CN102352327A

CN102352327A - Marine actinomycete L131 and metabolin, preparation method and application of metabolin

Info

Publication number: CN102352327A
Application number: CN2011102615990A
Authority: CN
Inventors: 赵心清; 王玉梅; 吴毅; 陈德玲; 谢萌; 方文晶; 沈俊涛
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2011-09-06
Filing date: 2011-09-06
Publication date: 2012-02-15

Abstract

The invention discloses marine streptomyces L131 and application of antifungal activity thereof. A kind of marine streptomyces L131 is separated from a sea mud sample, and the purification culture medium is a Bennett culture medium; the marine streptomyces L131 colony protrudes, the surface is corrugated and is provided with white spores, and the marine streptomyces generates faint yellow soluble pigments. As shown in an inhibition zone test method, the fermentation broth of the marine actinomycete L131 of the invention has significant inhibition on the fulvia fulva. The invention also discloses a preparation method of metabolin crude extract with fulvia fulva inhibition of marine actinomycete. The preparation method comprises the steps of controlling the fermentation conditions of the marine actinomycete L131, and separating and purifying the active substance for inhibiting fulvia fulva through porous adsorption resin. The invention has the advantages that the antibacterial metabolin is simple and easy to be prepared and the cost is low, so that the invention provides environmental-friendly pesticide with good effect.

Description

The method for making and the application of one strain marine actinomycete L131 and metabolite thereof, metabolite

Technical field

The present invention relates to microorganism field, marine actinomycete L131 particularly also relates to the method for making of bacterial strain metabolite and in addition in the application of biological pesticide research field.

Background technology

Ocean environment occupies 70.8% of the earth total area; Containing abundant Microbial resources; The environmental quality that the ocean is unique; Like low temperature, low nutrition, high pressure, high salinity etc., brought up distinctive metabolic way of marine microorganism and meta-bolites, actinomycetes are because of having the ability that complicated form atomization has the abundant secondary metabolite of synthetic kind.In recent years, along with the development of Protocols in Molecular Biology, increasing marine actinomycete comes to light; Many secondary metabolites that come from marine actinomycete also identified have antitumor; Antibiotic, antimalarial, multiple bioactive functions such as insecticidal function and enzyme inhibitors.The novel structure that marine actinomycete produced, the active substance of diverse in function become the important source of natural radioactivity product and new drug development.

Tomato is one of the most general fruit and vegetable of whole world cultivation.Leaf muld of tomato ( Fulvia fulva) be a kind of serious plant disease that threatens protection ground tomato production, leaf muld of tomato main harm blade, harm stem, flower, fruit etc. when serious.Scab is born in blade back more, irregular shape or the oval faint yellow green statin that moves back occur, the leaf back layer that just mildews, and later mould layer becomes beige or chocolate is velvet-like.After fruit is caught an illness, near base of fruit, form circular black scab, sclerosis is depression slightly, and anorexia is used.Therefore, the infection of this germ can directly cause the tomato underproduction, and has a strong impact on Tomato Quality, this sick generation in a single day, and the meeting rapid spread brings tremendous loss to tomato production.

Prevent and treat to leaf muld of tomato normal chemical pesticide that uses on producing, field spray medicine is a control leaf muld of tomato popular important means, adopts dust or smog dispenser and spray method usually.Dust dispenser dust agent commonly used has 5% m-tetrachlorophthalodinitrile dust agent, 5% Jia Ruinong dust agent, the clean dust agent in 7% blade face, 25% this gram WP etc., and fumicants commonly used has 45% chlorothalonil smoke.Spray pesticide is many with 150 times of liquid of 2% Astromicin aqua, 500 times of liquid of 50% carbendazol wettable powder, 800-1000 times of liquid of 70% thiophanate methyl wettable powder, 600-800 times of liquid of 47% Jia Ruinong wettable powder etc.The control effect of chemical agent is obvious; The speed of preventing and treating is fast, but that life-time service can make the dependency of chemical agent is increasing, and field season of growth medication number of times constantly increases; Pathogenic bacteria is constantly strengthened using the agricultural chemicals resistance; Control effect constantly descends, and the residual pollution that also can cause environment of medicament influences human beings'health simultaneously.Therefore, must explore new highly effective and safe controlling way.

In recent years; Biological control research becomes focus gradually; The application of biological control becomes the important means of comprehensive regulation plant pest day by day, but the antimycotic microbiotic of efficient, the low toxicity that obtains at present, low residue is still less, therefore new antimycotic microbiotic and zymotechnique efficiently; To become and preserve the ecological environment, realize the strong guarantee of agricultural sustainable development.

Summary of the invention

The object of the present invention is to provide a strain marine actinomycete streptomycete ( StreptomycesSp.) L131, its metabolite have the effect of significant inhibition tomato leaf mould, disclose the preparation method of metabolite, and the application in the biological control agricultural chemicals.

A strain marine actinomycete streptomyces strain of the present invention is from the ooze sample of little flat island, Dalian, to separate the streptomycete L131 that obtains, and it is well-grown on the Bennett substratum, and bacterium colony is protruding, surface folding, and the adularescent spore produces faint yellow soluble pigment;

Consisting of of said Bennett substratum: glucose 10 g, peptone 2 g, yeast powder 1 g, Carnis Bovis seu Bubali cream 1 g, agar 15 g, zero(ppm) water 1L, 7.2,121 ℃ of moist heat sterilization 20min of pH.

Said marine streptomyces L131 metabolite makes through following preparation method:

(1) fermentation of streptomycete L131

The preparation of A, streptomycete L131 seed liquor: mycelia on the flat board and spore are inserted in the YMG substratum, and 30 ℃, 150 rpm cultivate 24 h and obtain seed liquor;

The fermentation of B, streptomycete L131: streptomycete L131 seed liquor is inserted the fermentation optimized with in the substratum with 2% inoculum size, 30 ℃, cultivation 168 h under the 150 r/min conditions;

Described YMG substratum is formed: glucose 4 g, and malt extract 10 g, yeast extract 4 g, zero(ppm) water l L, pH 5.5 ± 0.2; The fermentation of optimizing is formed with substratum: Semen Maydis powder 15 g, analysis for soybean powder 15 g, yeast extract 1.5 g, CaCO ₃0.5 g, water is settled to 1L, 121 ℃ of moist heat sterilization 20 min;

(2) streptomycete L131 to fungi inhibited the preparation of metabolite

Fermented liquid is centrifugal, and centrifugal 10 min of 6000 r/min get supernatant and pretreated macroporous adsorbent resin AB-8 after centrifugal with 10:3 (v:m) mixed; Place shaking table 150 r/min to adsorb 24 h, use equal-volume zero(ppm) water, 40% washed with methanol then respectively; Use equal-volume 100% methyl alcohol desorb, 150 r/min desorb 24 h, and stripping liquid uses 35 ℃ of evaporates to dryness of rotary evaporation in vacuo appearance; With little volume methyl alcohol residuum is dissolved again concentratedly again, it is the metabolite of streptomycete L131.

Said marine streptomyces ( Streptomyces) application of metabolite in the agricultural chemicals of biocontrol of plant disease of L131.

Said marine streptomyces ( Streptomyces) application of L131 in preventing and treating leaf muld of tomato agricultural chemicals or preparation.

Said a kind of preparation of preventing and treating the tomato leaf mould comprises effective constituent and ancillary component, its effective constituent mainly be marine streptomyces ( Streptomyces) metabolite of L131.

The present invention one strain marine actinomycete, the inhibition zone method test shows that its fermented liquid has significant inhibitory effect to the tomato leaf mould.The invention also discloses having of this marine actinomycete the tomato leaf mould is had inhibiting metabolite; Its preparation method is through the fermentation condition of control marine streptomyces L131; Use macroporous adsorbent resin that the active substance that suppresses the tomato leaf mould is carried out separation and purification, antibiotic metabolite prepares simple, and cost is low; Have the effect that suppresses leaf muld of tomato, belong to effect environment friendly agricultural preferably.

Description of drawings

What Fig. 1 showed is the colonial morphology of bacterial strain L131;

What Fig. 2 showed is the flat board inhibition tomato leaf mould effect of bacterial strain L131.

What Fig. 3 showed is bacterial strain L131 blade experimental result.

Embodiment

Below in conjunction with specific embodiment, the present invention is described further.

The evaluation of embodiment 1 bacterial strain L131

The morphological specificity of comprehensive streptomycete, physiological and biochemical property, 16S rDNA sequence etc. are accredited as streptomyces with it.Concrete qualification result is following:

1. thalli morphology

Bacterial strain L131 is well-grown on the Bennett substratum, and bacterium colony is protruding, surface folding, and the adularescent spore produces faint yellow soluble pigment (see figure 1).

2. physiological and biochemical property

Shown in following two forms of bacterial strain L131 physiological and biochemical property (table 1,2): bacterial strain L131 can not produce H ₂S; It is positive that gelatine liquefication becomes; Milk peptonizes and is reacted into feminine gender; Can not produce amylorrhexis starch; Can glucose, sucrose, SANMALT-S, inositol, sorbyl alcohol, semi-lactosi grows as sole carbon source.The pH value can be grown in the scope of 6-12, pH well-grown and produce spore in the scope of 7-11; Salt concn can be grown at 0-11%, and salt concn is at the 0-8% well-grown and produce spore.

Table 1 physiological and biochemical property

Figure 2011102615990100002DEST_PATH_IMAGE001

Annotate: "+" expression is positive, and "-" expression is negative

Actinomycetic pH growth scope of table 2 and salt tolerance

Cultural characteristic	L131
		pH=5	-
pH=6	+
		pH=7	+++
pH=8	+++
		pH=9	+++
pH=10	+++
		pH=11	++
pH=12	+
		pH=13	-
The pH growth scope	6-12
		The righttest growth scope of pH	7-11
NaCl? 4%	+++
		NaCl? 6%	++
NaCl? 8%	++
		NaCl? 9%	+
NaCl? 10%	+
		NaCl? 11%	+
NaCl? 12%	-
		Salt concn growth scope (%)	0-11
The righttest growth scope of salt concn (%)	0-8

3.16S rDNA identifies

The 16S rDNA complete nucleotide sequence length overall of streptomycete L131 is 1421bp, and sequence is as follows:

GGCGGCGTGCTTACACATGCAAGTCGAACGATGAACCGCTTTCGGGCGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGACCGTCTGCCGCATGGTGGATGGTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAGTGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGTCTGGAGACAGGCGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGTGGA

According to the phylogenetic tree comparative analysis, this bacterial strain and silver color streptomycete Streptomyces argenteolus(CGMCC 4.1681) demonstrate the highest sequence homology.

Embodiment 2 preparations have the metabolite of inhibiting marine streptomyces L131 to leaf muld of tomato:

(1) fermentation of streptomycete L131

The fermentation of described optimization is formed with substratum: Semen Maydis powder 15 g, analysis for soybean powder 15 g, yeast extract 1.5 g, CaCO ₃0.5 g, water is settled to 1L, 121 ℃ of moist heat sterilization 20 min;

(2) the tomato leaf mould there is the preparation of the metabolite of inhibiting streptomycete L131

Macroporous adsorbent resin is carried out pre-treatment: macroporous adsorbent resin is earlier with distilled water flushing 3-5 time; Incline suspended substance and crushed particles; Again with dress as 250 mL triangular flasks behind 95% alcohol immersion, 24 h; At first wash 4 h with 3-5 times of volume ethanol, ethanol is washed till the effluent muddiness that is not white in color, and zero(ppm) water is cleaned ethanol; 1.0 mol/LHC1 with 3 times of volumes wash 4 h then, and zero(ppm) water is washed till neutrality; Wash 4 h with 1.0 mol/LNaOH solution of 3 times of volumes at last, zero(ppm) water is washed till neutrality, then with the containers for future use of packing into after the resin weighing of handling well.

Fermented liquid is centrifugal, and centrifugal 10 min of 6000 r/min get supernatant and pretreated macroporous adsorbent resin AB-8 (Tianjin Nankai Hecheng S&T Co., Ltd.) after centrifugal with 10:3 (v:m) mixed; Place shaking table 150 r/min to adsorb 24 h; Use equal-volume zero(ppm) water, 40% washed with methanol then respectively, use equal-volume 100% methyl alcohol desorb, 150 r/min desorb 24 h; Stripping liquid uses 35 ℃ of evaporates to dryness of rotary evaporation in vacuo appearance; With little volume methyl alcohol residuum is dissolved again concentratedly again, it is the metabolite of streptomycete L131, and the tomato leaf mould is had remarkable restraining effect.

(3) inhibition zone method suppresses the experiment of tomato leaf mould

Preparation tomato leaf mould test slab: after the substratum PDA sterilization, pour petridish into, each petridish 20 mL treats after the substratum condensation tomato leaf mould spores to be applied to media surface uniformly.Use punch tool punching back to add 100 μ l test fluid, detect inhibition zone.Anti-bacteria test result is seen accompanying drawing 2.

Wherein the PDA substratum is formed: yam 200 g; Sucrose 20 g; Agar 20 g; Zero(ppm) water is settled to 1 L; The pH nature; Peeling potatoes boils 30min after the stripping and slicing, use filtered through gauze then, sugaring and agar again, and water is supplied volume, 121 ℃ of moist heat sterilization 20 min.

The experiment of embodiment 3 blades

Choose tomato branch upside blade, experiment is divided into experimental group (fermented liquid+germ), positive group (germ+clear water), control group (clear water).Each group is provided with two repetitions, and each repeats 2 leaves.The tomato leaf of plucking was soaked the impurity such as earth of flush away blade surface 10 minutes in clear water.Four leaves of experimental group are put into fermented liquid and were soaked 30 minutes, dry.Rest blade directly dries, and puts into control group respectively and positive group plate is for use.After drying,,, the bacterium cake is positioned over the central authorities of experimental group and positive group blade, and adds clear water and preserve moisture according to the principle of 1 bacterium cake of every leaf from the bacterium cake of the flat board of an inoculating tomato leaf mycete peek diameter 10mm.The blank group does not connect the bacterium cake, adds clear water and preserves moisture.Each group all places 26 ℃ to cultivate 3 days.Experimental result is seen accompanying drawing 3.The blank group is not fallen ill, and the experimental group and the fermentation liquor treatment group of inoculation bacterium cake are all fallen ill.Visible by Fig. 3, the leaf spot lesion of catching an illness is concentrated, and color is obviously darker; Black in color; And there is obvious browning sign at the blade site of pathological change back side, and with the test group region of disease lighter color that fermentation liquor treatment is crossed, is light brown; The flavescence phenomenon does not appear in the site of pathological change back side, has than big-difference with actual disease symptom.See that from onset state fermentation liquor treatment is to the obvious restraining effect of having of leaf muld of tomato.

Claims

1. marine streptomyces L131 is characterized in that: separate in the ooze sample, the purifying substratum is the Bennett substratum, and marine streptomyces L131 bacterium colony is protruding, surface folding, and the adularescent spore produces faint yellow soluble pigment.

2. the said a kind of marine streptomyces L131 of claim 1 is characterized in that: the consisting of of said Bennett substratum: glucose 10 g, peptone 2 g; Yeast powder 1 g, Carnis Bovis seu Bubali cream 1 g, agar 15 g; Zero(ppm) water is settled to 1L, 7.2,121 ℃ of moist heat sterilization 20min of pH.

3. a marine streptomyces L131 is characterized in that, its 16S rDNA complete nucleotide sequence, and length overall is 1421bp, sequence is as follows:

GGCGGCGTGCTTACACATGCAAGTCGAACGATGAACCGCTTTCGGGCGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGACCGTCTGCCGCATGGTGGATGGTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAGTGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGTCTGGAGACAGGCGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGTGGA。

4. the said a kind of marine streptomyces L131 metabolite of claim 1 is characterized in that: make through following preparation method:

(1) fermentation of streptomycete L131

Described YMG substratum is formed: glucose 4 g, and malt extract 10 g, yeast extract 4 g, zero(ppm) water is settled to 1L, and pH 5.5 ± 0.2,121 ℃ of moist heat sterilization 20 min, pH 5.5 ± 0.2;

The fermentation of optimizing is formed with substratum: Semen Maydis powder 15 g, analysis for soybean powder 15 g, yeast extract 1.5 g, CaCO ₃0.5 g, zero(ppm) water is settled to 1L, 121 ℃ of moist heat sterilization 20 min;

With centrifugal 10 min of fermented liquid 6000 r/min; Get supernatant and pretreated macroporous adsorbent resin AB-8 after centrifugal with 10:3 (v:m) mixed, place shaking table 150 r/min to adsorb 24 h, use equal-volume zero(ppm) water then respectively; 40% washed with methanol; Use equal-volume 100% methyl alcohol desorb, 150 r/min desorption, 24 h, stripping liquid uses 35 ℃ of evaporates to dryness of rotary evaporation in vacuo appearance; With the pure methyl alcohol of 1/10 volume residuum is dissolved again concentratedly again, obtain the tomato leaf mould is had the metabolite of remarkable inhibiting streptomycete L131.

5. the said a kind of marine streptomyces L131 metabolite of claim 4 is in the agricultural chemicals of leaf muld of tomato control or the application in the preparation.

6. the agricultural chemicals or the preparation of the control of the said leaf muld of tomato of claim 5, it is characterized in that: comprise effective constituent and ancillary component, its effective constituent mainly is the metabolite of marine streptomyces L131.