CN116496909A - Endophytic fungus sessile spore shell fungus and application thereof in dendrobium candidum cultivation - Google Patents
Endophytic fungus sessile spore shell fungus and application thereof in dendrobium candidum cultivation Download PDFInfo
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- 241000026010 Dendrobium candidum Species 0.000 title claims abstract description 31
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- 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
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- 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
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- C12N1/145—Fungal isolates
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Abstract
The invention discloses endophytic fungus aschersonia aleyrodis and application thereof in dendrobium candidum cultivation. The classification is named as the ascomycetes Zopfield sp, the classification is ascomycetes order Sphaerochaetes, the family Sphaerochaetes, the strain number is JNLSHDC-3, the strain is preserved in China general microbiological culture Collection center (CGMCC) No.18562, and the preservation date is 2019, 09 and 02. The endophytic fungus sessile fungus Zopfield sp. Strain JNLSHDC-3 has obvious effects on promoting the growth of dendrobium candidum seedlings and increasing stress resistance, and simultaneously has the advantages of artificial culture, simple culture conditions, easy mass production and good development and application prospect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to endophytic fungus aschersonia aleyrodis and application thereof in dendrobium candidum cultivation.
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 Dendrobium officinale and Dendrobium nobile are loaded in the "Chinese pharmacopoeia" 2020 edition, and are one of the rare Chinese medicinal materials in China.
The wild dendrobium has strict requirements on the growth environment, is mostly attached to stone walls or stone seams of cliff walls, grows slowly, has no endosperm, is 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, with the application of a tissue culture rapid propagation technology in dendrobium officinale seedling breeding, the artificial cultivation of dendrobium officinale is mature, but the tissue culture seedlings are formed by heterotrophic growth, and after transplanting in a field, proper fungi do not form symbiosis with the tissue culture seedlings, so that the problems of low transplanting survival rate, slow growth, low content of effective components and the like exist.
Research shows that mycorrhizal fungi of dendrobium candidum and dendrobium candidum are symbiotic, so that the germination of 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, and various nutrients required by the growth of the plants can be provided for the plants, so that the growth and development of the plants can be promoted. In recent years, research strength in the aspect of dendrobe mycorrhizas is gradually increased, and how to screen mycorrhizal bactericides with remarkable effects to further promote the growth of dendrobe is an important application research direction for green and healthy development of the dendrobe industry.
Disclosure of Invention
The invention aims to: aiming at the defects and the shortcomings of the prior art, one of the purposes of the invention is to provide a strain JNLSHDC-3 of aschersonia aleyrodis Zopfield sp and a microbial agent produced by the strain JNLSHDC-3. The invention also aims to provide the application of the strain agent in dendrobium candidum cultivation.
The technical scheme is as follows: the endophytic fungus of the invention is named as the aschersonia zopfila sp, the classification status is ascomycetes order sphaerotheca, the strain number is JNLSHDC-3, the endophytic fungus is preserved in China general microbiological culture Collection center, the preservation number is CGMCC No.18562, 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.
The main biological characteristics of the strain are as follows: the colony is cultured on PDA culture medium for 7d, the diameter of the colony is 7.2cm, and the growth is moderate. The bacterial colony is flat, hyphae are grey white, grow in a radial shape, have irregular edges, are zigzag and grey white on the opposite surface, do not precipitate pigment, and are not easy to produce spores under the condition of artificial culture.
The ITS region complete sequence was amplified and sequenced, and the PCR amplified 16sRNA complete sequence (SEQ ID NO. 1) GTAAGTGGGGGTTTTTACGGCAGGCGCCCGCGCCACTCCGCAGCGAGGTTGTCTACTACGCTCGGTGTGGACAGCGAGCCCGCCACTGGTTTTCGGGGCCTGCGGTCGGACGCAGGTCCCCAACACAAGCCCGGGGGCTTGATGGTTGAAATGACGCTCGGACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGACTCATTTTAAGTACAGTACTCAGAGAGGCCGGTAAAAAAGCAATGGTTTGGGTGCCTCCGGCAGGCCCTCGGCGGCCCCCCGCGGGTGGGCGGGGGGCCGGCCTGCCGAAGCAACTGTTCAGTGGTTCGTTCGCGATGGTTTGTGGGAGTTTTGCAACTCTTTAATGATCCCTCCGCAGGTTC.
The invention relates to application of endophytic fungus sessile spore shell fungus in dendrobium candidum cultivation.
The microbial agent produced by the strain JNLSHDC-3.
The preparation method of the microbial agent produced by the strain JNLSHDC-3 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 JNLSHDC-3 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 JNLSHDC-3 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 beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the endophytic fungus sessileella sp. Strain JSLSHDC-3 has obvious growth promoting effect on dendrobium candidum seedlings, can obviously improve the chlorophyll content of the dendrobium candidum seedlings after application, and reduces the Malondialdehyde (MDA) content of the dendrobium candidum seedlings, thereby promoting the growth and enhancing the stress resistance 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:
the strain JNLSHDC-3 is obtained by separating root systems of dendrobium huoshanense 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 aphyllum roots, washing the substrate on the surfaces of the root sections with flowing tap water, sucking the water on the surfaces of the root sections by using filter paper in an ultra-clean workbench, immersing the root sections in 75% alcohol 45s for several times, and immersing in 0.1% secondary mercury solution for disinfection for 5min. After the disinfection is finished, the disinfection is carried out by washing 3 to 4 times with sterilized deionized water to thoroughly remove the disinfectant, 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 to 1mm by a sterilized scalpel. The tissue sections were transferred to PDA media plates with streptomycin sulfate (50-100. Mu.g/mL) and 5 tissue sections were inoculated on each plate, 15 plates per medium. The endophytic fungi obtained through separation are subjected to tieback test to obtain the strain, the strain is named as JNLSHDC-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 JSNLSHDC-3 were as follows: the strain is cultured on PDA culture medium for 7d, colony diameter is 7.2cm, and growth is moderate. The bacterial colony is flat, hyphae are grey white, grow in a radial shape, have irregular edges, are zigzag and grey white on the opposite surface, do not precipitate pigment, and are not easy to produce spores under the condition of artificial culture.
The ITS region complete sequence was amplified and sequenced, and the PCR amplified 16sRNA complete sequence (SEQ ID NO. 1) GTAAGTGGGGGTTTTTACGGCAGGCGCCCGCGCCACTCCGCAGCGAGGTTGTCTACTACGCTCGGTGTGGACAGCGAGCCCGCCACTGGTTTTCGGGGCCTGCGGTCGGACGCAGGTCCCCAACACAAGCCCGGGGGCTTGATGGTTGAAATGACGCTCGGACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGACTCATTTTAAGTACAGTACTCAGAGAGGCCGGTAAAAAAGCAATGGTTTGGGTGCCTCCGGCAGGCCCTCGGCGGCCCCCCGCGGGTGGGCGGGGGGCCGGCCTGCCGAAGCAACTGTTCAGTGGTTCGTTCGCGATGGTTTGTGGGAGTTTTGCAACTCTTTAATGATCCCTCCGCAGGTTC.
Morphological and molecular biological identification shows that the strain is aschersonia ales (Zopfield sp.) and the separation position is ascomycetes, sphaerochaete, phaeodactylicaceae, the strain number is JNLSHDC-3, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.18562 and the 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.
Example 2:
preparation of solid microbial agent of strain JNLSHDC-3.
1) Taking corn flour as a culture medium, ammonium sulfate as an exogenous nitrogen source and glucose as an exogenous carbon source, preparing a solid culture medium, regulating the pH to 5.5-6.5, sterilizing, cooling the culture medium, and inoculating a aschersonia aleyrodis (Zopfield sp.) strain JNLSHDC-3;
2) Culturing for 20-30 d under the dark culture condition of illumination for 12h at the temperature of 25-30 ℃.
3) And (3) crushing the solid microbial inoculum cultured in the step (2) into powder for later use.
In the step 1), corn flour, ammonium sulfate and glucose are added with 50g of purified water per 100g of the mixture in the mass ratio of 40:5:9, and the mixture is uniformly mixed and sterilized to prepare a solid culture medium.
Sterilizing in step 1) at 121deg.C under high pressure of 0.11MP for 30min.
Example 3:
application of a sessile stemona (Zopfield sp.) strain JNLSHDC-3 microbial agent in dendrobium candidum cultivation.
1) The solid microbial agent cultured in the embodiment 2 is inoculated into dendrobium candidum seedling culture medium, and the inoculation amount of the solid microbial agent per cubic dendrobium candidum culture medium is 50g. After application, the relative water content of the dendrobium candidum culture medium is maintained to be more than 10 days at about 70%.
2) The dendrobium candidum tissue culture seedlings with consistent size and shape are selected as cultivation materials, the dendrobium candidum seedlings are cleaned, aired until the roots turn white, weighed, planted in the inoculation cultivation substrate according to the planting density of 5 plants/cluster and 30 clusters/square meter, and the non-inoculation substrate is set with a non-contrast (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 is analyzed as follows:
1. chlorophyll a, chlorophyll b and chlorophyll a+b content: weighing about 0.5g of healthy dendrobium candidum leaves, cutting, adding 25ml of mixed leaching solution of 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 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.
The strain JNLSHDC-3 analyzes the dendrobium candidum tissue culture seedling promoter as follows:
2. and (3) measuring the content of malondialdehyde: weighing about 1g of healthy dendrobium candidum leaves, shearing, adding 2ml of 10% TCA and a small amount of quartz sand, grinding to homogenate, adding 8ml of TCA, grinding, centrifuging at 4000r/min for 10min, taking the supernatant as a sample extracting solution, sucking 2ml of the obtained extracting solution (2 ml of distilled water is added in comparison), adding 2ml of 0.6% TBA solution, reacting the mixture on a boiling water bath for 15min, and centrifuging after rapid cooling. The supernatants were taken to determine extinction at 532, 600 and 450 wavelengths.
The content of the measured data is calculated according to the following formula.
C1＝11.71D450；
C2＝{6.45(D532-D600)-0.56D450}NW -1 ；
D450, D532, D600 represent extinction values at wavelengths of 450, 532 and 600nm, respectively. N represents total volume (ml) of the extracting solution, and W represents fresh weight of plant tissue.
TABLE 1 influence of JNLSHDC-3 on Dendrobium officinale growth
The results show that after the solid microbial inoculum prepared by SNLSHDC-3 is inoculated, the chlorophyll content of the solid microbial inoculum is obviously improved compared with that of a control, and the photosynthesis capacity of the solid microbial inoculum is enhanced, so that the growth of the solid microbial inoculum is promoted; in addition, the measurement of the malondialdehyde content shows that the malondialdehyde content of the inoculation plant is obviously reduced, and the stress resistance is improved to a limited extent.
Claims (8)
1. The endophytic fungus sessile fungus is named sessile fungus Zopfield sp, and has strain number JSLSHDC-3 and preservation number CGMCC No.18562 and preservation date of 2019, 09 and 02.
2. The use of endophytic fungus, aschersonia aleyrodis, according to claim 1, in dendrobium officinale cultivation.
3. The microbial agent produced by the strain JNLSHDC-3 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 JNLSHDC-3 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 6, 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.
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| CN117363489A (en) * | 2023-10-13 | 2024-01-09 | 中科净土(广州)技术服务有限公司 | Sphaerotheca longifolia with cucumber growth promoting and disease resisting functions and application thereof |
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| CN117363489A (en) * | 2023-10-13 | 2024-01-09 | 中科净土(广州)技术服务有限公司 | Sphaerotheca longifolia with cucumber growth promoting and disease resisting functions and application thereof |
| CN117363489B (en) * | 2023-10-13 | 2024-05-14 | 中科净土(广州)技术服务有限公司 | Sphaerotheca longifolia with cucumber growth promoting and disease resisting functions and application thereof |
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