CN102653720B - Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a - Google Patents
Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a Download PDFInfo
- Publication number
- CN102653720B CN102653720B CN 201110050525 CN201110050525A CN102653720B CN 102653720 B CN102653720 B CN 102653720B CN 201110050525 CN201110050525 CN 201110050525 CN 201110050525 A CN201110050525 A CN 201110050525A CN 102653720 B CN102653720 B CN 102653720B
- Authority
- CN
- China
- Prior art keywords
- huperzine
- selagine
- strain
- endophytic fungi
- anthrax
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The invention belongs to the technical fields of medicinal plants and agricultural microorganism and particularly relates to an endophytic fungi which is separated from a medicinal plant huperzia serrata, can generate huperzine a or analogues, and can be used in mass production of the huperzine a or analogues. The separated endophytic fungi ES026 is identified as colletotrichum gloeosporioides bymolecular biology and morphology, and the strain has been preserved in China Center For Type Culture Collection (CCTCC), with the preservation number of CCTCC NO: M2011046. The endophytic fungi is significantly characterized in that after liquid fermentation, the strain can generate an active substance with reversibility which can inhibit acetylcholinesterase, and high-performance liquid chromatography and mass spectrum analysis shows that the compound is huperzine a (HupA), thus a new medicinal herb source for treating alzheimer's disease is provided.
Description
Technical field
The invention belongs to medicinal plant and agriculture microbial technology field, be specifically related to from the medicinal plant Herba Lycopodii serrati, separate a strain endogenetic fungus, this bacterial strain is that anthrax belongs to glue spore kind (C.gloeosporioides), it can produce selagine or analogue, can be used for mass production selagine or analogue.
Background technology
Along with the world population aging, alzheimer's disease (Alzheimers ' disease, AD, senile dementia) has become global public health problem.Senile dementia morbidity height, the course of disease are long, are one of diseases that disability rate is the highest in all chronic diseases, and this will all bring great disease burden to family and society.According to the up-to-date prediction of international Alzheimer federation issue in 2009, by 2010, global dementia patients number will reach 3,560 ten thousand, and the year two thousand thirty reaches 6,570 ten thousand, the year two thousand fifty dementia patients quantity will reach 1.154 hundred million, wherein 59% patient is in the Asia.In decades, along with the understanding of people to AD, the transmission that strengthens cholinergic nerve has become and has slowed down one of main strategy of relevant cognitive disorder symptom.As E2020, tacrine or lycoremine, on clinical treatment, be proved to be and had the treatment potentiality, be used for the treatment of AD by the U.S. and the approval of some European countries.Separate self-traditional Chinese medicines with alkaloid selagine (the huperzine A of plant Herba Lycopodii serrati (Hupperzia serrata) (being commonly called as Herba Lycopodii serrati), Hup A) similar or be better than the AChE inhibitor of those clinical applications that gone through, having characteristics such as long-acting, low toxicity, oral availability height, is the prospect medicine for the treatment of AD.
Selagine production at present still depends on from the Herba Lycopodii serrati plant extracts, have following shortcoming: 1. Herba Lycopodii serrati belongs to high pteridophyte, because its poor growth, spore germination rate is low, make wild resource scarcity (Ma X, et al.The Lycopodium alkaloids[J] .Nat.Prod.Rep., 2004,21:752-772.).2. artificial cultivation technique is not captured (Li N.Study on the effect of Promote growth on host and fermentation Products of endophytic fungi from Huperzia serrata[D] .2007.) as yet.In the plant body HupA content relatively low (Wang Jun is etc. selagine Study on content [J] in Hunan Province's plants of Huperzia. Chinese Pharmaceutical Journal, 2005,21:1616-1618.).The nineties in 20th century, started the upsurge of synthetic Hup A, though obtain certain achievement, but low because of synthetic selagine activity, cost is high, toxic side effect greatly can't suitability for industrialized production (Chen Yegao, Deng. the new development [J] of lycopsida alkaloid component research. Yunnan Normal University's journal, 2010,30 (6): 12-24.).Above factor finally causes the HupA source very short, and therefore seeking the selagine source new drugs becomes necessary.
There is separation obtains from the Huperziaceae plant endophyte to produce the relevant report (seeing Table 1) of selagine and analogue thereof at present, these studies show that utilizing microbial fermentation to produce selagine becomes a kind of possibility, and adopt plant endogenesis epiphyte fermentative production selagine to solve the green approach of selagine raw material sources beyond doubt.But all there is a bottleneck problem in the present bacterial strain selagine output of reporting: yield poorly.This and the needed milligram level of realization suitability for industrialized production still have a segment distance.Compare with the bacterial strain that produces selagine in other report, it is obviously higher that anthrax belongs to glue spore kind (C.gloeosporioides) selagine output among the present invention, to realizing that suitability for industrialized production is more advanced in years further.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, from Herba Lycopodii serrati, separate and obtain a strain endogenetic fungus ES026, this bacterial strain is that anthrax belongs to glue spore kind (C.gloeosporioides), and it can produce selagine or analogue, can be used for mass production selagine or analogue.
Technical scheme of the present invention is as follows:
The present invention adopts reverse method for screening, from Huperziaceae plant Herba Lycopodii serrati live plant, separate the candidate bacterium ES026 that obtains endophyte of plant, identify and this bacterial strain of microbiology assay certificate belongs to anthrax and belongs to glue spore kind (Colletotrichum.gloeosporioides) through 18S rDNA, on February 23rd, 2011 delivered this bacterial strain to the Chinese typical culture collection center (CCTCC) that is deposited in the Wuhan University of Wuhan City, Hubei Province, and deposit number is CCTCC NO:M2011046.
Particularly, it is as follows that anthrax of the present invention belongs to separation and the screening process of glue spore kind (C.gloeosporioides) ES026 bacterial strain:
Gather Herba Lycopodii serrati live body plant from open-air (Lichuan City, Chinese Hubei Province) earlier, connect soil and take back the laboratory together, select healthy intact plant to carry out cleaning surfaces and sterilization again, under the aseptic condition segment is carried out at the different tissues position, then be positioned in the culturing bottle that contains PDA and cultivate, observe the colony growth situation, can see that tissue segments had filamentous fungus to grow in general 5-7 days; To carrying out liquid fermentation and culture after the bacterium colony separation and purification cultivation that grows, extract the total alkaloids in the mycelium, do contrast with the standard substance selagine, carry out HPLC and detect, the bacterial strain of selagine analogue is produced in screening according to collection of illustrative plates; Doubtful strain fermentation is cultivated, and extract total alkaloids utilizes the selagine analogue in the total alkaloids of HSCCC to do separation and purification again, carries out LC-MS and analyzes; Determine that to analyzing the back bacterial strain that produces selagine carries out systematics research, comprises the evaluation of morphology and molecular biology level.
Strain classification is learned the evaluation of status:
1. solid culture feature:
Adopt PDA substratum (composition according to a conventional method: potato 200g, cut and add less water behind the fritter and boil, glucose 20g, agar 15g adds water and is settled to 1L) to cultivate anthrax and belong to glue spore kind ES026 bacterial strain.
Colonial morphology: cultivated 3 days for 25 ℃, colony diameter is 30mm, covers with whole culture dish in the time of 7 days substantially, and bacterium colony be circular, and be white early stage, after become Steel Gray; Dark grey in the middle of the bacterium colony, the fine hair shape, it is fluffy a little to grow, and colony edge aligns mutually, bacterium colony back side visible black color concentric wheel stripe, outer rim presents tawny.
Mycelia form: colourless septate hypha.
Produce the spore feature: conidiogenous cell bottleneck formula, conidium round shape or oblong, an end is slightly little sometimes, and is unicellular colourless, has 1～2 oily ball; On the PDA substratum, sporulation quantity is low, and the spore size is 10.62～21.38 (15.21) * 4.65～6.02 (5.16) μ m.
2.ITS sequencing:
1) extracts the total DNA of mycelium (seeing embodiment 1).
2) be primer with ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATAGC-3 '), amplify 18S rDNA sequence, through order-checking, obtain the nucleotide sequence shown in sequence table SEQ ID NO:1.
3) sequencing result is submitted
Http:// blast.ncbi.nlm.nih.gov/BlastCarry out sequence alignment, reach a conclusion according to BLAST result and in conjunction with its morphological feature, prove that this strain separated belongs to anthrax and belongs to glue spore kind (C.gloeosporioides).
Effect of the present invention
1. the present invention provides a strain new microorganism for the exploitation of source new drugs selagine.
2. object of the present invention belongs to microorganism field, and it is rapid to have a growth and breeding, not limited by season, weather and region, advantage such as the output room for promotion is big.
3. the present invention successfully avoids excessively excavating the Herba Lycopodii serrati wild plant resource, effectively preserves the ecological environment, and realizes environmentally friendly and resource sustainable application resource-conserving.
The present invention has potential development prospect, and its invention effect is shown in Table 1.
The comparison of table 1 the present invention and prior art
Description of drawings
Sequence table SEQ ID NO:1 is that the anthrax that the present invention separates belongs to glue spore kind ES02618S rDNA gene order.
Fig. 1: the Herba Lycopodii serrati plant picture of field acquisition.
Fig. 2: the filamentous fungus that grows from Herba Lycopodii serrati stem under the isolated condition.
Fig. 3: anthrax belongs to glue spore kind (C.gloeosporioides) ES026 total alkaloids HPLC detected result (peak 2 is the selagine standard substance, and peak 1 and 3 is the sample peak).
Fig. 4: anthrax belongs to one-level mass spectrum and the second order ms figure of glue spore kind (C.gloeosporioides) ES026 tunning.Wherein:
Fig. 4 A is the one-level mass spectrum; Fig. 4 B is second order ms.
Fig. 5: anthrax belongs to the ITS PCR product electrophorogram of glue spore kind (C.gloeosporioides) ES026.
Fig. 6: anthrax belongs to glue spore kind (C.gloeosporioides) ES026 cluster analysis figure.
Fig. 7: anthrax belongs to the colonial morphology of glue spore kind (C.gloeosporioides) ES026 on the PSA substratum.
Fig. 8: anthrax belongs to the microstructure of glue spore kind (C.gloeosporioides) ES026 under 100 times of oily mirror visuals field.
Embodiment
1, gathers the wild plant of Herba Lycopodii serrati from Lichuan City, Chinese Hubei Province, take back the laboratory culture (see figure 1) together with earth.
2, select health, the flushing of plant height 6-10cm plant flowing water 30 minutes, remove surperficial dirt.
3, carry out surface sterilization, step is as follows:
1) uses alcohol-pickled 2 times of 70%-75%, each 2-3 minute earlier.
2) then use 0.1%HgCl
2Soaked jog 3 times 5 minutes.
3) drain liquid on the plant, embathe 5 times with sterilized water again, fully clean to avoid HgCl
2Residual, and coat and contain the PDA substratum (composition according to a conventional method: potato 200g cuts and adds less water behind the fritter and boil glucose 20g, agar 15g/L, pH6.5 before the sterilization with embathing liquid for the last time; Add water and be settled to 1L, under 121 ℃ of high pressure steam the sterilization 20 minutes) plate on.According to 200 (embathing liquid for the last time) ul/ ware, prepare 3-5 ware (control group: to get rid of the interference that surface sterilization does not thoroughly bring).
4) blot residual liquid with aseptic filter paper, the stem section of plant is cut to 1cm, or root, stem, leaf blocked respectively place the culturing bottle that the PDA substratum is housed.
5) explant with step 4) places under 25 ℃ of conditions together with control group, and a week is cultivated in artificial lighting every day (intensity of illumination is 2000lux).
4, be separated to 128 strain endogenetic fungus (see figure 2)s altogether on the explant that obtains from step 3; according to ordinary method (Zhang Hao; Deng. a kind of monospore of gibberellic hypha simply fast separation method---dull and stereotyped dilution setting-out partition method [J]. plant protection; 2008,34 (6): 134-136.) obtained strains is carried out separation and purification.
5, the bacterial strain behind the purifying is carried out fermentation culture with the PDB liquid nutrient medium, collect mycelium.
Wherein: the prescription of PDB liquid nutrient medium is:
Potato (stripping and slicing) 200g; Glucose 20g; Replenish distilled water to 1000ml, transfer pH to 6.5 before the sterilization.
The making method of PDB liquid nutrient medium is: will clean the potato chopping of back peeling, adding water 1000ml boiled about half hour, go potato with 4 layers of gauzes worry, add glucose 20g then, add water and complement to 1000ml, packing sterilization after the stirring and dissolving (sterilization is 20 minutes under 121 ℃ of high pressure steam).
Anthrax belongs to the mycelial acquisition of glue spore kind ES026, may further comprise the steps:
1) preparation seed culture fluid: anthrax is belonged to glue spore kind ES026 be inoculated in the Erlenmeyer flask that contains 100ml PDB substratum, in 25 ℃, 160rpm cultivated 24 hours.
2) enlarged culturing: anthrax is belonged to glue spore kind ES026 seed liquor be inoculated in the enlarged culturing liquid with 5% (volume ratio) inoculum size, in 25 ℃, 160rpm cultivated 120 hours, obtained spherical mycelium.
3) culture collection mycelium: with step 2) is crossed 60 mesh filter screens, uses distilled water flushing 3 times, is positioned in the loft drier after extracting, and is dried to constant weight under 40 ℃.
6, utilize soda acid pH value method (the concrete step of face as follows) alternately to extract endobacillary total alkaloids.
The extraction of total alkaloids, its step is as follows:
1) mycelium behind the mill-drying is extremely Powdered under the liquid nitrogen condition;
2) hydrochloric acid soln with pH1.5 soaks mycelium in 1: 30 (g/v) ratio, supersound process (Jiangsu, the KQ5200DE of Kunshan Ultrasonic Instruments Co., Ltd.), and 40KHZ, 160W after 1 hour, spends the night.
3) centrifugal acid solution is collected the supernatant liquor body portion.
4) with strong aqua adjust pH to 9.0, left standstill 2-3 hour.
5) with equal-volume chloroform extracting 3 times, merge organic phase.
6) use Rotary Evaporators (Shanghai Ai Lang Instr Ltd., model: N-1100) concentrate organic phase to thick under 40 ℃ of conditions, use dissolve with methanol again.
7, detect the total alkaloids that step 6 is extracted with high performance liquid chromatograph (HPLC), and do contrast with selagine (HupA) standard substance (available from Shanghai Tongtian Biotechnology Co., Ltd.).Technical qualification are as follows: moving phase: methyl alcohol-80mM ammonium acetate (volume ratio 3: 7); Flow velocity: 1.0ml/min; Temperature: 25 ℃; Detect wavelength: 310nm; Pillar: Eclipse XDB-C18-USP4.6*250mm.
8, the data of step 7 gained are analyzed (see figure 3), the result shows that a strain endogenetic fungus that is separated to from stem produces the HupA analogue.
9, adopt the total alkaloids of high speed adverse current chromatogram (HSCCC, Shanghai Tongtian Biotechnology Co., Ltd.) purification procedures 1～6 gained, collect this analogue.
10, the component that step 9 is separated to is delivered to test center of the Central China University of Science and Technology, and HPLC-MS determines that according to the peak spectrum signature this component is the HupA (see figure 4) after detecting.
11, the component that simultaneously step 9 is separated to is carried out acetylcholine esterase active inhibition test (Yin Shuaiwen, Deng. the evaluation [J] of different pteridophyte extract inhibiting activity of acetylcholinesterase. research and development of natural products, 2009,21:854-857.), standard substance HupA does contrast, concentration range be 0.01mg/ml to 0.2mg/ml, the gained linear equation is: y=0.1317x-0.1073R
2=0.994).
12, behind the active function that obtains this microbial fermentation product, again this microorganism to be carried out taxonomy and identify, its evaluation comprises traditional mycology feature (as previously mentioned) evaluation and molecular biology identification.Concrete grammar is:
Anthrax belongs to the total DNA extraction of glue spore kind (C.gloeosporioides) ES026:
1) grinds: in the small-sized mortar of sterilization, add the fresh anthrax of 0.2g and belong to glue spore kind ES026 mycelia, add a small amount of 2 * CTAB extracting solution (100mmol/L Tris-HCl pH 8.0 again, 1.4mol/LNaCl, 20mmol/L EDTA, 2%CTAB, the 0.1%-2% beta-mercaptoethanol), grind on ice, the time should not be long to prevent dna degradation.Mixture is transferred to the 2.0mL centrifuge tube, and it is measured eventually is 1000 μ L.
2) water-bath: ground thick anthrax is belonged to glue spore kind ES026 mycelia mixed solution put into 65 ℃ of water-bath 1h.
3) collect supernatant: after water-bath finished, the centrifugal 10min of 12,000r/min got supernatant liquor 800 μ L and is transferred in the 2.0mL centrifuge tube of a sterilization.
4) extracting: add the saturated phenol of isopyknic chloroform/Tris-(volume ratio is 1: 1) mixed solution, the centrifugal 10min of 12,000r/min gets the 2.0mL centrifuge tube that supernatant liquor is put into another sterilization.Repeat 2 times.
5) bring up again: add isopyknic chloroform/primary isoamyl alcohol (volume ratio is 24: 1) mixed solution in supernatant liquor, the centrifugal 10min of 12,000r/min gets the 1.5mL centrifuge tube that supernatant liquor is put into another new sterilization.
6) precipitation: add 3mol/L Potassium ethanoate 80-100 μ L in supernatant liquor, add the ice-cold Virahol of equal-volume again, mixing is placed in-20 ℃ or the 4 ℃ of refrigerators precipitation 1h or spends the night, and the centrifugal 10min of 12,000r/min abandons supernatant liquor, collecting precipitation.
7) washing: with the freshly prepared 70% alcohol washing precipitation of 500 μ L, the centrifugal 10min of 12,000r/min abandons supernatant liquor (prevent refluence), repeats 2 times, and collecting precipitation is put dry 30min in 37 ℃ the thermostat container.
8) dissolving: in dried centrifuge tube, add 50 μ L deionized water or 1 * TE, fully put into 4 ℃ of refrigerators after the dissolving and treat electrophoresis detection.
9) preserve: will put into-20 ℃ of refrigerators by total DNA of electrophoresis detection and preserve standby.
Pcr amplification system and reaction conditions:
The PCR reaction system comprises 10 * buffer, 2.5 μ L, dNTP 2 μ L (2.5mmol/L), each 1 μ L (10 μ m) of primer I TS1 and ITS4, TaqDNA polysaccharase 0.2 μ L (5U/ μ L), dna profiling 1 μ L, ddH
2O supplies 25 μ L.Reaction conditions is to enter circulation after 95.0 ℃ of 5min sex change, and loop parameter is: 95.0 ℃ of 1min, and 56.0 ℃ of 1min, 72.0 ℃ of 1min, 35 circulations are extended 10min for back 72.0 ℃.After finishing, reaction with PCR product 1.2% agarose electrophoresis, obtains the gene fragment that size is about 530bp, (see figure 5).Sequencing result is shown in sequence table SEQ ID NO:1.
Sequencing result is forwarded to http://blast.ncbi.nih.gov/Blast. compares, the institute of recycling software MEGA3.1 and BJOEDIT cloned sequence carries out the cluster analysis (see figure 6), learn feature (seeing Fig. 7 and Fig. 8) in conjunction with strain morphology, this bacterium be accredited as anthrax belong to glue spore kind (C.gloeosporioides) (referring to Wei Jingchao. fungi identification handbook [M]. Shanghai science tech publishing house, 1979.)
The anthrax that the present invention separates belongs to glue spore kind ES026 and can produce selagine or analogue, and its biologic activity is can the reversibility acetylcholine esterase inhibition, for the mass production selagine provides new source.Enforcement of the present invention can adopt conventional biofermentation method (for example adopting the method for fermentor tank) to produce.
Claims (2)
1. a strain separates the endogenetic fungus that obtains from plants of Huperzia Herba Lycopodii serrati plant living body, it is characterized in that this bacterial strain is colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides) ES026, be deposited in Chinese typical culture collection center, its preserving number is: CCTCCNO:M2011046, this bacterial strain produces selagine.
2. the application of the described colletotrichum gloeosporioides Penz of claim 1 in extracting selagine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110050525 CN102653720B (en) | 2011-03-02 | 2011-03-02 | Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110050525 CN102653720B (en) | 2011-03-02 | 2011-03-02 | Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102653720A CN102653720A (en) | 2012-09-05 |
| CN102653720B true CN102653720B (en) | 2013-09-25 |
Family
ID=46729451
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110050525 Expired - Fee Related CN102653720B (en) | 2011-03-02 | 2011-03-02 | Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102653720B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060386B (en) * | 2012-10-22 | 2016-01-06 | 青岛农业大学 | A mould Pt-2 of sprouting in Portunus trituberculatus Miers suppresses secondary metabolite of AchE and preparation method thereof |
| CN103834577B (en) * | 2014-02-07 | 2016-01-06 | 福建中医药大学 | The methods and applications of phlegmariurus mycorrhizal fungi and product selagine thereof |
| CN105191657A (en) * | 2015-11-06 | 2015-12-30 | 长沙慧日生物技术有限公司 | Rapid breeding method of aseptic huperzia serrata seedling |
| CN106966887B (en) * | 2017-03-28 | 2020-06-05 | 兰州理工大学 | Compound separated from colletotrichum gloeosporioides, preparation method and application thereof |
| CN109402160A (en) * | 2018-11-15 | 2019-03-01 | 华中农业大学 | A kind of Agrobacterium tumefaciens mediated glue born of the same parents' anthrax-bacilus genetic transforming method |
| CN110468055B (en) * | 2019-07-29 | 2021-09-14 | 西北大学 | Huperzia serrata colletotrichum and application thereof |
| CN114164123B (en) * | 2021-12-17 | 2022-10-04 | 福建农林大学 | Endophytic fungus S24 capable of promoting growth of China fir |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195804A (en) * | 2006-12-05 | 2008-06-11 | 上海中医药大学 | Huperzia serrata endogenetic epiphyte and uses thereof |
| CN101265451A (en) * | 2007-03-13 | 2008-09-17 | 张晶 | Endogenetic fungus for Huperzia Serrata (Thunb.)Trev |
| CN101942393A (en) * | 2009-12-30 | 2011-01-12 | 朱笃 | Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A |
-
2011
- 2011-03-02 CN CN 201110050525 patent/CN102653720B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195804A (en) * | 2006-12-05 | 2008-06-11 | 上海中医药大学 | Huperzia serrata endogenetic epiphyte and uses thereof |
| CN101265451A (en) * | 2007-03-13 | 2008-09-17 | 张晶 | Endogenetic fungus for Huperzia Serrata (Thunb.)Trev |
| CN101942393A (en) * | 2009-12-30 | 2011-01-12 | 朱笃 | Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A |
Non-Patent Citations (2)
| Title |
|---|
| 张君诚 等.蕨类植物内生菌研究进展.《中国农学通报》.2010,第26卷(第20期),70-72. |
| 蕨类植物内生菌研究进展;张君诚 等;《中国农学通报》;20101231;第26卷(第20期);70-72 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102653720A (en) | 2012-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102653720B (en) | Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a | |
| CN101933460B (en) | Inonotus obliquus and method for extracting triterpennoids from same | |
| CN101942393B (en) | Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A | |
| CN103667070A (en) | Plant endophytic fungi of huperzia serrata and application thereof in preparing huperzine A | |
| CN103911293B (en) | The endogenetic fungus that one plant height is paclitaxel produced and apply this bacterial strain and produce the method for taxol | |
| Zhang et al. | Microdochium paspali, a new species causing seashore paspalum disease in southern China | |
| CN102660467B (en) | Liquorice endophytic fungus for producing glycyrrhetinic acids | |
| CN103667072B (en) | A kind of Huperzia serrata endogenetic epiphyte and the application at preparation 8 α, 15 α-epoxidation selagine thereof | |
| CN102220247B (en) | Glycyrrhiza endophytic fungi for producing glycyrrhetic acid | |
| CN103468579B (en) | New lecanicillium bacteria genus fungi specie providing pathogenicity for diaphorina citri | |
| Wan et al. | Leaf spot of Ligustrum japonicum caused by Diaporthe eres newly reported in China | |
| CN105462854B (en) | Application of the sophora tonkinensis Gapnep endogenetic fungus SDTE-P in prevention and treatment Radix Notoginseng anthracnose | |
| CN102154123B (en) | Dioscorea nipponica Makino Fusarium sp. and application thereof | |
| Hao et al. | Isolation and identification of swainsonine-producing fungi found in locoweeds and their rhizosphere soil | |
| CN105462893A (en) | Application of sophora tonkinensis endophytic bacterium B29 in preventing and controlling panax notoginseng root rot | |
| CN105462847A (en) | Application of sophora tonkinensis endophytic fungus TRXY-46 in preventing and controlling panax notoginseng root rot | |
| CN109161488A (en) | One plant height produces Irpex lacteus strain and its cultural method of cordycepin | |
| CN101914452B (en) | Huperzine A high yield strain TCM-01 | |
| CN109234175A (en) | The Fusarium oxysporum DCLZJ-4 bacterial strain and application thereof of one plant of production chonglou saponin | |
| CN108085273A (en) | One plant of streptomyces antifungus and its metabolin, metabolin preparation method and application | |
| CN101418341A (en) | Method for quickly screening taxol-producing endophytic fungi | |
| CN102408998B (en) | Salt-tolerant marine mangrove endophytic fungi with neurodegenerative disease control activity | |
| Guo MengYuan et al. | Identification and bioactive potential of endogenetic fungi isolated from medicinal caterpillar fungus Ophiocordyceps sinensis from Tibetan plateau. | |
| CN105462894B (en) | Application of the sophora tonkinensis Gapnep endogenetic bacteria B29 in prevention and treatment Radix Notoginseng anthracnose | |
| CN102907253B (en) | Huperzia serrata endophyte and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130925 Termination date: 20170302 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |