CN101691540B - Muscodor endophytic fungi ZJLQ070 and application thereof and fungicide - Google Patents
Muscodor endophytic fungi ZJLQ070 and application thereof and fungicide Download PDFInfo
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Abstract
The invention discloses endophytic fungi Muscodor sp. The preservation name of the strain is ZJLQ070, the preservation unit is China General Microbiological Culture Collection Center, the preservation date is January 8, 2009 and the preservation number is CGMCC 2864. The endophytic fungi can be prepared into living preparations and can be used for suppressing or killing pathogenic microorganisms or musty microorganisms. The invention simultaneously discloses a fungicide comprising 2-methyl-methyl propionate, 2-methylpropionate and the like, especially comprising alpha-phellandrene, beta-phellandrene, trans1-methyl-4-(1-methyl ethyl)-2-cyclohexene-1-alcohol, cis1- methyl-4-(1-methyl ethyl)-2-cyclohexene-1-alcohol, trans3-methyl-6-(1-methyl ethyl)-2-cyclohexene-1-alcohol and cis3-methyl-6-(1-methyl ethyl)-2-cyclohexene-1-alcohol.
Description
Technical field
The present invention relates to microbiology and sterilant field, specifically, the present invention relates to a kind of new Muscodor platymiscium endogenetic fungus and based on or be derived from its volatility mixture and their purposes thereof.
Background technology
Fungi is a huge family in the microorganism, and fungi is very close with human relation, and fungi can be in order to make important product in the industrial or agricultural such as medicine, food, chemical industry.Famous penicillin and cephamycin are that fungi brings one of most precious medicine of the world of medicine up to now, also are present most important microbiotic, are widely used in the treatment of bacterial infective diseases, and have saved thousands upon thousands mankind's life.
Plant endogenesis epiphyte (endophytic fungi) is the important monoid of fungi, estimates that according to Dreyfuss and Chapel the endogenetic fungus sum reaches 1,000,000 kinds.Existing a few is used to medicine, agronomy production or industrial application aspect now, but the plant endogenesis epiphyte resource is not also fully excavated far away.Endogenetic fungus has diversified ecological function, and the ecological function of endogenetic fungus is likely relevant with its diversity, and the diversity of endogenetic fungus means its Chemical Diversity [3].Endogenetic fungus can produce various secondary metabolites, and they have the important use potentiality in human lives, production, can produce antineoplastic compound such as taxol as endogenetic fungus; Novel anti-tumor actives such as oreganic acid; New compound cryptocin, the cryptocandin etc. of pathogenic fungies such as anti-candida albicans, alpha fungus and trichophyton purpureatum; Generation has periconicins, the rhizoctonic acid etc. of pathogenetic bacterias such as anti-helicobacter pylori, streptococcus aureus, kerekou pneumonia Salmonella and Salmonella typhimurium.
Now found that multiple different microorganism has the biologically active substance that produces the controlling plant disease, comprises sterilant, sterilant and plant-growth regulator etc.Research for the pathogenic micro-organism of controlling pathogenic micro-organism, material of construction mildew micro-organism and the mankind or animal living enviroment in the farm crop production process at present also has many, and important progress is arranged, but mainly still rely on chemical synthesis process to produce sterilant or sterilant.Use the chemosynthesis medicament to bring a series of serious consequence: 1) high residue: because the chemosynthesis medicament comprises that its multiple starting material have carinogenicity and toxicity, enter and be not easy eliminating in the animal and plant body, the environment degradable ability causes the pesticide residue height; 2) destroy the eubiosis: biological long-term evolution and adaptation, the various biological relative populations of occurring in nature reach running balance, use chemical agent to cause the natural enemy quantity of harmful organism to significantly reduce; 3) drug-fast generation: the pathogenic bacteria of occurring in nature and the gene of insects have very strong sudden change ability, and non-rational use of drug, the medication of the region between the heart and the diaphragm order impel drug-fast rapid generation.Because above-mentioned all shortcomings, the use of chemosynthesis medicament is also more and more restricted, develop new, the mechanism of action is unique and people and animals and the harmless biotechnological formulation of ecotope more and more are much accounted of, and also becomes the important directions that modern pesticide industry develops.
Many kinds of fungies can produce some light-concentration volatile gases, and some volatile gases also has antibiotic, desinsection or viricidal function, as Dennis﹠amp; The volatile gases material that discoveries such as Webster wood mould (Trichoderma) produces can suppress the growth of dry thread Pyrenomycetes (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), ultimate corruption mould (Pythium ultimum) etc.Strobel etc. are separated to from tropical xylophyta can produce volatility compounding substances (volatile compounds, VOCs) four kinds of Muscodor belong to endogenetic fungus, and they are respectively cinnamon Ceylon cinnamon (Cinnamomum zeylanicum) endogenetic fungus Muscodor albus, fern leaf silvery birch (Grevilleapteridifolia) endogenetic fungus Muscodor roseus, torrid zone bejuco (Paullinia paullinioides) endogenetic fungus Muscodor vitigentis, Ananas plant Ananas ananassoides endogenetic fungus Muscodorcrispans.The VOCs that Muscodor albus, Muscodor roseus, Muscodor vitigentis and Muscodor crispans produce has inhibitory or killing effect to fungi, bacterium and virus etc., in control or kill pathogenic micro-organism in pathogenic micro-organism, material of construction mildew micro-organism and the mankind or the animal living enviroment in the farm crop production process and have important use and be worth, they and also carried out relevant commercial development.
Utilize the applied research of mycetogenetic VOCs at present also rare, Strobel etc. are about achievement in research and the Application and Development of living fungi inside the Pass the Muscodor symbolic animal of the birth year, it is the important channel of seeking novel cpd, newtype drug or newtype drug intermediate that proof Muscodor belongs to mycetogenetic VOCs, also is the valuable source of development of new biotechnological formulation.Utilize mycetogenetic VOCs to control the pathogenic micro-organism of pathogenic micro-organism, material of construction mildew micro-organism and the mankind or animal living enviroment in the farm crop production process, have the non-target organism toxicological harmless, free from environmental pollution, do not destroy the eubiosis, not the advantage that can induce harmful microorganism, virus and insect etc. to develop immunity to drugs.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of VOCs and produces fast, as to suppress the volatility thing of novel Muscodor genus fungal bacterial strain of various plants pathogenic fungi, bacterium and zymic and generation thereof lastingly combination.
In order to solve the problems of the technologies described above, the invention provides a kind of plant endogenesis epiphyte, this plant endogenesis epiphyte Muscodor sp. bacterial strain preservation name is called: ZJLQ070, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on January 8th, 2009, preservation address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preserving number: CGMCC 2864.
The present invention also provides the live body preparation that utilizes above-mentioned plant endogenesis epiphyte to make simultaneously, and it is the stable live body preparation that comprises the compositions such as cultivation viable bacteria, ventilative and hygroscopicity solid carrier, stablizer, trace element, carbon nitrogen nutrition source and growth hormone of Muscodor genus fungi ZJLQ070.
Above-mentioned live body preparation can adopt following method to make: with the activation culture of endogenetic fungus Muscodor sp.ZJLQ070, insert seed culture fluid, after with stirrer mycelium being blended, add seed culture fluid and make thalline recover growth, insert solid medium, pack after the vacuum lyophilization.
The present invention also provides the purposes of above-mentioned plant endogenesis epiphyte or its live body preparation simultaneously, it is characterized in that: be used for inhibition or pathogenic microbe killing or mildew micro-organism.
Improvement as the purposes of plant endogenesis epiphyte of the present invention or live body preparation: pathogenic micro-organism is pathogenic micro-organism in the farm crop production process or the pathogenic micro-organism in the mankind or the animal living enviroment; Described mildew micro-organism is the material of construction mildew micro-organism.
The present invention also provides a kind of sterilant simultaneously, this sterilant comprises 0.65%～10.2%2-methyl-methyl propionate, 28%～78%2-methylpropanoic acid, 0.6%～2.5%1-vinyl-1-methyl-2.4-two (1-methyl ethylene)-hexanaphthene, 0.10%～0.4% caryophyllene, 0.06%～1%2,6-dimethyl-6-(4-methyl-3-amylene) two ring [3.1.1] hept-2-ene"s, 0.8%～3.5%1,2,3,4,5,6,7,8-octahydroization-1,4-dimethyl-7-(1-methyl ethylidene) Azulene, 5.8%～11%1,2,3,4,4a, 5,6,8a-octahydroization-4a, 8-dimethyl-2-(1-methylethyl) naphthalene, 0.05%～0.2% 3,3,7,11-tetramethyl-three ring [6.3.0.0 (2,4)] 11 carbon-8-alkene, 0.3%～2.8%a-phellandrene, 4%～45% β-phellandrene, 0.1%～0.6% trans 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.08%～0.4% cis 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.25%～4% trans 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.03%～0.28% cis 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol; All the other are solvent; Above per-cent is volume percent.
Improvement as sterilant of the present invention: sterilant comprises 7.8%～8.2%2-methyl-methyl propionate, 76.5%～78%2-methylpropanoic acid, 0.63%～0.7%1-vinyl-1-methyl-2.4-two (1-methyl ethylene)-hexanaphthene, 0.10%～0.12% caryophyllene, 0.06%～0.08%2,6-dimethyl-6-(4-methyl-3-amylene) two ring [3.1.1] hept-2-ene"s, 0.8%～0.9%1,2,3,4,5,6,7,8-octahydroization-1,4-dimethyl-7-(1-methyl ethylidene) Azulene, 5.8%～6.2%1,2,3,4,4a, 5,6,8a-octahydroization-4a, 8-dimethyl-2-(1-methylethyl) naphthalene, 0.05%～0.07% 3,3,7,11-tetramethyl-three ring [6.3.0.0 (2,4)] 11 carbon-8-alkene, 0.3%～0.4%a-phellandrene, 4%～4.5% β-phellandrene, 0.1%～0.15% trans 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.08%～0.12% cis 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.3%～0.35% trans 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.03%～0.05% cis 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol; All the other are solvent (for conventional solvent, as: acetone, cyclohexane, methyl alcohol etc.).
The VOCs that endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 produces is effective sterilant, to dry thread Pyrenomycetes, ultimate corruption is mould, Fusarium oxysporum, alternaria tenuis (Alternaria alternata), aspergillus (Aspergillus sp), Botrytis cinerea (Botrytis cinerea), broad bean grape spore (Botrytis fabae), circular thorn dish spore (Colletotrichumorbiculare), the bryonia Asia is every spore shell (Didymella bryoniae), Fulvia fulva (Fulvia fulva), Paecilomyces lilacinus (Paecilomyces lilacinus), Penicillium digitatum (Penicillum digitatum), persimmon dish stey (Pestalotiadiospyri), piricularia oryzae (Pyricularia oryzae), Sclerotium rolfsii (Sclerotium rolfsii), sclerotinite (Sclerotinia sclerotiorum), big beautiful Verticillium (Verticillium dahliae), yeast saccharomyces cerevisiae (Saccharomycescerevisiae), intestinal bacteria (Escherichia coli) and leaf bacillus multiple cause of disease filamentous funguss such as (Phyllobacterium sp.), yeast and Gram-negative bacteria and gram-positive microorganism all have inhibition or killing action.
Endogenetic fungus Muscodorsp. bacterial strain ZJLQ070 has that to produce VOCs speed fast, the characteristics that long-lasting is long, the mould cell walls with piricularia oryzae of the ultimate corruption of degrade, and this VOCs have a uniqueness make the joyful fragrance of people.
Endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 can utilize barley meal, fine rice bran, millet powder and sucrose etc. to be carbon source, can utilize peptone, yeast powder, yeast extract paste, wheat bran, soybean cake powder and Dried Corn Steep Liquor Powder etc. to be nitrogenous source, can on cereal or wheat class, potato dextrose agar, oat agar glucose or wort agar glucose, grow, and produce VOCs with bacteriostatic activity.
Endogenetic fungus Muscodorsp. bacterial strain ZJLQ070 can not be a sole carbon source with any in glucose, fructose, maltose, Zulkovsky starch, dextrin, sodium acetate, Sodium Propionate, ammonium tartrate, Trisodium Citrate, Sodium.alpha.-ketopropionate and the glycerol, can not be only nitrogen source with any in urea, SODIUMNITRATE, ammonium nitrate, the ammonium sulfate.
Endogenetic fungus Muscodorsp. bacterial strain ZJLQ070 ribosomal deoxyribonucleic acid internal transcribed spacer district (Internaltranscribed spacer of ribosomal DNA, ITS rDNA), the big subunit of ribosomal deoxyribonucleic acid (28SrDNA), ribonucleic acid polymerase II subunit (RNA polymerase II subunit, RPB2), 'beta '-tubulin (beta-tubulin) gene has unique sequence.
The active carrier preparation of endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 has the business development purposes, the VOCs of ZJLQ070 culture and generation thereof can be used for handling or preventing material of construction and the poisonous mould of buildings, can be used for the fresh-keeping mildew-resistant of fruit and food, can be used for preventing or killing harmful mould of grain and feed, processing, flowers or the fruit that can be used for agriculture production soil are dredged the control of disease.
VOCs of the present invention can obtain from the novel endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 culture separation that is grown on cereal or wheat class, potato dextrose agar, oat agar glucose or the wort agar glucose.
VOCs can be used as fumigant as the inhibition of the pathogenic micro-organism in pathogenic micro-organism, material of construction mildew micro-organism and the mankind or the animal living enviroment in the farm crop production process or kill, and has the great potential of therefrom developing novel, efficient, nontoxic natural bactericidal agent.
The VOCs that produces in Muscodor sp.ZJLQ070 live body preparation of the present invention or the endogenetic fungus Muscodor sp ZJLQ070 culturing process and comprise one or more phellandrenes or the purposes of the synthesizing fungicide of 2-tetrahydrobenzene-1-alcohol derivate.They are applicable to industry, agricultural, aquaculture and service industry etc.For example they can handle or prevent poisonous mould in material of construction and the buildings; Can be used for the fresh-keeping mildew-resistant of fruit and food; Can be used for preventing or killing harmful mould of grain and feed; Processing, flowers or the fruit that can be used for agriculture production soil are dredged the control of disease.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is Muscodor sp.ZJLQ070 bacterium colony figure;
Fig. 2 is a Muscodor sp.ZJLQ070 mycelia sem photograph;
Fig. 3 bacterial strain ZJLQ070 rrna rDNA internal transcribed spacer district (Internal Transcribed Spacer, ITSrDNA) evolutionary tree of Gou Jianing;
The evolutionary tree that Fig. 4 bacterial strain ZJLQ070 large ribosomal subunit (28SrDNA) makes up;
Fig. 5 bacterial strain ZJLQ070RNA polymerase II (RNA polymerase II subunit, the RPB2) evolutionary tree of Gou Jianing;
The evolutionary tree that Fig. 6 bacterial strain ZJLQ070beta-tubulin gene makes up;
Fig. 7 is the influence (scanning electron microscope picture) of the volatile gases (VOCs) of Muscodor sp.ZJLQ070 generation to plant pathogenic fungi:
A: the mould Pythium ultimum of ultimate corruption, B: piricularia oryzae Pyricularia oryzae, C:Muscodor sp.ZJLQ070 and Pyricularia oryzae, D:Muscodor sp.ZJLQ070 and Pythium ultimum,
Fig. 8 is that Muscodor.sp bacterial strain ZJLQ070 active carrier preparation and Muscodor.sp bacterial strain ZJLQ070 barley corn culture contrast the phytopathogen inhibiting rate;
A, aspergillus Aspergillus.SP; B, Fusarium oxysporum (Fusarium oxysporum); C, Botrytis cinerea Botrytiscinerea; D, ultimate corruption mould (Pythium ultimum);
A, aspergillus Aspergillus.SP; B, Fusarium oxysporum (Fusarium oxysporum); C, Botrytis cinerea Botrytiscinerea; D, ultimate corruption mould (Pythium ultimum);
Annotate: wherein capitalize A, B, C, D and be pathogenic bacteria and the Muscodorsp. bacterial strain ZJLQ070 barley corn culture cultivation results that stands facing each other; Small letter a, b, c, d are pathogenic bacteria and Muscodorsp. bacterial strain ZJLQ070 active carrier preparation face-off cultivation results;
Fig. 9 is that fruit rotted to test comparison diagram after Muscodor sp bacterial strain ZJLQ070 volatility mixture VOCs control was gathered;
A, Muscodorsp bacterial strain ZJLQ070 culture; B, blank.
Embodiment
The separation and purification of embodiment 1, endogenetic fungal bacterial strain
(1) collection of plant tissue specimens: (it is domestic that Fengyangshan Nature Reserve is positioned at the west and south, Zhejiang Province Longquan City from man of Chinese Zhejiang Fengyang mountain country wilderness area; the geographical position is 119 ° 06 '～119 ° 15 ' E; 27 ° 46 '～27 ° 58 ' N; weather is the maritime monsoon climate of typical middle subtropical zone); at height above sea level 1200～1500m herborization sample; comprising A.chinensis Planch. (Chinese goosebeery) and camplotheca acuminata (Camptothecaacuminata); gather the position and comprise root; stem; leaf and fruit; be stored in freshness protection package after the collection, be no more than 48h 4 ℃ of following shelf times of condition.
(2) separation and Culture of endogenetic fungus: the herbarium tissue of step (1) is washed surperficial grieshoch and dirt settling with tap water, and airing surface moisture content is through 75% dehydrated alcohol and 0.5% clorox surface sterilization.Root, stem, leaf and fruit are controlled at 30-60s according to face tissue with 75% dehydrated alcohol disinfectant time, and 0.5% clorox disinfecting time is 2-10min.Use aseptic water washing three times after the surface sterilization, aseptic filter paper blots the tissue that is cut into 3 * 3mm size behind the surperficial moisture content with the aseptic operation cutter, place potato dextrose agar (potato dextrose agar, PDA) (contain penbritin 100 μ g/ml and Vetstrep 60 μ g/ml) on the substratum, connect 12 block organization's sheets in the culture dish of 90mm approximately.Cultivate in 25 ℃ of biochemical incubators, around plant tissue, grow mycelium, picking mycelia tip places on the fresh PDA substratum, be stored in after the culture purified in PDA slant medium and the liquid freezing substratum, PDA slant medium culture adds whiteruss normal temperature to be preserved, and culture is stored in-80 ℃ of super low-temperature refrigerators in the liquid freezing substratum.The material that surface sterilization was handled is not done shearing and is directly planted to check in contrast on the PDA flat board whether surface sterilization is thorough, guarantees to separate endogenetic fungus that the fungi that obtains is a plant but not surperficial epiphyte.The inventor places the insulation ice chest to take back the laboratory sample of gathering to separate, be divided into from obtaining 158 strain endogenetic fungal bacterial strains.
Embodiment 2, the endogenetic fungus screening of producing volatility mixture VOCs
Employing has lattice culture dish face-off culture method, poured in the lattice plastic culture dish (90mm) melting good PDA substratum, the stainless steel punch tool of diameter 5mm cuts endogenetic fungus bacterium cake, insert culture dish one side, behind 25 ℃ of cultivation 2d-5d, insert the cause of disease indicator at the dull and stereotyped opposite side of PDA, be contrast only to connect the cause of disease indicator simultaneously, sealing the film film with parafilm seals, indicator to be contrasted is covered with culture dish one rear flank, observe growth and the antibacterial situation of endogenetic fungus, measure the indicator colony diameter, filter out the endogenetic fungus that can produce volatility mixture VOCs and can suppress the pathogenic bacteria growing indication pathogenic bacteria bacterium colony.
158 strain endogenetic fungal bacterial strains after the separation and purification have been carried out bioactivity screening, respectively with Botrytis cinerea, dry thread Pyrenomycetes, ultimate corruption is mould to be indicator, 5 bacterial strain ZJLQ023 belonging to of Muscodorsp. wherein, ZJLQ024, ZJLQ070, ZJLQ151, ZJLQ374 produces the VOCs with anti-microbial activity, to three kinds of indicator Botrytis cinereas, dry thread Pyrenomycetes, ultimate corruption is mould all to have very strong inhibition activity, particularly ZJLQ023, ZJLQ024, the volatility mixture VOCs that three bacterial strains of ZJLQ070 produce can kill Botrytis cinerea fully, dry thread Pyrenomycetes, ultimate corruption is mould.Muscodor sp. bacterial strain ZJLQ070 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on January 8th, 2009, preserving number: CGMCC 2864.
The test of embodiment 3, Muscodor sp. bacterial strain ZJLQ070 bacteriostatic activity spectrum
With endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 and Muscodor albus (200480025237.X informs as patent) the activation 10-15d standby (culture temperature is 25 ℃) that on the PDA substratum, grows.Respectively that Botrytis cinerea, dry thread Pyrenomycetes, alternaria tenuis, neat microsolerotium, ultimate corruption is mould, Fusarium oxysporum cucumber specialized form, circular thorn dish spore, big beautiful Verticillium, sclerotinite, persimmon dish stey, piricularia oryzae, Penicillium digitatum, broad bean grape spore, Fulvia fulva, bryonia Asia activate on the PDA substratum every spore shell, aspergillus, Paecilomyces lilacinus, switching is gone on the PDA fresh culture, uses behind the growth 3-7d down at 25 ℃; Yeast saccharomyces cerevisiae is rule on the YEPD solid medium, chooses single bacterium colony, is inoculated in to shake bacterium (200r/min, 30 ℃) in the YEPD liquid nutrient solution, uses behind the 48h; Intestinal bacteria, leaf bacillus are rule on the LB solid medium earlier, choose single colony inoculation in the LB liquid medium, and 37 ℃ of cultivation 24h are standby.
Poured in the lattice plastic culture dish (90mm) melting good PDA substratum, with diameter is that the stainless steel punch tool of 5mm cuts endogenetic fungus Muscodorsp. bacterial strain ZJLQ070 bacterium cake, insert culture dish one side with inoculating needle, after cultivating 3d, 5d, 7d respectively under 25 ℃ of conditions, insert the cause of disease indicator at the dull and stereotyped opposite side of PDA, be contrast only to connect the cause of disease indicator simultaneously, parafilm seals film and seals, and cultivates the growth and the antibacterial situation of observing bacterium colony behind the certain hour.(wherein yeast saccharomyces cerevisiae, intestinal bacteria, leaf bacillus are adopted the method for scoring inoculation, and all the other fungies are all adopted bacterium cake inoculation method), with the positive contrast of Muscodoralbus bacterial strain, test result sees Table 1.
Table 1Muscodorsp. bacterial strain ZJLQ070 and Muscodoralbus are to the bacteriostatic activity of indicator
Annotate: data are the survival rate of indicator in the table.
Muscodorsp. do not suppress the other side's growth between bacterial strain ZJLQ070 and the Muscodoralbus mutually, Muscodorsp. bacterial strain ZJLQ070 also can not influence the growth of wooden mould Trichoderma sp..Muscodorsp. bacterial strain ZJLQ070 to the inhibition of Fusarium oxysporum and Paecilomyces lilacinus relative a little less than, Muscodoralbus is not strong to the inhibition of Fusarium oxysporum and Paecilomyces lilacinus and Penicillium digitatum, and Muscodorsp. bacterial strain ZJLQ070 can suppress the growth of Penicillium digitatum fully, and the inhibition of Fusarium oxysporum and Paecilomyces lilacinus also is eager to excel than Muscodoralbus.Muscodorsp. bacterial strain ZJLQ070 and Muscodor albus have very strong inhibition/killing action to other selected indicator.
The VOCs that Muscodor sp. bacterial strain ZJLQ070 produces can decompose the mould hyphal cell wall with piricularia oryzae of ultimate corruption, thereby cause ultimate corruption mould deadly with the collapse of piricularia oryzae thalline, but Muscodoralbus can only suppress the pathogenic bacteria growth, and the ultimate corruption of can not degrading is mould and the piricularia oryzae mycelium.(scanning electronmicroscopy, SEM) VOCs of shooting Muscodor sp. bacterial strain ZJLQ070 generation cell walls mould to ultimate corruption and piricularia oryzae decomposes situation with the vacuum freezing scanning electronic microscope.Microscope is HITACHI S-3000N, and refrigeration system is ALTO2100.Ready material is cut into size about 0.8cm * 0.8cm (substratum try one's best book), stick on the sample table with glue special, place liquid nitrogen to carry out cryofixation, temperature is-189 ℃; Move into the distillation that heats up in the ALTO2100 system of vacuum state afterwards, controlled temperature is at-96 ℃, time 5min; When beginning to cool to below-140 ℃ after distillation finishes, the gold-plated 2min of electron spray(ES); Afterwards sample is slowly sent on the stationary platform, (Fig. 7) observes in the function software that opens HITACHI S-3000N microscopic system behind the adjustment position.
The culture of Muscodor sp. bacterial strain ZJLQ070 and Muscodor albus cultivation different time compares the restraining effect of pathogenic bacteria, the result shows: Muscodor sp. bacterial strain ZJLQ070 cultivates 3d and inoculates the cause of disease indicator after the time, it is obviously strong than Muscodor albus that it suppresses effect, not obviously difference between them behind cultivation 5d and the 7d, it is fast to illustrate that ZJLQ070 produces active VOCs, can suppress growth of pathogenic bacteria faster, and have the characteristics of a specified duration of imitating of holding.
Embodiment 4, endogenetic fungus ZJLQ070 morphology and Physiology and biochemistry are identified
Bacterial strain ZJLQ070 identifies by the means such as VOCs component of morphology, molecular systematics and generation.Reference literature [1-2] method, adopt PDA, wort agar (malt extract agar, MEA) and short spore substratum carry out inserted sheet and cultivate, inducible strain ZJLQ070 produces spore, but induces mode (illumination and chemical reagent) all not produce conidium and conidial fructification through multiple short spore.Colonial morphology, color, growth velocity and microscopic morphology feature to endogenetic fungus ZJLQ070 are observed, at PDA, MEA and short spore substratum (KH2PO41g, KNO31g, MgSO47H200.5g, KCl 0.5g, Zulkovsky starch 0.2g, glucose 0.2g, sucrose 0.2g, agar 15g, water 1000mL, natural pH value) on all do not secrete pigment, the bacterium colony densification (see figure 1) that is white in color does not have conidium or conidial fructification to form.Utilize the vacuum freezing scanning electronic microscope that the mycelia form of bacterial strain ZJLQ070 is observed, mycelia is cord-like and twines (as shown in Figure 2).
[reference]
[1] Wei Jingchao, the fungi identification handbook. Shanghai science tech publishing house, 1979
[2] Hawksworth DL, Kirk PM, Sutton BC, et al.1995.Ainsworth﹠amp; Bisby ' sDictionary of the Fungi.8th edn.Cambridge University Press, London. fungi dictionary (the 8th edition), London: Cambridge press
At basic medium (ammonium nitrate 2g, potassium primary phosphate 1g, Repone K 0.5g, sal epsom 0.5g, ferrous sulfate 0.01g, glucose 30g, agar 20g) on, add glucose, fructose, maltose, Zulkovsky starch, dextrin, sodium acetate, Sodium Propionate, ammonium tartrate, Trisodium Citrate, when wherein any in Sodium.alpha.-ketopropionate and the glycerol is sole carbon source, Muscodorsp. bacterial strain ZJLQ070 all can not normal growth, add urea, SODIUMNITRATE, ammonium nitrate, when wherein any in the ammonium sulfate was only nitrogen source, Muscodorsp. bacterial strain ZJLQ070 also all can not normal growth.But with barley meal, fine rice bran, millet powder and sucrose is carbon source, when being nitrogenous source with peptone, yeast powder, yeast extract paste, wheat bran, soybean cake powder and Dried Corn Steep Liquor Powder, Muscodorsp. bacterial strain ZJLQ070 all can well grow (carbon nitrogen source utilization and growing state see Table 2).
Table 2Muscodor sp. bacterial strain ZJLQ070 carbon nitrogen source utilizes situation
Annotate: "-" expression bacterial strain can not be grown in the table
The test of endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 optimum growth temperature, on the PDA substratum, the endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 in the frozen pipe is activated, the activation back cuts the bacterium cake of diameter 5mm with aseptic stainless steel punch tool, transfer respectively on PDA and MEA substratum, move in the biochemical incubator under 10,15,20,25,28,30,35 and 40 ℃ of conditions, measure the colony growth diameter behind the 15d.Endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 can not grow when being higher than 30 ℃, best growth temperature is 25 ℃, on the MEA substratum, 25 ℃ of colony diameters of cultivating 15d are 23-28mm, on the PDA substratum, 25 ℃ of colony diameters of cultivating 15d are 26-35mm, are contrast with bacterial strain Muscodoralbus during test, and test result is as described in Table 3.
Table 3 endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 optimum growth temp test (unit: mm)
Annotate: the N representative can not be grown.
The high temperature resistant test of endogenetic fungus Muscodorsp. bacterial strain ZJLQ070: on the PDA substratum, the endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 in the frozen pipe is activated, the activation back cuts the bacterium cake of diameter 5mm with aseptic stainless steel punch tool, transfer respectively on PDA and MEA substratum, move into 30, handle 24h respectively in the biochemical incubator under 35 and 40 ℃ of conditions, 48h, 72h and 120h, after the processing culture dish taking-up is placed 25 ℃ biochemical incubator 10d, observing the colony growth situation afterwards again and measure the colony growth diameter, is contrast with bacterial strain Muscodor albus during test.Though endogenetic fungus Muscodorsp. bacterial strain ZJLQ070 can not normal growth under 30 ℃ of conditions, transfer to after 120 hours under 25 ℃ of conditions still can normal growth again but handle, Muscodorsp. the bacterial strain ZJLQ070 high temperature of ability more than 35 ℃ not, test result is as described in Table 4.
The high temperature resistant test of table 4 endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 (unit: mm)
Annotate: the N representative can not be grown.
Embodiment 5Muscodor sp. endogenetic fungus ZJLQ070 molecular systematics is analyzed
Muscodor sp. endogenetic fungus ZJLQ070 molecular systematics characteristic has mainly been analyzed its ITS rDNA, 28SrDNA, RPB2 and beta-tubulin gene, and carries out Phylogenetic Analysis.
At first be the total DNA extraction of Muscodor sp. endogenetic fungus ZJLQ070: the ZJLQ070 inoculation is cultivated 3-5d for 25 ℃ on the PDA substratum, moves to be connected in the PDB liquid nutrient medium again, and 25 ℃, 150rpm/min are cultivated 4d.With filter paper leaching mycelia, behind the thieving paper wipe dry in liquid nitrogen grinding powder, be sub-packed in the 1.5mL centrifuge tube by the amount of the about 100mg of every pipe.Adopt DNeasy Plant Mini Kits (QIAGEN), extract genomic dna by the program of manufacturer.In 100mg thalline powder, add 400 μ l lysis buffer AP1 and 4 μ l 100mg/ml RNase A stock solutions, thermal agitation mixing 60s, 65 ℃ of insulation 10min lysing cell are every 3min vibration mixing 1 time; Add 130 μ l buffer A P2 in the lysate, mixing, the centrifugal 5min of 14000rpm/min behind the ice bath 5min; Get supernatant liquor to the centrifugal post of QIAshredder, the centrifugal 2min of 14000rpm/min; Filtered solution is moved to 1.5ml eppendorf pipe, add 1.5 times of precipitation buffering liquid AP3/E, inhale with the rifle head and beat mixing; Get 650 μ l reaction solutions to the DNeasy micro-column, the centrifugal 1min of 11000rpm/min discards the filtrate in the collection tube; The reaction solution of remainder is moved to the DNeasy micro-column, and the centrifugal 1min of 11000rpm/min discards collection tube and filtrate; The DNeasy micro-column is placed another collection tube, add 500 μ l buffer A W to DNeasy micro-columns, the centrifugal 1min of 11000rpm/min discards the filtrate in the collection tube; The DNeasy micro-column is placed collection tube again, add 500 μ l buffer A W to DNeasy micro-columns, the centrifugal 2min of 14000rpm/min discards collection tube and filtrate; The DNeasy micro-column is placed on another 1.5ml eppendorf pipe, add on buffer A E to the DNeasy film of 100 μ l preheatings (65 ℃), the static 5min of room temperature, the centrifugal 1min of 11000rpm/min collects filtrate and is genomic dna.-20 ℃ of preservations are standby.Get 5 μ l samples electrophoresis on 1.4%Agarose glue, detect the molecular size of DNA, get 50 times of 1 μ l dilutions simultaneously, measure OD260/OD280, detect dna content and quality.
The amplification employing universal primer ITS1 of ITS rDNA (5 '-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 '), the PCR system: dna profiling (100ng/ μ L) 1.0 μ L, 10 * PCR damping fluid (contains 25mmol/L MgCl
2) 5.0 μ L, dNTPs (5mmol/L) 1.0 μ L, each 0.5 μ L of primer (50 μ g/mL), Taq archaeal dna polymerase (2U/ μ L) 1 μ L, add water and supply 50 μ L, react PCR reaction conditions: 94 ℃ of 3min at the enterprising performing PCR of the Minicycler PTC-150 of MJ Research company type PCR instrument behind the mixing; 94 ℃ of 40s, 55 ℃ of 50s, 72 ℃ of 60s, 30 circulations; 72 ℃ of 10min.Pcr amplification product is through 1.0% agarose gel electrophoresis, and pcr amplification product is with the QIA quick PCR purification kit purifying of QIAGEN company: add the PB damping fluid of 5 times of volumes in the PCR reaction product, the vortex mixing; Above-mentioned mixed solution is gone in the QIAquick micro-column, and centrifugal post places on the 2mL collection tube, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector; Add 0.75mL PE damping fluid to micro-column, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector the centrifugal 1min of 14000rpm; Micro-column is placed on the 1.5mL centrifuge tube, add 30 μ L EB damping fluid or water, the centrifugal 1min of 13000rpm behind the static 1min collects the PCR product that filtrate is purifying.℃ preservation of PCR product-20 behind the purifying is standby.Get 2 μ L filtrates electrophoresis on sepharose, estimated concentration.
The amplification employing universal primer LROR of 28S rDNA gene (5 '-ACCCGCTGAACTTAAGC-3 ') and LR5 (5 '-ATCCTGAGGGAAACTTC-3 '), the PCR system: dna profiling (100ng/ μ L) 5.0 μ L, 10 * PCR damping fluid (contains 25mmol/L MgCl
2) 5.0 μ L, dNTPs (5mmol/L) 1.0 μ L, each 0.5 μ L of primer (50 μ g/mL), Taq archaeal dna polymerase (2U/ μ L) 1 μ L, add water and supply 50 μ L, react PCR reaction conditions: 94 ℃ of 3min at the enterprising performing PCR of the Minicycler PTC-150 of MJ Research company type PCR instrument behind the mixing; 94 ℃ of 30s, 52 ℃ of 50s, 72 ℃ of 60s, 35 circulations; 72 ℃ of 10min.Pcr amplification product is through 1.0% agarose gel electrophoresis, and pcr amplification product is with the QIA quick PCR purification kit purifying of QIAGEN company: add the PB damping fluid of 5 times of volumes in the PCR reaction product, the vortex mixing; Above-mentioned mixed solution is gone in the QIAquick micro-column, and centrifugal post places on the 2mL collection tube, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector; Add 0.75mL PE damping fluid to micro-column, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector the centrifugal 1min of 14000rpm; Micro-column is placed on the 1.5mL centrifuge tube, add 30 μ L EB damping fluid or water, the centrifugal 1min of 13000rpm behind the static 1min collects the PCR product that filtrate is purifying.℃ preservation of PCR product-20 behind the purifying is standby.Get 2 μ L filtrates electrophoresis on sepharose, estimated concentration.
The amplification employing universal primer frpb2-5f of RPB2 gene (5 '-GAYGAYMGWGATCAYTTYGG-3 ') and frpb2-7cr (5 '-CCCATRGCTTGYTTRCCCAT-3 '), the PCR system: dna profiling (100ng/ μ L) 5.0 μ L, 10 * PCR damping fluid (contains 25mmol/L MgCl
2) 5.0 μ L, dNTPs (5mmol/L) 1.0 μ L, each 0.5 μ L of primer (50 μ g/mL), Taq archaeal dna polymerase (2U/ μ L) 1 μ L, add water and supply 50 μ L, react PCR reaction conditions: 94 ℃ of 3min at the enterprising performing PCR of the Minicycler PTC-150 of MJ Research company type PCR instrument behind the mixing; 94 ℃ of 30s, 53 ℃ of 50s, 72 ℃ of 90s, 35 circulations; 72 ℃ of 10min.Pcr amplification product is through 1.0% agarose gel electrophoresis, and pcr amplification product is with the QIA quick PCR purification kit purifying of QIAGEN company: add the PB damping fluid of 5 times of volumes in the PCR reaction product, the vortex mixing; Above-mentioned mixed solution is gone in the QIAquick micro-column, and centrifugal post places on the 2mL collection tube, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector; Add 0.75mL PE damping fluid to micro-column, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector the centrifugal 1min of 14000rpm; Micro-column is placed on the 1.5mL centrifuge tube, add 30 μ L EB damping fluid or water, the centrifugal 1min of 13000rpm behind the static 1min collects the PCR product that filtrate is purifying.℃ preservation of PCR product-20 behind the purifying is standby.Get 2 μ L filtrates electrophoresis on sepharose, estimated concentration.
The amplification employing universal primer BT1 of beta-tubulin gene (5 '-AACATGCGTGAGATTGTAAGT-3 ') and BT22 (5 '-TCTGGATGTTGTTGGGAATCC-3 '), the PCR system: dna profiling (100ng/ μ L) 5.0 μ L, 10 * PCR damping fluid (contains 25mmol/L MgCl
2) 5.0 μ L, dNTPs (5mmol/L) 1.0 μ L, each 0.5 μ L of primer (50 μ g/mL), Taq archaeal dna polymerase (2U/ μ L) 1 μ L, add water and supply 50 μ L, react PCR reaction conditions: 94 ℃ of 3min at the enterprising performing PCR of the Minicycler PTC-150 of MJ Research company type PCR instrument behind the mixing; 94 ℃ of 30s, 53 ℃ of 50s, 72 ℃ of 90s, 35 circulations; 72 ℃ of 10min.Pcr amplification product is through 1.0% agarose gel electrophoresis, and pcr amplification product is with the QIA quick PCR purification kit purifying of QIAGEN company: add the PB damping fluid of 5 times of volumes in the PCR reaction product, the vortex mixing; Above-mentioned mixed solution is gone in the QIAquick micro-column, and centrifugal post places on the 2mL collection tube, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector; Add 0.75mL PE damping fluid to micro-column, the centrifugal 30-60s of 13000rpm; Discard the filtrate in the collection tube, micro-column is relay reclaim in the collector the centrifugal 1min of 14000rpm; Micro-column is placed on the 1.5mL centrifuge tube, add 30 μ L EB damping fluid or water, the centrifugal 1min of 13000rpm behind the static 1min collects the PCR product that filtrate is purifying.℃ preservation of PCR product-20 behind the purifying is standby.Get 2 μ L filtrates electrophoresis on sepharose, estimated concentration.
PCR product behind the purifying carries out ligation, and (10 μ L) is as follows for linked system: 2 * T4 ligase enzyme damping fluid, 5 μ L, PCR purified product 2 μ L, PGEM-T carrier 1 μ L, T4 ligase enzyme 1 μ L, ddH
2O 1 μ L.The mixing sample, 4 ℃ of connections are spent the night.10 μ L are connected mixed solution transformed competence colibacillus cell, and the Transformation Program of competent cell is as follows: get 100 μ L competent cell suspensions from-80 ℃ of Ultralow Temperature Freezers, place and thaw on ice; Add DNA and connect liquid 10 μ L, shake up gently, place 30min on ice; Thermal shock 90s in 42 ℃ of water-baths places cooled on ice 3-5min rapidly behind the thermal shock; Add 0.8mL LB liquid nutrient medium (not containing Amp) in centrifuge tube, 37 ℃, 80rpm shaking culture 1h behind the mixing make bacterium the restore normal growth state and the antibiotics resistance gene of expression plasmid coding; With the centrifugal 30s of above-mentioned bacterium liquid 5000rpm, outwell the part substratum, keep about 300 μ L, getting 100 μ L after shaking up coats dull and stereotyped the going up of blue hickie screening that contain 100 μ g/ μ LAmp and (drips 40 μ L 3%X-gal and 7 μ L 20%IPTG. containing suitably antibiotic prefabricated 90mm agar plate central authorities, smoothen with an aseptic spreader, 37 ℃ of temperature are bathed until whole liquid-absorbent), face up and place 0.5h, treat that bacterium liquid is absorbed the back by substratum fully and is inverted culture dish, cultivate 16-24h for 37 ℃; Picking white clone, the LB liquid culture is preserved, and inserts fragment checking and sequencing analysis.CTAB method extracting plasmid: contain in the LB liquid nutrient medium of 100 μ g/mLAmp to 5ml with the isolating single colony inoculation of aseptic toothpick picking, 37 ℃ of shaking culture 12h are to the logarithmic growth later stage, 1.5mL incubated overnight bacterium liquid is added in the 1.5mL centrifuge tube, the centrifugal 30s of 12000rpm, abandoning supernatant, supernatant liquor is all flow to end the centrifuge tube inversion, add 200 μ L STET (containing sugar), abundant suspension thalline, adding 4 μ L concentration is the N,O-Diacetylmuramidase of 10mg/mL, put upside down mixing, room temperature is placed 5min, boiling water boils 50s, the centrifugal 10min of 12000rpm removes precipitation with toothpick, and supernatant adds the RNase of 5 μ L10mg/mL, put upside down mixing, 68 ℃ of water-bath 10min add 10 μ l 5% (w/v) CTAB, and the mixing room temperature is placed 3min, the centrifugal 5min of 12000rpm, precipitation is suspended in the 300 μ L1.2mol/L NaCl solution, and vortex adds 750 μ L dehydrated alcohols, room temperature is placed 5min, the centrifugal 2min of 12000rpm precipitates with 500 μ L, 70% alcohol flushing, the centrifugal 2min of 12000rpm, be deposited in air or the incubator after the drying, be dissolved in 20 μ l TE or the distilled water.After the plasmid empirical tests that extracts, send company's order-checking, order-checking is given birth to worker's biotechnology Services Co., Ltd by Shanghai and is finished.
ITS rDNA, 28S rDNA, RPB2 and the beta-tubulin gene order of endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 is as (SEQ ID NO:1---SEQ ID NO:4) as described in the annex sequence table.
The ITS rDNA of endogenetic fungus ZJLQ070,28S rDNA, RPB2 and beta-tubulin gene order are carried out blast search on GenBank.Based on BLAST result, bacterial strain ZJLQ070 gene order is carried out Phylogenetic Analysis with comprising the relevant genus that Muscodor belongs to software Clustal * 1.81.The structure of phylogenetic tree is by software PAUP
*4.0b10 finish, use heuristic search method in the maximum parsimony principle and carry out system and analyze, specifically be provided with as follows: generate tree with additive process progressively, add stochastic sequence, repeat 1000 times; The tree-to branch-reclosing method as branch commutative operation rule; Single breach is treated as the 5th kind of base in the sequence, and the sequence of insertion is if homology is then not deleted with other sequences; Use the reliability of bootstrapping analysis and evaluation tree, it is 1000 repetitions that the bootstrapping value is set.Determine that from the phylogenetic tree result who makes up ZJLQ070 is the novel species that Muscodor belongs to, belong to fungi Muscodor albus with known Muscodor, Muscodor roseus, Muscodor vitigentis are different with Muscodor crispans.
The VOCs that embodiment 6 endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 produce analyzes
The VOCs that endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 produces adopts Solid-Phase Extraction/gas chromatography/mass spectrometry technology, and (Solid phase microextraction/Gas chromatograph/Mass spetra SPME/GC/MS) analyzes.With Muscodor sp. bacterial strain ZJLQ070 inoculation on the PDA substratum, cultivate 3-5d for 25 ℃, cutting the bacterium cake is transferred on PDA or the MEA substratum, with sealing film the disposable plastic culture dish is sealed, cultivate certain hour (3d, 5d, 10d) in 25 ℃ of biochemical incubators, bore an aperture from the culture dish edge with the fine needle head.Solid-phase microextraction Stainless Steel protection needle tubing is inserted the Sample Room of GC/MS; release extracting head; extracting head is exposed in the GC vaporizer; 20 fens kinds of 240 ℃ of activation; afterwards with extracting head withdrawal protection needle tubing; insert immediately in the off-the-shelf culture dish to be measured, release extracting head, extracting head is exposed in the culture dish; can not run into mycelia; extraction immediately with extracting head withdrawal protection needle tubing, is inserted the Sample Room of GC/MS after 45 minutes immediately under the room temperature; release extracting head; extracting head exposes in the GC vaporizer, and chromatographic determination is carried out in 240 ℃ of desorbs 30 seconds.Analysis operation condition and equipment are as follows:
Instrument, reagent and GC/MS condition: solid-phase micro-extracting device (U.S. Supelco company), extracting head coating are 50/30 μ M DVB/CAR/PDMS; Gas chromatograph-mass spectrograph (Agilent 6890N GC/5975B inert XLMS), quartz capillary column HP-5MS (30m * 250 μ m * 0.25 μ m); Data retrieval is used the NIST05 database.
Chromatographic condition: carrier gas is a helium, post flow 1.0mLmin
-1Temperature of vaporization chamber: 240 ℃, 30 ℃ of chromatographic column initial temperatures keep 3min, with 5 ℃ of min
-1Temperature rise rate rises to 220 ℃.
The mass spectrum condition: the EI ionizer, ionization voltage 70eV, 230 ℃ of ion source temperatures, 150 ℃ of quadrupole temperature, 280 ℃ of interface temperature, sweep limit: 20-450amu does not shunt.
Analyze by GC/MS, obtain the total ion current figure of each composition, determine the chemical ingredients of each component according to standard diagram and relevant document, and calculate the relative percentage composition of each component by the peak area normalization method, Measurement results sees Table 5: table 5 endogenetic fungus Muscodor sp. bacterial strain ZJLQ070 and Muscodor albus cultivate the VOCs that 5d produces
Main antibacterial active compounds by:
(a) 2-methyl-methyl propionate (Propanoic acid, 2-methyl-, methyl ester);
(b) 2 Methylpropionic acid (Propanoic acid, 2-methyl-);
(c) 1-vinyl-1-methyl-2.4-two (1-methyl ethylene)-hexanaphthene (Cyclohexane, 1-ethenyl-1-methyl-2,4-bis (1-methylethenyl)-, [1S-(1.alpha., 2.beta., 4.beta.)]-);
(d) caryophyllene (Caryophyllene);
(e) 2,6-dimethyl-6-(4-methyl-3-amylene) two ring [3.1.1] hept-2-ene"s (Bicyclo[3.1.1] hept-2-ene, 2,6-dimethyl-6-(4-methyl-3-pentenyl)-);
(f) 1,2,3,4,5,6,7,8-octahydroization-1,4-dimethyl-7-(1-methyl ethylidene) Azulene (Azulene, 1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1S-(1.alpha., 4.alpha., 7.alpha.)]-);
(g) 1,2,3,4,4a, 5,6,8a-octahydroization-4a, 8-dimethyl-2-(1-methylethyl) naphthalene (Naphthalene, 1,2,3,4,4a, 5,6,8a-octahyd ro-4a, 8-dimethyl-2-(1-methylethenyl)-, [2R-(2.alpha., 4a.alpha., 8a.beta.)]-);
(h) 3,3,7,11-tetramethyl-three ring [6.3.0.0 (2,4)] 11 carbon-8-alkene (Tricyclo[6.3.0.0 (2,4)] undec-8-ene, 3,3,7,11-tetramethyl-), importantly contain:
(i) a-phellandrene (alpha.-Phellandrene);
(j) β-phellandrene (beta.-Phellandrene);
(k) trans 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, (2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, trans-)
(l) cis 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol (2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis-)
(m) trans 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol (2-Cyclohexen-1-ol, 3-methyl-6-(1-methylethyl)-, trans-)
(n) cis 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol (2-Cyclohexen-1-ol, 3-methyl-6-(1-methylethyl)-, cis-) class isoreactivity material.
The preparation of embodiment 7Muscodor sp. bacterial strain ZJLQ070 active carrier preparation
The present invention prepares the method for Muscodorsp. bacterial strain ZJLQ070 active carrier preparation, this method comprises the preparation of (1) Muscodorsp. bacterial strain ZJLQ070 seed culture fluid, (2) Muscodorsp. bacterial strain ZJLQ070 seed liquor is inoculated in the solid support medium, (3) Muscodorsp. bacterial strain ZJLQ070 incubation growth in solid support medium, the drying of (4) Muscodorsp. bacterial strain ZJLQ070 carrier culture packs.Suitable solid carrier is little molehill stone, perlite or diatomite, most preferably is diatomite; Suitable medium is the composition that comprises calcium ion, magnesium ion, iron ion, phosphate ion and other trace element, peptone, yeast extract, soybean-cake flour, glucose, small rice grain, barley meal, wheat bran, murphy juice and corn steep liquor; Stablizer comprises sucrose, glucose, glycerine or lactose, most preferably is glycerine; And suitable culture condition, comprise pH, temperature, humidity and incubation time.
Specific as follows:
1) in preferred embodiments Muscodor sp. bacterial strain ZJLQ070 seed liquor preparation be by with bacterial strain ZJLQ070 after activation on the PDA substratum, will the bacterium cake insert in the liquid nutrient medium after optimizing and prepare seed liquor.Preferred seed liquid nutrient medium be the potato glucose nutrient solution (potato dextrose broth, PDB), MID changes nutrient solution (every liter contains: sucrose 30.00g, peptone 1.00g, yeast powder 0.25g, ammonium tartrate 5.00g, Ca (NO
3) 20.28g, KNO
30.08g, KCl 0.06g, MgSO
40.36g, NaH
2PO
4H
2O 0.02g, FeCl
36H
2O2.0mg, MnSO
45.0mg, ZnSO
47H
2O 2.5mg, H
3BO
31.4mg KI 0.7mg, pH 5.5), (every liter contains barley meal yeast powder substratum: barley meal 30g, yeast powder 5.0g, peptone 3.0g, Ca (NO
3)
20.4g, KNO
30.12g, KCl 0.2g, MgSO
40.25g, FeCl
36H
2O 5.0mg, MnSO
45.0mg, ZnSO
47H
2O 5.0mg, H
3BO
32.0mg, KI 1.0mg, pH5.5), (every liter contains millet powder yeast powder substratum: millet powder 30g, yeast powder 5.0g, peptone 3.0g, Ca (NO
3)
20.4g, KNO
30.12g, KCl 0.2g, MgSO
40.25g, FeCl
36H
2O 5.0mg, MnSO
45.0mg, ZnSO
47H
2O 5.0mg, H
3BO
32.0mg, KI 1.0mg, pH5.5) (every liter contains: fine rice bran 30g, yeast powder 5.0g, peptone 3.0g, Ca (NO with fine rice bran yeast powder substratum
3)
20.4g, KNO
30.12g, KCl 0.2g, MgSO
40.25g, FeCl
36H
2O 5.0mg, MnSO
45.0mg, ZnSO
47H
2O 5.0mg, H
3BO
32.0mg, KI 1.0mg, pH5.5), more preferably barley meal yeast powder substratum, millet powder yeast powder substratum and fine rice bran yeast powder substratum most preferably are millet powder yeast powder substratum.The preparation of seed culture fluid is grown under the temperature of controlling and pH and rotating speed, to obtain the culture of high-cell density.Preferred temperature more preferably between 20-28 ℃, most preferably is 25 ℃ between 10-30 ℃.Preferred pH is 3-8, preferred 4-7, more preferably 5.5.Preferred rotating speed is between the 50-300rpm, and is more excellent in 100-200rpm, most preferably is 150rpm.Incubation time is preferably 2-10d, and more preferably 3-8d most preferably is 5d, whole seed culture fluids of gathering after cultivating.
2) the Muscodorsp bacterial strain ZJLQ070 seed culture fluid for preparing is inserted solid support medium, under the culture condition after the optimization, grow.Preferred solid carrier is little molehill stone, perlite or diatomite, most preferably is diatomite; Preferred culture medium comprises the composition of calcium ion, magnesium ion, iron ion, phosphate ion and other trace element, peptone, yeast extract, soybean-cake flour, sucrose, Zulkovsky starch, glucose, small rice grain, barley meal, wheat bran, fine rice bran, murphy juice and corn steep liquor; The composition of nitrocalcite, sal epsom, potassium primary phosphate, Repone K, zinc sulfate, manganous sulfate, iron(ic) chloride, boric acid, potassiumiodide, peptone, yeast extract, soybean-cake flour, glucose, small rice grain, barley meal, wheat bran, fine rice bran, murphy juice and corn steep liquor more preferably most preferably is the composition of nitrocalcite, sal epsom, potassium primary phosphate, Repone K, zinc sulfate, manganous sulfate, iron(ic) chloride, boric acid, potassiumiodide, peptone, yeast extract, soybean-cake flour, glucose, small rice grain, wheat bran, fine rice bran, murphy juice.Preferred solid carrier diatomite ratio 50-90% of (butt meter) in solid support medium, more excellent is 70-90%, optimum is 85%.Improvement as substratum of the present invention, consisting of of this solid support medium: diatomite 380g/kg, nitrocalcite 0.4g/kg, sal epsom 0.25g/kg, potassium primary phosphate 1.0g/kg, Repone K 0.2g/kg, zinc sulfate 5mg/kg, manganous sulfate 5mg/kg, iron(ic) chloride 5mg/kg, boric acid 2mg/kg, potassiumiodide 1mg/kg, peptone 5.0g/kg, yeast extract 3.0g/kg, soybean-cake flour 5.0g/kg, glucose 10.0g/kg, small rice grain 20.0g/kg, wheat bran 30.0g/kg, fine rice bran 20.0g/kg, all the other are murphy juice, and humidity is controlled at 45%.That is to say: the composition of sour calcium, sal epsom, potassium primary phosphate, Repone K, zinc sulfate, manganous sulfate, iron(ic) chloride, boric acid, potassiumiodide, peptone, yeast extract, soybean-cake flour, glucose, small rice grain, wheat bran, fine rice bran is dissolved in forms mixed solution in the murphy juice, mix with diatomite again.Preferred temperature is 20-30 ℃, more preferably 22-28 ℃, most preferably is 25 ℃.Preferred growth time is 1-15d, and more preferably 3-12d most preferably is 7d.Preferred humidity is controlled at 20-80%, and more excellent is 30-70%, and optimum is 55%.The ratio of seed culture fluid and solid support medium is 1-20% (weight ratio) during preferred inoculation, and preferred ratio is 5-15%, most preferably 10%.
3) add stablizer in the active carrier preparation, for example sucrose, glucose, glycerine or lactose etc. are to keep the activity of somatic cells, in preferred scheme, stablizer is sucrose, glucose, glycerine or lactose, and more preferably sucrose, glucose, glycerine most preferably are glycerine.The time of adding stablizer is that solid fermentation is cultivated adding when finishing, and in stablizer add-on preferred version, preferred ratio is that (stablizer: the weight ratio of active carrier preparation), more preferably 10-15% most preferably is 10% to 5-20%.Carry out dry packing afterwards again, adopt the low freeze techniques of low temperature when dry, to suppress the growth metabolism activity of Muscodorsp. bacterial strain ZJLQ070 somatic cells.The moisture controlled index of preferred commodity active carrier preparation is 10-50%, more is optimized for 15-35%, and optimum is 20%.Packing is the storage and use microenvironment for a stability and safety being provided for the active carrier preparation; the present invention packs dried active carrier preparation in the environment of air-moisture-permeable into; the protection of outer employing non-woven fabrics; in inner layer glass film bag, be equipped with quantitative activation nutritive medium in advance; as long as with the hand extruding nutritive medium is added in the active carrier preparation when using; so that bacterial strain ZJLQ070 somatic cells obtains moisture and nutrition is grown with recovery, discharge the volatility mixture VOCs of anti-microbial activity.The activation nutritive medium comprises water, peptone, yeast extract, biological growth element, glucose and trace element (for known technology).
Adopting two compartment plate face-off culture method test Muscodor sp. bacterial strain ZJLQ070 active carrier preparation to suppress the activity of phytopathogen, is blank not add Muscodorsp. bacterial strain ZJLQ070 culture.Cause of disease indicator: Botrytis cinerea, Fusarium oxysporum, mould, the aspergillus of ultimate corruption.The cause of disease indicator is connected on the PDA substratum activates, cut mycelia piece (diameter 5mm) (aspergillus is inoculated with spore suspension) and be inoculated in PDA substratum (90mm has every culture dish) first, Muscodor sp. bacterial strain ZJLQ070 culture is placed culture dish opposite side (no substratum), seal with the parafilm film, 25 ℃ of biochemical incubators are cultivated.Treat that the pathogenic bacteria bacterium colony grows to when expiring ware soon in the blank ware, measure the growth-inhibiting of the volatility compounding substances VOCs of Muscodorsp. bacterial strain ZJLQ070 culture generation, calculate inhibiting rate the cause of disease indicator.The pathogenic bacteria bacterium cake piece taking-up of the not growth after the test is transferred on the fresh PDA substratum, 25 ℃ of biochemical incubators are cultivated, check whether it can grow, determine whether fully kill pathogenic bacteria (Fig. 8) of volatility compounding substances VOCs that Muscodorsp. bacterial strain ZJLQ070 culture produces.
Test result shows: Muscodor sp. bacterial strain ZJLQ070 active carrier preparation is strong to the inhibition specific activity Muscodor sp. bacterial strain ZJLQ070 barley corn culture of cause of disease indicator Fusarium oxysporum, because the VOCs that Muscodor sp. bacterial strain ZJLQ070 produces is mould relatively more responsive to aspergillus, Botrytis cinerea and ultimate corruption, two kinds of cultures can both kill them fully.
The VOCs control that embodiment 8Muscodor sp. bacterial strain ZJLQ070 produces is gathered, and back fruit is rotten to be tested
In order to test VOCs that Muscodor sp. bacterial strain ZJLQ070 produces to adopting the septic preventive and therapeutic effect of back fruit, embodiment is as follows: will control the rotten problem of the back fruit of gathering with the active carrier preparation Muscodor sp. bacterial strain ZJLQ070 that is grown on the PDA substratum or prepares.Particularly, the fruit (Xinjiang bergamot pear or honey peach) of no wound and insect pest after picking is gathered, place fruit conserving case (salable), the standardized gently wound of scalpel, on wound, insert brown rot germ (Monilinia fructicola), and apply moistening filter paper at wound and be used to preserve moisture, the active carrier preparation Muscodor sp. bacterial strain ZJLQ070 that is grown on the PDA substratum or prepares is positioned in the fruit conserving case, be used to suppress growth of pathogenic bacteria, seal, put into 25 ℃ growth cabinet, keep humidity 85%.Every processing 3 repeats, and not add Muscodor sp. bacterial strain ZJLQ070 culture for to blank, takes out fruit behind the 3-5d, investigation incidence and Taking Pictures recording.The result shows that the VOCs that Muscodor sp. bacterial strain ZJLQ070 culture produces can control the rotten problem (Fig. 9) of back fruit of gathering preferably.
The VOCs that embodiment 9Muscodor sp. bacterial strain ZJLQ070 produces is to the preventive and therapeutic effect of seedling disease
Measure the preventive and therapeutic effect of the VOCs of Muscodor sp. bacterial strain ZJLQ070 generation with the radish seedling growth method to the seedling disease.With grind Hou De Bite stone pack into diameter 50mm, the height 400mm test tube in, the height of Zhuan Ru Bite stone is about 10cm, 121 ℃ of moist heat sterilization 20min.Insert the ultimate pythium spp cake of pathogenic bacteria (diameter 8mm) 5-6 piece ， Yong Bite stone and cover in each test tube, 10 seeds (ten No. three radish of short leaf, Guangdong Province's breeding is introduced service company and provided) are provided on it, Zai Yong Bite stone covers.Hanging up Muscodor sp. bacterial strain ZJLQ070 active carrier preparation (pack with gauze the outside) in the test group test tube, is blank only to connect pathogenic bacteria, and test tube seals with ventilative cork.The immigration growth cabinet (25 ℃, illumination: dark=12h: cultivate 12h), regularly add up the incidence of percentage of germination and observation seedling.
The VOCs that table 6Muscodor sp. bacterial strain ZJLQ070 produces is to the preventive and therapeutic effect of seedling disease
The VOCs that Muscodor sp bacterial strain ZJLQ070 active carrier preparation produces can suppress the mould influence to radish seed sprouting, growth of seedling of ultimate corruption effectively.
The VOCs that embodiment 10Muscodor sp. bacterial strain ZJLQ070 active carrier preparation produces suppresses the test of air microbe population in the environment for human survival
In order to test VOCs that Muscodorsp. bacterial strain ZJLQ070 active carrier preparation produces, adopt the plate culture test to microbial status in the influence of microbe population in the air and the environment.Pouring the PDA substratum of 15-20 milliliter into diameter is the 90mm sterile petri dish, treats to re-use after the culture medium solidifying.Test is carried out in laboratory, Zhejiang University biotechnology research institute biotechnology building (25 ℃ of room temperatures), separates two little (every 1-2m with sheet glass
2), place Muscodor sp. bacterial strain ZJLQ070 active carrier preparation 100g between wherein handling, do not place Muscodor sp. bacterial strain ZJLQ070 active carrier preparation between contrast, behind 48h, 72h and the 120h ready culture dish placed between processing and between contrast, and uncap, make it in air, expose 60min, the culture dish lid back of closing is moved in 25 ℃ the biochemical incubator and cultivates, test airborne microbe population.
Table 7Muscodor sp. bacterial strain ZJLQ070 active carrier preparation suppresses the test result of air microbe population in the environment for human survival
Muscodor sp. bacterial strain ZJLQ070 active carrier preparation can reduce microbial numbers in the ambient air effectively, Muscodor sp. bacterial strain ZJLQ070 active carrier preparation after exposing 72h to air in the inhibiting rate of microorganism reach 70.5%, and expose 120h and do not have notable difference.
The synthetic mixture sterilant of the separable VOCs that produces from Muscodor sp. bacterial strain ZJLQ070 culture of embodiment 11
The applicant finds that (1) Muscodor sp. bacterial strain ZJLQ070 culture produces the basic all the components of VOCs; (2) Muscodor sp. bacterial strain ZJLQ070 culture produces the combination of part composition among the VOCs, or a kind of synthetic VOCs of composition has the sterilization idiocratic of Muscodor sp. bacterial strain ZJLQ070 among the generation VOCs of (3) Muscodorsp bacterial strain ZJLQ070 culture.
In order to test relative biological activity of classes of compounds and classes of compounds optimal concentration and ratio in whole mixtures, all mixtures are the detection mixture of the 100 μ L in every 50mL spatial domain on the culture in the type culture flat board.For example, β-phellandrene accounts for identifies 4.37% of volatile matter mixture, with the test of 4.37 μ L/50mL (0.0874 μ L/mL) spatial domain β-phellandrene is tested, and other compound also is with identical standard procedure.
With 8.02%2-methyl-methyl propionate, the 77.08%2-methylpropanoic acid, 0.67%1-vinyl-1-methyl-2.4-two (1-methyl ethylene)-hexanaphthene, 0.11% caryophyllene, 0.07%2,6-dimethyl-6-(4-methyl-3-amylene) two ring [3.1.1] hept-2-ene"s, 0.84%1,2,3,4,5,6,7,8-octahydroization-1,4-dimethyl-7-(1-methyl ethylidene) Azulene, 6.02%1,2,3,4,4a, 5,6,8a-octahydroization-4a, 8-dimethyl-2-(1-methyl ethylidene) naphthalene, 0.06%3,3,7,11-tetramethyl-three ring [6.3.0.0 (2,4)] 11 carbon-8-alkene, 7.13% acetone is mixed with organic mixture (being called composition 1).If the composition 1 of basic, normal, high three levels mixes with 0.36%a-phellandrene, 4.37% β-phellandrene, 0.13% trans 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.10% cis 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.32% trans 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.04% cis 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, waving property of test mixing compound is to the inhibition of indication pathogenic bacteria, mould with ultimate corruption is the cause of disease indicator, and test result sees Table 8.
The synthetic volatility mixture inhibiting rate mould that composition 1 and other compound of three different levelss of table 8 is made into to ultimate corruption
From test result as can be seen, high, in, the composition 1 and the 0.36%a-phellandrene of low three levels, 4.37% β-phellandrene, 0.13% trans 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.10% cis 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, 0.32% trans 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol, the inhibiting rate difference that the volatile gases of 0.04% cis 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol mixture is mould to ultimate corruption, low-level inhibiting rate constantly is minimum, and middle level and high-level inhibiting rate all reach 100%.
The sterilant of embodiment 12, synthetic, it comprises the composition of volume percent as described in Table 9, surplus is an acetone.
3 embodiment of table 9, sterilant
| N1 | N2 | N3 | |
| 2-methyl-methyl propionate | 10.18 | 0.7 | 8.02 |
| 2 Methylpropionic acid | 60.42 | 28.26 | 77.08 |
| 1-vinyl-1-methyl-2.4-two (1-methyl ethylene)-hexanaphthene | 2.04 | 2.23 | 0.67 |
| Caryophyllene | 0.38 | 0.12 | 0.11 |
| 2,6-dimethyl-6-(4-methyl-3-amylene) two ring [3.1.1] hept-2-ene"s | 0.95 | 0.15 | 0.07 |
| 1,2,3,4,5,6,7,8-octahydroization-1,4-dimethyl-7-(1-methyl ethylidene) Azulene | 3.35 | 2.27 | 0.84 |
| 1,2,3,4,4a, 5,6,8a-octahydroization-4a, 8-dimethyl-2-(1-methylethyl) naphthalene | 6.93 | 10.46 | 6.02 |
| 3,3,7,11-tetramethyl-three ring [6.3.0.0 (2,4)] 11 carbon-8-alkene | 0.15 | 0.16 | 0.06 |
| The a-phellandrene | 0.32 | 2.58 | 0.36 |
| β-phellandrene | 5.88 | 44.25 | 4.37 |
| Trans 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol | 0.22 | 0.52 | 0.13 |
| Cis 1-methyl-4-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol | 0.10 | 0.36 | 0.10 |
| Trans 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol | 0.29 | 3.91 | 0.32 |
| Cis 3-methyl-6-(1-methylethyl)-2-tetrahydrobenzene-1-alcohol | 0.06 | 0.24 | 0.04 |
Employing two compartment plate culture is tested with N1～N3 and is equal to the activity experiment of showing to cultivate in 5 Muscodor albus VOCs (as a comparison case) the inhibition phytopathogen of 5d generation.The cause of disease indicator: Botrytis cinerea, dry thread Pyrenomycetes, alternaria tenuis, neat microsolerotium, ultimate corruption are mould, Fusarium oxysporum cucumber specialized form, circular thorn dish spore, big beautiful Verticillium, sclerotinite, persimmon dish stey, piricularia oryzae, Penicillium digitatum, broad bean grape spore, Fulvia fulva, bryonia Asia be every spore shell, aspergillus, Paecilomyces lilacinus.The cause of disease indicator is connected on the PDA substratum activates, cut mycelia piece (diameter 5mm) (aspergillus is inoculated with spore suspension) and be inoculated in PDA substratum (90mm has every a culture dish) side, to add culture dish opposite side (every kind of amount of mixture is 100 μ L respectively) respectively with 4 kinds of mixtures of all cpds ratio among the VOCs (as a comparison case) that cultivates the 5d generation according to N1～N3 and the Muscodor albus that is equal to table 5, seal with the parafilm film, 25 ℃ of biochemical incubators are cultivated.Treat that the pathogenic bacteria bacterium colony grows to when expiring ware soon in the blank ware, measure the growth-inhibiting of the volatility compounding substances VOCs of synthetic to the cause of disease indicator, calculate inhibiting rate, the result is as shown in table 10.
Table 10
Sequence table
SEQ ID NO：1
Rrna rDNA internal transcribed spacer district (Internal Transcribed Spacer, ITS rDNA) gene order
cctttgtgaa cctaccatcg ttgcttcggc ggcggaggtg ctacgctgca aggcgctacc 60
ctgtagttac cctgtagtcc cagggagctg tcatcagctc tttaggggag cccacagcct 120
agcgacgttt tcgttacagg gctgtagctc cggactgccc tccccgccgg cggccaacta 180
aactctgttt tctttggaac tctgaatcat aaacttaata agttaaaact ttcaacaacg 240
gatctcttgg ttctggcatc gatgaagaac gcagcgaaat gcgataagta atgtgaattg 300
cagaattcag tgaatcatcg aatctttgaa cgcacattgc gcccattagc attctagtgg 360
gcatgcctgt tcgagcgtca tttcaccact taagccctgt tgcttagcgt tgggggccta 420
cggcacagcc tgtagcccct taaagtgatt ggcggagttg gttctatctc taagcgtagt 480
aatttcttct cgcttctgca gtagtgctgg cccccgccgt aaaa 524
SEQ ID NO：2
Large ribosomal subunit (28S rDNA) gene order
ttgccctagt aacggcgagt gaagcggcaa cagctcaaat ttgaaatctg gctctcgggc 60
ccgagttgta atttgtagag gatgattttg gcgcggtgcc ttccgagttc cctggaacgg 120
gacgccttag agggtgagag ccccgtacgg ttggacacca agcctctgta aatctccttc 180
gacgagtcga gtagtttggg aatgctgctc taaatgggag gtaaatttct tctaaagcta 240
aataccggcc agagaccgat agcgcacaag tagagtgatc gaaagatgaa aagcactttg 300
aaaagagggt taaatagcac gtgaaattgt tgaaagggaa gcatttacta ccagacctct 360
gccctgcgga tcatgtggtg ttctcaccgc tgcacttcgc ttggtttagg ccagcatcgg 420
tttttgtagg gggataaaag ccttaggaac gtagctccct cgggagtgtt atagcctttt 480
gcataatacc cttacgggga ccgaggaccg cgcttcggca aggatgctgg cataatggta 540
gtcaatgacc cgtcttgaaa cacggaccaa ggagtcgaac atttgtgcga gtgtttgggt 600
gttaaaccct cacgcgtaat gaaagtgaac gtaggtgaga gcccttacgg gtgcatcatc 660
gaccgatctt gatgtcttcg gatggatttg agtaagagca taactgttcg gacccgaaag 720
atggtgaact atgcgtggat agggtgaagc cagaggaaac tctggtggag gctcgcagcg 780
gttctgacgt gcaaatcgat cgtcaaatct gcgcatgggg gcgaaagact tatcgaacca 840
ttaaaccagc ggtaaaatga cttactaggt ggttaagagg ccccccgcga ggggagaggc 900
ctaggggggt tacccggtag ctgctaccct gcacttccgg gtggccgccc gacatcgcta 960
aattgcgagg acatcccacc aaggccaggg gttaccgccg cccgctgaaa agcgccgcgg 1020
caccgagagt agcgctcttg ggtacggtaa aaacgccccc ggtagcggac gacttgcagc 1080
caaccccgca ctacagggga aggttcacag actaaacagc gatgggtcgg cgcgctgccg 1140
gcctaagaca tagtcgacct ggggcctgag aaggtgccct gcagcgtggc gtacaggcg 1199
SEQ ID NO：3
Rna plymerase ii (RNA polymerase II subunit, RPB2) gene order
cttttccgta acatcgtccg ccgcatgacg caggaggtct tgtcccactt gaagcgaagc 60
atcgagcagg gcaagcagtt caacatcgcc ctcgccgtca aggccaatat tattactagc 120
ggtctcaagt actcgctcgc caccggtaac tggggtgatc agaagaaagc catgagctcc 180
accgctggcg tgtcccaggt gctcaacaga tatactttcg cgtccacgct ctcgcatttg 240
cgacgaacca acacgcccgt aggtcgagat ggcaagctcg ccaagccccg ccagcttcac 300
aacacccact ggggcctagt ttgtcctgcc gagacccccg agggtcaggc ctgtggtctg 360
gtgaagaact tgtctcttat gtgctccgtc agtgtcggca cctcgacgga gcccatcatc 420
gagtacatgg cgtcccggaa catggagatt ctcgaagaat acgaacccca gcgctatccc 480
aatgccacca agatctttct caacggctca tggatcggcg tccaccacga tcccaagtct 540
ctcgtgagag atgtccagca gctacgccga accaatcaga tccctgcaga agtatcattg 600
gttcgcgaca tccgtgatcg cgagttcaag atcttctcgg acgctggccg agtcatgcga 660
cccttgtttg tcgtcgaaca agaggacacg cccgaccgcc tgaagggaca gctcgctctc 720
accaaagaaa tggccaagaa aatcgaagcc gatcaagatc cggcaacgat agaaaggaac 780
gaatattatg gttgggaggg gttggtcgat gacggcgcca ttgagtatct ggatgccgag 840
gaggaagaga cggccatgat ttgcatgacg cccgaaga 878
SEQ ID NO：4
'beta '-tubulin (beta-tubulin) gene order
aacatgcgtg agattgtaag tcccaaccgt cgcgcgcgcc acgcccgacc gccccccgac 60
ccctctgttt acttgcccga ggagcccaac cagaccacct ccaaaccacc tcttccactc 120
gcctccccat ccttggacgc gtccgaatcc agtcactctg ggtcgaatgc ctctccacac 180
ctatatctcc ctcatcatat cagcaggttg ggcagttgct gccaacccgt gtcccagtcc 240
gcccagagcc aaacatatgt cgagaactct gctaacccgt gtatctttcg catctaggtt 300
cacctccaga ccggtcagtg cgtaagtcaa tcctccatct caacatgatc ctgtcgaaaa 360
tgccgttgta ctaacgtatc gctacagggt aaccaaattg gtgctgcttt ctggtgtgta 420
ccacaaacgc gaaacacggt tacaggccat caatactgac tgttgtgcta caggcaacag 480
atctccggcg agcacggtct cgacggcaat ggagtgtatg ttgctgtcgc ttgactgcca 540
ctgcttgaac cccatgactg acatacgaaa cagctacaat ggcacctccg agctccagct 600
cgagcgcatg agcgtctact tcaacgaggt atgcttccac ctaaccgacc taatgccaca 660
ggcaattcga agaaaaagat ccacctgact gatacgcgat gtgcagggtg ccggcaacaa 720
gtacgtcccc cgtgccgtcc tcgtcgatct cgagcccggt accatggacg ccgtccgcgc 780
cggtcccttc ggccagctct tccgccccga caacttcgtc ttcggccagt ccggtgctgg 840
caacaactgg gccaagggtc actacaccga gggtgccgag ctcgtcgacc aggtcctcga 900
tgtcgtccgt cgcgaggccg agggctgcga ctgcctccag ggcttccaga tcacccactc 960
gctcggtggt ggtaccggtg ccggtagggc acgctcctca tctccaagat ccgcgaggag 1020
ttccccgacc gcatgatggc caccttctcc gtcgtcccct cccccaaggt ctccgacacc 1080
gtcgtcgagc cctacaacgc caccctctcc gtccaccagc tcgtcgagaa ctcggacgag 1140
accttctgca tcgacaacga ggccctctac gacatctgca tgcgtaccct caagttgtcc 1200
aacccctcgt acggcgacct gaaccacctc gtctccgccg tcagtcgggc gtcaccacct 1260
gtctgcgttt ccccggccag ctcaactctg acctgcgcaa gttggctgtc aacatggtgc 1320
ccttcccccg tctccacttc ttcatggtcg gctttgctcc tctgaccagc cgcggcgcca 1380
gtgccttccg cgctgtcacc gttcccgagt tgacccagca gatgttcgac cccaagaaca 1440
tgatggctgc ctctgacttc cgcaacggtc gctacctcac ttgctctgcc atcttgtaag 1500
cgaccaaccc gtattctctt aaaaggacat ttcaatgcta acacaactgt gccagccgtg 1560
gtaaggtctc catgaaggag gtcgaggacc agatgcgcaa cgtccagaac aagaactcgt 1620
cctacttcgt cgagtggatt cccaacaaca tccaga 1656
Claims (3)
1. plant endogenesis epiphyte, it is characterized in that: this plant endogenesis epiphyte Muscodor sp. bacterial strain preservation name is called: ZJLQ070, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on January 8th, 2009, preserving number: CGMCC 2864.
2. the live body preparation that utilizes plant endogenesis epiphyte as claimed in claim 1 to make.
3. the purposes of plant endogenesis epiphyte as claimed in claim 1 or the described live body preparation of claim 2 is characterized in that: be used for suppressing or killing Botrytis cinerea Botrytis cinerea, dry thread Pyrenomycetes Rhizoctonia solani, alternaria tenuis Alternaria alternata, neat microsolerotium Sclerotium rolfsii, the mould Pythium ultimum of ultimate corruption, circular thorn dish spore Colletotrichum orbiculare, big beautiful Verticillium Verticillium dahliae, sclerotinite Sclerotinia sclerotiorum, persimmon dish stey Pestalotia diospyri, piricularia oryzae Pyricularia oryzae, Penicillium digitatum Penicillum digitatum, aspergillus Aspergillus sp., Saccharomyces Cerevisiae in S accharomyces cerevisiae, intestinal bacteria Escherichia coli or leaf bacillus Phyllobacterium sp.
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| CN106538526A (en) * | 2016-09-27 | 2017-03-29 | 中国中医科学院中药研究所 | The application of Fusarium oxysporum antibacterial and its application process and caryophyllene |
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| CN103215313B (en) * | 2013-03-31 | 2015-07-08 | 浙江大学 | Fungus solid fermentation method |
| CN104419649B (en) * | 2013-09-06 | 2017-09-15 | 中国医学科学院药用植物研究所 | The brave foster aerogenesis acted on biological control is mould |
| CN104419647B (en) * | 2013-09-06 | 2017-06-09 | 中国医学科学院药用植物研究所 | Malipo aerogenesis with biological control effect is mould |
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| CN108795773B (en) * | 2017-04-28 | 2022-09-20 | 贵州中烟工业有限责任公司 | Musk mould strain and perfume prepared from same |
| CN108795771B (en) * | 2017-04-28 | 2022-09-20 | 贵州中烟工业有限责任公司 | Musk mould strain and perfume prepared from same |
| CN109486870A (en) * | 2018-11-29 | 2019-03-19 | 华南农业大学 | A kind of collection of fungus Trichoderma volatile matter and identification method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004034785A2 (en) * | 2002-10-15 | 2004-04-29 | Montana State University | Methods and compositions relating to the production of insect repellents by a novel endophytic fungus |
| WO2004089084A2 (en) * | 2003-04-04 | 2004-10-21 | Montana State University | The application of volatile antibiotics and non-volatile inhibitors from muscodor spp. to control harmful microbes in human and animal wastes |
| CN1845676A (en) * | 2003-07-17 | 2006-10-11 | 加里·A·斯特罗贝尔 | Compositions related to a novel endophytic fungi and methods of use |
-
2009
- 2009-09-30 CN CN2009101535116A patent/CN101691540B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004034785A2 (en) * | 2002-10-15 | 2004-04-29 | Montana State University | Methods and compositions relating to the production of insect repellents by a novel endophytic fungus |
| WO2004089084A2 (en) * | 2003-04-04 | 2004-10-21 | Montana State University | The application of volatile antibiotics and non-volatile inhibitors from muscodor spp. to control harmful microbes in human and animal wastes |
| CN1845676A (en) * | 2003-07-17 | 2006-10-11 | 加里·A·斯特罗贝尔 | Compositions related to a novel endophytic fungi and methods of use |
Non-Patent Citations (3)
| Title |
|---|
| Ezra D 等.《New endophytic isolates of muscodor albus, a volatile-antibiotic-producing fungus》.《Microbiology》.2004,第150卷全文. * |
| EzraD等.《Newendophyticisolatesofmuscodoralbus a volatile-antibiotic-producing fungus》.《Microbiology》.2004 |
| Scrobel GA 等.《Volatile antimicrobials, from Muscodor albus,a novel endophytic fungus》.《Microbiology》.2001,第147卷(第11期),全文. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106538526A (en) * | 2016-09-27 | 2017-03-29 | 中国中医科学院中药研究所 | The application of Fusarium oxysporum antibacterial and its application process and caryophyllene |
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