization;
2) Culturing for 20-30 d under the conditions of 25-30 ℃ and 12h illumination/12 h dark culture;
3) And (3) crushing the solid microbial agent cultured in the step (2) into powder to obtain the microbial agent.
5. The method of manufacturing according to claim 4, wherein: the mass ratio of corn flour to ammonium sulfate to glucose in the step 1) is 40-50:5:8-10; adding 45-55 g of purified water into each 100g of the mixture in the mass ratio, uniformly mixing and sterilizing to prepare a solid culture medium.
6. The use of the microbial agent of claim 3 in dendrobium officinale cultivation.
7. The use according to claim 6, characterized in that: the inoculation amount of the inoculation microbial inoculum of each cubic dendrobium candidum culture medium is 20 g-50 g.
8. The use according to claim 7, characterized in that: the relative water content of the dendrobium candidum culture medium is maintained to be more than 10 days between 60 and 90 percent.
9. The use according to claim 7, characterized in that: the dendrobium candidum tissue culture seedlings are selected as cultivation materials, the dendrobium candidum seedlings are cleaned, dried until the roots turn white, weighed, and planted in a inoculation cultivation matrix according to the planting density of 5 plants/cluster and 30 clusters/square meter.
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| CN202211488708.7A CN116731866A (en) | 2022-11-25 | 2022-11-25 | Pediopside parvulum for promoting dendrobium candidum growth and application thereof |
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| CN202211488708.7A CN116731866A (en) | 2022-11-25 | 2022-11-25 | Pediopside parvulum for promoting dendrobium candidum growth and application thereof |
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