CN103966108A - Method for quickly screening out antibacterial active filamentous fungi - Google Patents
Method for quickly screening out antibacterial active filamentous fungi Download PDFInfo
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- CN103966108A CN103966108A CN201410212913.XA CN201410212913A CN103966108A CN 103966108 A CN103966108 A CN 103966108A CN 201410212913 A CN201410212913 A CN 201410212913A CN 103966108 A CN103966108 A CN 103966108A
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Abstract
The invention relates to a method for quickly screening out antibacterial active filamentous fungi. The method comprises the following steps: (1), collecting samples; (2), preparing a culture medium; (3), preparing an intercepting suction head; (4), preparing a co-culture bacterial strain; (5), separating and co-culturing filamentous fungi; and (6), screening out the antibacterial active bacterial strain. According to the method disclosed by the invention, the antibacterial active filamentous fungi is quickly screened out by a co-culture way, and can increase screening probability of novel antibacterial active bacterial strain and antibacterial active substance in comparison with a mode of screening antibacterial active filamentous fungi by pure culture; and moreover, the whole screening process is quick, simple to operate, and capable of quickly, intuitively and efficiently screening out antibacterial active filamentous fungi, so that great convenience is provided for screening and researching the antibacterial active filamentous fungi.
Description
Technical field
The present invention relates to the fast separating process of a kind of microorganism, say more specifically a kind of method of rapid screening anti-microbial activity filamentous fungus.
Background technology
Antibiotic being widely used for a long time causes resistance pathogenetic bacteria kind to increase, even occur " superbacteria ", new infectious diseases also continues to bring out, cause the extremely urgent (Li Yuezhong of research and development of novel antibacterial material, Chen Qi. Marine Microorganisms and the research that produces biologically active metabolite product thereof, biotechnology progress, 2000,20 (5): 28-31).All the time, actinomycetes are considered to the main source of active secondary metabolites, but recent research shows, the potentiality of filamentous fungus product active secondary metabolites are also very huge, therefore screen filamentous fungus active secondary metabolites become study hotspot (Zhu Weiming, Wang Junfeng. the my humble opinion of thalassiomycetes biologically active substance research, fungus journal, 2011,30 (2): 218-228)
The screening method of microorganism active secondary metabolite is the committed step of the novel active secondary metabolites screening efficiency of restriction.Protocols in Molecular Biology is fast-developing, can avoid the sepn process of microorganism by building Metagenomic library screening new type natural product, has expanded natural product screening scope.But the extraction of environment large fragment DNA is difficult at present, gene is not expressed in host cell or expression amount is low, cannot carry out the practicality that the problems such as high-throughout screening bioactive compounds have restricted grand genomic library.By chemically modified or full chemosynthesis, can obtain antibiotic compounds, but building-up process is complicated, and causes environmental pollution.By genome, excavate the discovery efficiency that can improve natural product, but the Function Identification of natural product gene cluster is a unsolved difficult problem (white forests and streams still always, Deng Zixin. microbial secondary meta-bolites biological synthesis gene cluster and medicine innovation, China's microbiotic magazine, 2006,31 (2): 80-86).As can be seen here, microorganism is investigated, screened novel secondary metabolite producing bacterial strain, remain the antibiotic approach of a kind of important development of new.
Recent research shows, the common cultivation of two or more microorganisms can stimulate the secondary metabolite of microorganisms novelty, significant to the exploitation of novel materials, provides by cultivating altogether the possibility of the screening efficiency that improves novel antimicrobial substance.(Huang Bing, Liu Ning, Huang Ying, Chen Jingchun. the common cultivation of actinomycetes and subtilis and the impact on active secondary metabolite thereof, biotechnology journal, 2009,25 (6): 932-940).But, the screening method at present screening method of novel antimicrobial substance being carried out mainly with single pure strain.By common cultivation, screen novel antimicrobial substance and produce filamentous fungus, by traditional method, first separated filamentous fungus, to all bacterial strains and the common liquid culture fermentation of other bacterial strain, prepare fermented supernatant fluid or fermented liquid organic solvent extraction liquid, further with filter paper method, cup-plate method, punch method, measure bacteriostatic activity (Li little Jun, Cheng Lixia, Wu Yanbin, Bian Yanqing. the novel method that Antagonistic Fungi antimicrobial spectrum and fermented liquid antagonistic ability are measured, biotechnology, 2007,17 (1): 55-58).The method cost is high, and the cycle is long, and labour intensity is large.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, and a kind of method of training method screening anti-microbial activity filamentous fungus altogether of passing through that cost is low, simple to operate, the cycle is short, efficiency is high is provided.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is a kind of method of rapid screening anti-microbial activity filamentous fungus, is characterized in:
(1) sample collecting: gather water sample and soil sample with sterile sampling bottle, deliver to rapidly laboratory, 4 ℃ of preservations;
(2) substratum preparation: nutrient agar: extractum carnis 3g, peptone 10g, NaCl 5g, distilled water 1000 ml, pH7.2; PDA substratum: potato 200g, glucose 20g, distilled water 1000 ml, natural pH; If prepare solid medium flat board, add agar 20g/L; 121 ℃ of sterilizing 20min, treat that temperature is down to 60 ℃, add the Streptomycin sulphate of filtration sterilization and penbritin to final concentration to be respectively 100 mg/ml and 50 mg/ml, after mixing, are down flat plate; If separation is Oceanic Samples in above-mentioned cultivation in process for preparation, with seawater, replace distilled water;
(3) preparation of intercepting suction nozzle: get 1ml suction pipette head, with wide mouth end, be as the criterion, intercepting 12mm, rest part amputates, and edge polishes smooth, 121 ℃ of sterilizing 20min;
(4) cultivate altogether the preparation of bacterial strain: picking from inclined-plane
escherichia colipreserve bacterial classification, at thickness, be about three rides on 3mm nutrient agar flat board, 37 ℃ of inversions are cultured to and occur single bacterium colony; With the slot end of intercepting suction nozzle, exist
escherichia colion single bacterium colony, punch, make
escherichia colisingle bacterium colony and substratum enter in suction nozzle;
(5) filamentous fungus separation and altogether cultivation: institute's sample thief is carried out to gradient dilution, get the different dilution diluents of 50 μ L and be coated on the PDA flat board that thickness is about 4mm.Be inverted for 30 ℃ and cultivate 1-5d, at filamentous fungus bacterium colony, form the initial stage, colony diameter is less than before intercepting suction nozzle wide mouth end, with containing
escherichia colithe intercepting suction nozzle wide mouth end of single bacterium colony and substratum punches on substratum, and filamentous fungus bacterium colony and substratum are retained in intercepting suction nozzle wide mouth end, and makes both sides substratum leave the distance of about 3mm, and 30 ℃ are continued to cultivate 3d;
(6) anti-microbial activity bacterial strain screening: will cultivate altogether suction nozzle wide mouth end and be placed in downwards
staphylococcus aureus,
escherichia coli,
saccharomyces cerevisiae,
penicillium expansumdeng dull and stereotyped containing bacterium indication, and gently narrow a mouthful end substratum with sterilizing tweezers, make filamentous fungus in suction nozzle with
escherichia colicontact.Bacterial indicator substratum is nutrient agar, and fungi is PDA substratum.After the applicable temperature of indicator is cultivated, observe, and with vernier callipers, measure the size of inhibition zone, inhibition zone is larger, and bacterial strain anti-microbial activity is stronger, thereby rapid screening obtains anti-microbial activity filamentous fungus.
The bacterial strain relating in the present invention all comes from Chinese common micro-organisms DSMZ,
escherichia colipreserving number be CGMCC 1.487,
staphylococcus aureuspreserving number be CGMCC 1.2465,
saccharomyces cerevisiaepreserving number be CGMCC 2.114,
penicillium expansumpreserving number be CGMCC 3.3703.
In the step (5) of the method for a kind of rapid screening anti-microbial activity filamentous fungus of the present invention, will by intercepting suction nozzle
escherichia colicultivate altogether with filamentous fungus to be screened, and be put in indicator flat board, indicator is measured antibacterial circle diameter after cultivating, according to having or not and big or small rapid screening anti-microbial activity bacterial strain of inhibition zone.
Compared with prior art, the invention has the beneficial effects as follows: the inventive method is screened anti-microbial activity filamentous fungus rapidly by the mode of common cultivation, compare with purebred cultivation screening anti-microbial activity filamentous fungus, can increase the screening probability of novel anti-microbial activity bacterial strain and antibacterial substance, and whole screening process is quick, simple to operate, can screen fast, intuitively, efficiently the filamentous fungus of anti-microbial activity, for the screening study of anti-microbial activity filamentous fungus provides very big convenience.
Embodiment
By example, further illustrate the present invention below so that those skilled in the art the present invention may be better understood, and do not form the restriction to right of the present invention.
Embodiment 1, a kind of method of rapid screening anti-microbial activity filamentous fungus,
(1) sample collecting: gather seawater sample and soil sample with sterile sampling bottle, deliver to rapidly laboratory, 4 ℃ of preservations;
(2) substratum preparation: nutrient agar: extractum carnis 3g, peptone 10g, NaCl 5g, distilled water 1000 ml, pH7.2; PDA substratum: potato 200g, glucose 20g, distilled water 1000 ml, natural pH; If prepare solid medium flat board, add agar 20g/L; When separated for filamentous fungus, after PDA medium sterilization, treat that temperature is down to 60 ℃, add the Streptomycin sulphate of filtration sterilization and penbritin to final concentration to be respectively 100 mg/ml and 50 mg/ml, after mixing, be down flat plate; If Oceanic Samples in above-mentioned cultivation in process for preparation, replaces distilled water with seawater;
(3) preparation of intercepting suction nozzle: get 1ml suction pipette head, with wide mouth end, be as the criterion, intercepting 12mm, rest part amputates, and edge polishes smooth, 121 ℃ of sterilizing 20min;
(4) cultivate altogether the preparation of bacterial strain: picking from inclined-plane
escherichia colipreserve bacterial classification, at thickness, be about three rides on 3mm nutrient agar flat board, 37 ℃ of inversions are cultured to and occur single bacterium colony; With the slot end of intercepting suction nozzle, exist
escherichia colion single bacterium colony, punch, make
escherichia colisingle bacterium colony and substratum enter in suction nozzle;
(5) filamentous fungus separation and altogether cultivation: institute's sample thief is carried out to gradient dilution, get the different dilution diluents of 50 μ L and be coated on the PDA flat board that thickness is about 4mm.Be inverted for 30 ℃ and cultivate 1-5d, at filamentous fungus bacterium colony, form the initial stage, colony diameter is less than before intercepting suction nozzle wide mouth end, with containing
escherichia colithe intercepting suction nozzle wide mouth end of single bacterium colony and substratum punches on substratum, and filamentous fungus bacterium colony and substratum are retained in intercepting suction nozzle wide mouth end, and makes both sides substratum leave the distance of about 3mm, and 30 ℃ are continued to cultivate 3d;
(6) anti-microbial activity bacterial strain screening: will cultivate altogether suction nozzle wide mouth end and be placed in downwards
staphylococcus aureus,
escherichia coli,
saccharomyces cerevisiae,
penicillium expansumdeng dull and stereotyped containing bacterium indication, and gently narrow a mouthful end substratum with sterilizing tweezers, make filamentous fungus in suction nozzle with
escherichia colicontact.Bacterial indicator substratum is nutrient agar, and fungi is PDA substratum.After the applicable temperature of indicator is cultivated, observe, and with vernier callipers, measure the size of inhibition zone; Inhibition zone is larger, and bacterial strain anti-microbial activity is stronger; Thereby rapid screening obtains anti-microbial activity filamentous fungus.
(7) cultural characteristic of bacterial strain and Physiology and biochemistry are identified
The evaluation of fungi is with reference to < < fungi identification handbook > > (Wei Jingchao, Shanghai: Shanghai science tech publishing house, 1979) and document (permitted Yulin, Zheng Yuexia, Ye Bingying, Zhang Jisen, Chen Youqiang. screening and the evaluation of a strain cellulose degradation fungi, microorganism journal, 2013,40 (2): 220 227) carry out.
Embodiment 2, in the step (5) of the method for a kind of rapid screening anti-microbial activity filamentous fungus described in embodiment 1, and will by intercepting suction nozzle
escherichia colicultivate altogether with filamentous fungus to be screened, and be put in indicator flat board, indicator is measured antibacterial circle diameter after cultivating, according to having or not and big or small rapid screening anti-microbial activity bacterial strain of inhibition zone.
Embodiment 3, contrast experiment:
1. art methods screening.From marine site, Lianyun Harbour, Lian Dao gets seawater, ooze, gathers soil and fresh water sample on flowers and fruits mountain, by method of dilution butteron on plate, utilizes the separated filamentous fungus of PDA substratum, in Oceanic Samples, in filamentous fungus isolation medium, with seawater, replaces distilled water.By the single colony inoculation after purifying to 4 ℃ of preservations after PDA slant culture.Be divided into from obtaining filamentous fungus 122 strains.Different strains is seeded to PDA liquid nutrient medium, and 30 ℃, 160rpm cultivates 2-3d, and after the double-deck filter paper suction filtration of fermented liquid, supernatant liquor crosses 0. 22
μafter m millipore filtration, be testing sample, adopt cup-plate method to measure bacteriostatic activity.In each Oxford cup, add 200
μl fermented sample, employing contains
staphylococcus aureus,
escherichia coli,
saccharomyces cerevisiae,
penicillium expansumindicator is dull and stereotyped, after 30 ℃ of cultivations, observes, and obtains altogether anti-
staphylococcus aureusactive bacterial strain 18 strains, anti-
escherichia coliactive bacterial strain 13 strains, anti-
saccharomyces cerevisiaeactive bacterial strain 3 strains, anti-
penicillium expansumactive bacterial strain 8 strains.
2. the inventive method that adopts embodiment 1 to record is screened.With above-mentioned same Ji Lu source, ocean sample, by method of dilution butteron on plate, utilize the separated filamentous fungus of PDA substratum.Picking from inclined-plane
escherichia colipreserve bacterial classification, at thickness, be about three rides on 3mm nutrient agar flat board, 37 ℃ of inversions are cultured to and occur single bacterium colony; With the slot end of intercepting suction nozzle, exist
escherichia colion single bacterium colony, punch, make
escherichia colisingle bacterium colony and substratum enter in suction nozzle.At filamentous fungus bacterium colony, form the initial stage, colony diameter is less than before intercepting suction nozzle wide mouth end, with containing
escherichia colithe intercepting suction nozzle wide mouth end of single bacterium colony and substratum punches on substratum, and filamentous fungus bacterium colony and substratum are retained in intercepting suction nozzle wide mouth end, and makes both sides substratum leave the distance of about 3mm, and 30 ℃ are continued to cultivate 3d.The intercepting suction nozzle wide mouth end of this common culturing micro-organisms is placed in and is contained
staphylococcus aureus,
escherichia coli,
saccharomyces cerevisiae,
penicillium expansumindicator is dull and stereotyped, after 30 ℃ of cultivations, observes, and obtains altogether anti-
staphylococcus aureusactive bacterial strain 22 strains, anti-
escherichia coliactive bacterial strain 15 strains, anti-
saccharomyces cerevisiaeactive bacterial strain 6 strains, anti-
penicillium expansumactive bacterial strain 10 strains.
From above contrast screening experiment, can find out, the inventive method has improved the screening efficiency of anti-microbial activity filamentous fungus significantly.
Claims (2)
1. a method for rapid screening anti-microbial activity filamentous fungus, is characterized in that, its step is as follows:
(1) sample collecting: gather water sample and soil sample with sterile sampling bottle, deliver to rapidly laboratory, 4 ℃ of preservations;
(2) substratum preparation: nutrient agar: extractum carnis 3g, peptone 10g, NaCl 5g, distilled water 1000 ml, pH7.2; PDA substratum: potato 200g, glucose 20g, distilled water 1000 ml, natural pH; While preparing solid medium flat board, add agar 20g/L; When separated for filamentous fungus, after PDA medium sterilization, treat that temperature is down to 60 ℃, add the Streptomycin sulphate of filtration sterilization and penbritin to final concentration to be respectively 100 mg/ml and 50 mg/ml, after mixing, be down flat plate; If Oceanic Samples in above-mentioned substratum process for preparation, replaces distilled water with seawater;
(3) preparation of intercepting suction nozzle: get 1ml suction pipette head, with wide mouth end, be as the criterion, intercepting 12mm, rest part amputates, and edge polishes smooth, 121 ℃ of sterilizing 20min;
(4) cultivate altogether the preparation of bacterial strain: picking from inclined-plane
escherichia colipreserve bacterial classification, at thickness, be about three rides on 3mm nutrient agar flat board, 37 ℃ of inversions are cultured to and occur single bacterium colony; With intercepting suction nozzle slot end, exist
escherichia colion single bacterium colony, punch, make
escherichia colisingle bacterium colony and substratum enter in suction nozzle;
(5) filamentous fungus separation and altogether cultivation: institute's sample thief is carried out to gradient dilution, get the different dilution diluents of 50 μ L and be coated on the PDA flat board that thickness is 4mm; Be inverted for 30 ℃ and cultivate 1-5d, at filamentous fungus bacterium colony, form the initial stage, colony diameter is less than before intercepting suction nozzle wide mouth end, with containing
escherichia colithe intercepting suction nozzle wide mouth end of single bacterium colony and substratum punches on substratum, and filamentous fungus bacterium colony and substratum are retained in intercepting suction nozzle wide mouth end, and makes approximately 3mm of both sides substratum distance, and 30 ℃ are continued to cultivate 3d;
(6) anti-microbial activity bacterial strain screening: will cultivate altogether suction nozzle wide mouth end and be placed in downwards
staphylococcus aureus,
escherichia coli,
saccharomyces cerevisiae,
penicillium expansumdull and stereotyped containing bacterium indication, and gently narrow a mouthful end substratum with sterilizing tweezers, make filamentous fungus in suction nozzle with
escherichia colicontact; Bacterial indicator substratum is nutrient agar, and fungi is PDA substratum; After the applicable temperature of indicator is cultivated, observe, and with vernier callipers, measure the size of inhibition zone, inhibition zone is larger, and bacterial strain anti-microbial activity is stronger, thereby rapid screening obtains anti-microbial activity filamentous fungus.
2. the method for a kind of rapid screening anti-microbial activity filamentous fungus according to claim 1, is characterized in that: will by intercepting suction nozzle in step (5)
escherichia colicultivate altogether with filamentous fungus to be screened, and be put in indicator flat board, indicator is measured antibacterial circle diameter after cultivating, according to having or not and big or small rapid screening anti-microbial activity bacterial strain of inhibition zone.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105441336A (en) * | 2015-12-23 | 2016-03-30 | 上海市农业科学院 | Method for effectively preventing degradation of filamentous fungi and restoring spore production |
| CN108486031A (en) * | 2017-11-29 | 2018-09-04 | 安徽科技学院 | A kind of acclimation method of quick raising filamentous fungi heavy metal tolerance |
Citations (1)
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| CN101050438A (en) * | 2007-03-09 | 2007-10-10 | 吉林大学 | New method for separating and purifying strain of bacteria of sulfate reducting bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101050438A (en) * | 2007-03-09 | 2007-10-10 | 吉林大学 | New method for separating and purifying strain of bacteria of sulfate reducting bacteria |
Non-Patent Citations (3)
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| 刘姝: "海洋链霉菌GB-2的分离筛选及其抗菌物质的研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441336A (en) * | 2015-12-23 | 2016-03-30 | 上海市农业科学院 | Method for effectively preventing degradation of filamentous fungi and restoring spore production |
| CN105441336B (en) * | 2015-12-23 | 2019-09-10 | 上海市农业科学院 | A method of it effectively prevent filamentous fungi to degenerate and restore to produce spore |
| CN108486031A (en) * | 2017-11-29 | 2018-09-04 | 安徽科技学院 | A kind of acclimation method of quick raising filamentous fungi heavy metal tolerance |
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