CN101525587A - Streptomyces strain and application thereof - Google Patents
Streptomyces strain and application thereof Download PDFInfo
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- CN101525587A CN101525587A CN200910080902A CN200910080902A CN101525587A CN 101525587 A CN101525587 A CN 101525587A CN 200910080902 A CN200910080902 A CN 200910080902A CN 200910080902 A CN200910080902 A CN 200910080902A CN 101525587 A CN101525587 A CN 101525587A
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Abstract
The invention discloses a Streptomyces sp. strain and the application thereof. The strain provided by the invention is Streptomyces sp. YL04 CGMCC No.2884. The strain YL04 provided by the invention with ideal biocontrol effect and development value can be popularized in large-area fields and mainly solves the problem that increasingly serious damping-off and blight diseases of crop seedlings like vegetables and melons cause serious output reduction or reduced product quality. The invention has significant scientific meaning and application value in enriching strain resources for biocontrol of our country and provides effective measures for achieving high-output and high-quality modern agriculture. The invention meets the national standards of agricultural goods production safety and the need of constructing a non-polluted, green and organic agricultural production base and has certain economic and social benefits.
Description
Technical field
The present invention relates to a streptomyces strain and application thereof.
Background technology
For centuries, chemical pesticide is being brought into play positive effect in the preventing and controlling of the phytopathy Chinese caterpillar fungus plague of rats, for the stable yields of agricultural, increase production, retrieve a loss and improve quality and made significant contribution.But the excessive serious problems such as resistance that also caused soil fertility decline, environmental pollution, influenced non-target organism and harmful organism of using with pesticide abuse.Especially in recent years, the pesticide residue person poultry poisoning's incident that causes that exceeds standard constantly takes place in the agricultural-food, and the people's quality of life in serious threat, has also limited the foreign export of agricultural products in China.The wildness generation of soil-borne disease and the potentially contaminated of chemical bactericide have become the primary key issue in the agricultural safety in production.
Along with the development of social civilization, the agricultural sustainable development problem makes human understanding to resource and environment that new leap arranged, and agricultural chemicals has been proposed efficient, low toxicity, environmentally friendly requirement.Countries in the world are also in the use of minimizing chemical synthetic pesticide that takes practical steps, announce to cancel the registration of 91 kinds of chemical pesticides in the nineties in 20th century as U.S. EPA, Holland, Denmark have formulated 5 years and the ten year plan that reduces half agricultural chemicals usage quantity in nineteen ninety, European Union has also formulated similar plan, and China has cancelled the registration of tens kinds of chemical pesticides.In order to solve the contradiction that improves between agricultural output and the assurance agricultural product quality, reach the purpose of the great disease and pest of integrated control, minimizing chemical pesticide usage quantity, development biological control technology more and more receives national governments, scientific worker and the common people's concern.
Bacteria agent (BCA) have low toxicity, environment compatibility good, have and build ability, lasting period of group long, harmful organism at the crop seedling rhizosphere and be difficult for characteristics such as it develop immunity to drugs, thereby obtained paying close attention to widely and paying attention to.Advanced in the world at present farming company has played an active part in the research and the marketization of bacteria agent mostly, beneficial bacteria has been obtained certain progress to the control of soil biography, seed-borne disease, and have series product to obtain successful application, obtained remarkable economical and ecological benefits.Biological control is being brought into play more and more important effect as important measures of integrated control in the plant pest comprehensive regulation.
Biocontrol microorganisms is the research focus of biocontrol of plant disease as an important ring of biological control always.Biocontrol microorganisms of a great variety, widespread use has fungi, bacterium, actinomycetes, a virus etc. in the production.Wherein actinomycetes are that people are studied and are applied to the biological and ecological methods to prevent plant disease, pests, and erosion microorganism in the production the earliest.Actinomycetes (Actinomycete) are the gram positive bacteriums that a class has thread branch cell, gain the name because of bacterium colony is radial, and great majority have flourishing branch mycelia.The actinomycetes mycelia is very thin, and width is bordering on rod-shaped bacterium, about 0.5～1 μ m, and can be divided into vegetative hyphae and aerial hyphae: vegetative hyphae claims the matrix mycelia again, and major function is to absorb nutritive substance, and what have produces different pigments, is the important evidence of strain identification; Aerial hyphae is general storied on vegetative hyphae, claim the secondary mycelia again, differentiating on aerial hyphae can spore-bearing fibrillae of spores, the shape of fibrillae of spores and arrangement mode are different because of kind, produce the conidium of bunchiness on the sophisticated fibrillae of spores, the surface tissue of spore, shape and color are more stable under certain condition, are the important evidence of identifying bacterial classification.Actinomycetes are with the breeding of asexual spore and thalline fracture mode, and overwhelming majority is the heterotroph aerophil, and the kind that has is the organic matter of complexity such as decomposition of cellulose at high temperature.Actinomycetes are very wide in distributed in nature, and the overwhelming majority is saprophytic, the minority parasitism, and can produce miscellaneous microbiotic.According to estimates, have 2/3 to be that actinomycetes produce in more than the 6000 kind of microbiotic of having found.The important genus of actinomycetes comprises streptomyces, micromonospora and Nocardia etc.
Actinomycetes are antibiotic important biomolecule sources, and in the actinomycetales, streptomyces (Streptomyces) is widely distributed, generation metabolic substd ability is strong, and the microbiotic that is produced by its has accounted for 2/3 of all actinomycetes microbiotic quantity, and is therefore important.The microbiotic of being used widely aborning at present that is produced by streptomycete mainly comprises: (1) jingganmycin: be the main agricultural chemicals of control wheat hypochnus in the north, also the bacterial strain or the strain system that do not find jingganmycin is produced resistance at present; (2) multi-effect mycin:, Valsa mali, apple fruit rot germ and fruit white rot of grape bacterium etc. are had stronger bacteriostatic action according to test; (3) pesticide corrosion 120: once be used widely in recent years, be used to prevent and treat wheat powdery mildew, rice sheath blight disease, apple tree canker, leaf spot of peanut and watermelon blight etc. (Wang Guangliang, Yu Jinyou, Shi Yuping, etc., 2004).
Through the continuous exploration in more than 40 years, filtered out at present the streptomycete that the tool biological and ecological methods to prevent plant disease, pests, and erosion of kind more than 10 is worth in the world, and played enormous function in the different historical stages.So far seek with the antibiotic activity of studying different actinomycetes and generation thereof and be still important field in the control of plant disease, have great importance for the sustainable development of the ecological agriculture.
Description of drawings
Fig. 1 is a bacterial strain YL04 genome DNA electrophoretogram.
Fig. 2 is the electrophorogram to the pcr amplification product of YL04 16SrRNA.
Summary of the invention
The purpose of this invention is to provide a streptomyces strain and application thereof.
Streptomycete provided by the present invention (Streptomyces sp.), name is called YL04, belong to actinomycetales (Actinomycete) streptomyces (Streptomyces), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 19th, 2009 and (be called for short CGMCC, the address is: the Datun Road, Chaoyang District, Beijing City), preserving number is CGMCC No.2884.
The present invention also protects the metabolite of described streptomycete (Streptomyces sp.) YL04 CGMCC No.2884.
Described streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 and/or its metabolite can be applicable to suppress fungi, specifically can be applicable in the plant fungal disease resistance, as are applied to anti-vegetable sprout term samping off, wheat hypochnus or take-all etc.
The present invention also protects the cultural method of a kind of streptomycete (Streptomyces sp.) YL04 CGMCC No.2884; be with following culture medium culturing streptomycete (Streptomyces sp.) YL04 CGMCC No.2884: ammonium sulfate 2g; dipotassium hydrogen phosphate 1g; Zulkovsky starch 10g; bitter salt 1g, sodium-chlor 1g, lime carbonate 3g; agar powder 10g, water is settled to 1000ml.The condition of cultivating described streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 specifically can be: pH 5.0-8.0,7-10 days, 24-28 ℃.
The present invention also protects a kind of method for preparing streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 metabolite; be with following culture medium culturing streptomycete (Streptomyces sp.) YL04 CGMCC No.2884: yeast extract paste 5g; bitter salt 0.05g; lime carbonate 0.2g; sodium-chlor 0.4g, starch 10g, soyflour 10g; peptone 5g, water is settled to 1000ml.Cultivate described streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 and specifically can adopt liquid culture, the condition of described liquid culture is: pH 5.0-8.0, rotating speed 120-200rpm, 3-7 days, 24-28 ℃, 1% connect bacterium amount, 10-40% bottling amount (volumn concentration).The condition optimization of described liquid culture is: 26 ℃ of pH8.0, rotating speed 180rpm, 3 days, temperature, 1% connect bacterium amount, 20% bottling amount (volumn concentration).
The present invention also protects a kind of fungistat, and its activeconstituents is streptomycete (Streptomyces sp.) YL04CGMCC No.2884 and/or cultivates the metabolite that streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 obtains.Described fungistat can be the biological prevention and control agent of plant fungal disease resistance.
Described fungi specifically can be melon and fruit rotten mildew fungus (Pythium aphanidermatum), gaeumannomyces graminis (Geaumanomyces graminis), Valsa mali (Valsa mali), wheat dry thread Pyrenomycetes (Rhizoctonia cerealisbv), rice blast fungus (Pyricularia grisea), verticillium dahliae (Verticillium dahliae), cotton-wilt fusarium (Fusarium oxysporumf.sp.vasinfectum), fusarium graminearum (Fusarium graminearum), tomato early blight bacterium (Alternaria solani), Exserohilum turcicum (Exserohilum turcicum), southern corn leaf blight (Bipolaris maydis), Rhizoctonia solani Kuhn (Rhizoctonia solani) or botrytis cinerea (Botrytis cinerea).
The present invention is by studies have shown that, bacterial strain YL04 has in various degree restraining effect to all test plant pathogenic fungies, and wherein the activity to Valsa mali is the highest, and inhibiting rate reaches 89.7%; The rotten mould inhibiting rate of melon and fruit is reached 73.18%; To the inhibiting rate of wheat dry thread Pyrenomycetes, gaeumannomyces graminis, tomato gray mould and rice blast fungus nearly 60%; But all test plant pathogenetic bacterias are not all had activity, illustrate that YL04 has the anti-mycotic activity than wide spectrum.
The present invention has also studied the YL04 bacterial strain by the spray inoculation method under greenhouse experiment, irritate after three kinds of different methods inoculations of root inoculation method and method for soaking seed cucumber, tomato and the pimento influence to its seedling and plant strain growth.The result shows, this bacterial strain is to three kinds of crop safeties, equal no pathogenicities, and the effect that promotes growth is arranged.This research provides theoretical foundation for YL04 further application aborning.
The present invention is by field plot trial, the samping off that pythium spp causes in behind clear and definite several vegetables of YL04 inoculation and the wheat seed it being produced, microbial damping-off of miliary damping-off and microbial gaeumannomyces graminis disease of gaeumannomyces graminis disease all have good preventing and control action kou, and can promote plant strain growth and production-increasing function, have significant diseases prevention seedling protecting effect.
Bacterial strain YL04 provided by the invention has the ideal biocontrol effect and exploitation is worth, can promote in the field big area, emphasis solves harm such as vegetables, crops seedling stage samping off such as melon and damping-off to be increased the weight of day by day, causes the problem of the serious underproduction or reduction quality product.The present invention has important scientific meaning and using value to the strain resource of enriching China and being used for biological control, provides effective measure for realizing the high yield and high quality modern agriculture.The present invention meets requirement and nuisanceless, the green and the Organic farming construction of production base needs of agricultural products in China safety in production standard, has certain economic and social benefit.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
In following examples, the diameter of Oxford cup is 10mm.
The separation of embodiment 1, streptomycete bacterial strain YL04 and evaluation
One, the separation of bacterial strain
Streptomycete bacterial strain YL04 is the bacterial strain that the strain found when the target leaf that picks up from potato base, Keshan County, Heilungkiang being carried out strains separation in 2004 has potential biological and ecological methods to prevent plant disease, pests, and erosion effect, and concrete separating step is as follows:
1, the sick strong position that has a common boundary of clip sample was carried out surface sterilization in 5 seconds with soaking behind the aseptic water washing 2 times in 70% alcohol, use aseptic water washing again 1 time, was put on the absorbent filter of sterilization, dried naturally.
2, the sick sample after will sterilizing is inoculated on the rye sucrose nutrient agar, cultivates 7 days for 20 ℃.When finding on one of them substratum early blight bacterium mycelial growth after 7 days, there is another kind of bacterium colony to generate, on the substratum of this periphery of bacterial colonies, can produces tangible inhibition zone, tomato early blight bacterium is shown certain restraining effect.This strain growth is very slow, forms very little bacterium colony, many gauffers, and densification, drying can form the living mycelia of root hair shape base, the similar actinomycetes of morphological specificity.The meta-bolites of this bacterial strain has good bacteriostatic activity to multiple pathogenic bacteria under isolated condition, with this bacterial strain called after YL04.
Two, identification of strains
(1) form of bacterial strain YL04 and physiological characteristic are identified
With reference to the method in " general bacterium common method " and " classification of actinomycetes basis " (Ruan Jisheng, 1977), the biological characteristics that adopts morphological observation, multinomial physiological and biochemical index to detect YL04 carries out systematic study.
The morphological specificity of bacterial strain YL04: bacterium colony is little, the surface is rugged, many wrinkles, dense, dry, and colony structure can change with plant age and growth conditions.The base living mycelia root hair shape, do not rupture; Aerial hyphae becomes fibrillae of spores very soon, and the little song of fibrillae of spores fragments into spherical spore.Aerial mycelium covers the bacterium colony top layer, is powdery.
The physiological characteristic of bacterial strain YL04: catalase test produces bubble, reacting positive; Zona pellucida appears in hydrolyzed starch; Produce hydrogen sulfide and be determined as feminine gender; Do not produce melanochrome; Produce cellulase; Reduction nitrate becomes nitrite; It is blue that liquefy gelatin, litmus milk reaction and display reindeer moss become, and the milk clear shows that this bacterium produces alkali, peptonizes etc.
Form and physiological characteristic qualification result show that bacterial strain YL04 belongs to streptomyces (Streptomyces).
(2) Molecular Identification of bacterial strain YL04 (the 16SrRNA gene sequencing of YL04 bacterial strain)
1, the extraction of genomic dna
The bacterial genomes of Ke Ruitai in the application (Beijing) bio tech ltd is extracted the genome that test kit extracts bacterial strain YL04.Get 5 μ L DNA samples and carry out 0.8% agarose gel electrophoresis, the 260nm ultraviolet lamp is observed down and is taken a picture.Electrophoresis result as shown in Figure 1, among Fig. 1, the 1:YL04 complete genome DNA; M:DNA Marke (DL2000Plus DNA Marker:5000,3000,2000,1000,750,500,250,100bp).As seen from the figure, DNA is a specific band, does not have tangible degradation fragment and produces, and illustrates that the genomic dna that is extracted is complete, can be used as the template of PCR reaction.
2, pcr amplification
Adopting the primer sequence of having reported (Xu Ping, 2003), is that template is reacted at the enterprising performing PCR of Techne PCR instrument with the YL04 genomic dna, and response procedures is 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 56 ℃ of renaturation 1min, 72 ℃ are extended 2min and carry out 35 circulations; 72 ℃ are extended 5min.Get 3 μ L PCR products and carry out 1% agarose gel electrophoresis, the 260nm ultraviolet lamp is observed down and is taken a picture.Primer sequence is as follows:
Primer A (forward primer), 8-27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ';
Primer B (reverse primer), 1523-1504r:5 '-AAGGAGGTGATCCAGCCGCA-3 '.
PCR reaction system (50 μ L):
10×PCR buffer 6μL
10mmol/L dNTP 4μL
Forward primer (20 μ mol/L) 1 μ L
Reverse primer (20 μ mol/L) 1 μ L
Genomic dna＜100ng
Taq enzyme (5U/ μ L) 1 μ L
H
2O mends to 50 μ L
Electrophoresis result as shown in Figure 2.Among Fig. 2,1: to the pcr amplification product of YL04 16SrRNA gene; 2 swimming lanes are that PCR does not have the template contrast; M:DNA Marke (DL2000 Plus DNA Marker:5000,3000,2000,1000,750,500,250,100bp).Amplified the clear special band in the 1500bp left and right sides.
3, the recovery of PCR product
Reclaim the PCR product with Easypure Quik Gel Extraction Kit (TransGen Biotech).
4, the clone of PCR product and order-checking
(1) connects
Connect with pGM-T Vector test kit (TIANGEN Biotech (Beijing) Co., Ltd.).
Linked system (10 μ L):
PGM-T Vector (about 50ng/ μ L) 1 μ L
2×T4 DNA Rapid Ligation Buffer 5μL
T4 DNA Ligase(3U/μL) 1μL
Purpose fragment 1-3 μ L
Moisturizing to 10 μ L
Mixing is placed on 4 ℃ of connections and spends the night.
(2) transform
(a) 50 μ L competence intestinal bacteria (Escherichia coli) Top10 of freeze thawing on ice;
(b) add 5 μ L and connect product, mixing gently;
(c) place more than the 30min on ice;
(d) 42 ℃ of thermal shock 90s;
(e) leave standstill 2min on ice, add 500 μ L LB substratum;
(f) 37 ℃, 1500rpm shakes training 45min;
(g) get 40 μ L, 2% X-gal and 7 μ L 20%IPTG and be uniformly coated on (37 ℃ of placements are more than 1 hour) on the penbritin LB flat board that contains 50 μ g/mL, be coated with 100 μ L conversion products then in the LB flat board, 37 ℃ of overnight incubation are through blue hickie screening positive clone.
(3) evaluation of positive colony
PCR reaction system (25 μ L):
10×PCR buffer 3μL
10mmol/L dNTP 2μL
20 μ mol/L forward primers, 0.5 μ L
20 μ mol/L reverse primers, 0.5 μ L
Taq enzyme (5U/ μ L) 0.5 μ L
H
2O 18.5μL
Dip in aseptic rifle head and to get the microbiotic positive bacteria and fall within the PCR system, the PCR positive colony is by the order-checking of Beijing three rich polygala root biotechnology limited liability companys.Sequencing result shows that expanding fragment length is 1519bp, and its dna sequence dna is shown in the sequence 1 of sequence table.
5,16SrRNA gene sequencing
The 16SrRNA nucleotide sequence of bacterial strain YL04 is input to after the NCBI website carries out the similarity comparison, the sequence similarity that the 16SrRNA nucleotide sequence of discovery YL04 and streptomyces (Streptomyces) are a plurality of kinds reaches more than 99.4%, so determine that YL04 belongs to streptomyces (Streptomyces).
By the Molecular Identification result of bacterial strain YL04, confirm that further bacterial strain YL04 is streptomyces (Streptomyces).YL04 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center with streptomycete (Streptomyces sp.), and preserving number is CGMCC No.2884.
The substratum of embodiment 2, bacterial strain YL04 and culture condition screening
Actinomycetes substratum commonly used has multiple, as Gause I substratum, rolled oats nutrient agar, czapek's solution etc., but at the actinomycetes of different genera and different culturing purposes, the optimum medium difference.44 kinds of substratum of this test and Selection are as for the examination substratum, therefrom filter out the 1-2 kind the most suitable YL04 growth and produce plain substratum; Simultaneously, determined that to produce element be purpose when cultivating bacterial strain YL04, the suitableeest bottling amount, pH value, shaken and train rotating speed and incubation time.For further physico-chemical property, constructional feature, mechanism of action and the industrial fermentation production thereof of the antimicrobial substance of research YL04 generation provide reference.
One, the screening of growth medium
1, the bacterial strain YL04 that will preserve under cold condition is forwarded to the Gause I medium slant, cultivates 5d down for 28 ℃, the activation bacterial strain.In the good test tube of strain growth, add an amount of sterilized water, fully vibration, making concentration is 10
8The cfu/ml spore suspension, 10 times of stepwise dilutions then, the spore suspension of making serial gradient is standby.
44 kinds of substratum that 2, will prepare, 121 ℃ of following moist heat sterilization 20min pour the substratum of fusing in the sterilized culture dish into according to the amount of 15ml/ ware, and it is standby to make culture medium flat plate.
3, drawing concentration is the spore suspension of 103cfu/ml, amount according to 100 μ l/ wares adds respectively on 44 kinds of culture medium flat plates, coating evenly, after in Bechtop, drying the moisture of planar surface, be placed in 26 ± 2 ℃ the incubator and secretly cultivate, single colony number of growing on the every ware of record after 10 days and measure colony diameter.
Test is provided with three repetitions, results averaged.The result shows, 44 kinds in the examination substratum, has 25 kinds can supply the YL04 strain growth, and it is all right wherein to number on ten kinds of substratum of 1,2,4,5,20,35,37,38,40,44 strain growth, therefore preliminary selected its for for trying substratum.Further carry out postsearch screening, the result of postsearch screening shows that single colony number is higher than other substratum on No. 2 substratum; The actinomycetes growth is very fast on No. 35 substratum, show as single colony diameter maximum, but single colony counts is less; So selected No. 2 substratum (starch ammonium substratum) are the substratum of the most suitable bacterial strain YL04 growth.
No. 2 the culture medium preparation method is: ammonium sulfate 2g, dipotassium hydrogen phosphate 1g, Zulkovsky starch 10g, bitter salt 1g, sodium-chlor 1g, lime carbonate 3g, agar powder 10g, distilled water 1000ml.
Two, produce the screening of plain substratum
1, in the well-grown test tube slant of bacterial strain YL04, add an amount of sterilized water, fully vibration, making concentration is 10
8The cfu/ml spore suspension is standby.
It is in the tissue culture bottle of 450ml that 44 kinds of liquid nutrient mediums that 2, will prepare add volume, every bottle of 100ml, and 121 ℃ of moist heat sterilization 20min, standby.
3, respectively the spore suspension of step 1 preparation is inoculated in 44 kinds of substratum 1mL/ bottle, 26 ℃, the cultivation of 150rpm shaking table.Respectively 1,2,3,5, the 7d sampling, centrifugal (10000gpm, 2min), it is standby to get supernatant liquor with sample cultivation liquid.
4, make the PDA flat board, on flat board be that symmetric points are placed two Oxford cups with the central point, Oxford cup anomaly plate center 3cm, the supernatant liquor (in contrast) that in each Oxford cup, adds the preparation of 100 μ l steps 3 with the processing that adds sterilized water, 3 repetitions are established in each processing, leave standstill, inserting diameter in dull and stereotyped center position behind the 24h is the bacterium cake of the target bacterium (Valsa mali) of 5mm.To train in the incubator that the sample ware is placed on 26 ± 2 ℃ and secretly cultivate, measure the radius of bacterium colony behind the 3d, calculate the growth bacteriostasis rate.
Test-results shows, after bacterial strain YL04 cultivates 1d, do not detect nutrient solution the target bacterium is had bacteriostatic activity, the nutrient solution that can detect 11 kinds of substratum behind the 2d has bacteriostatic activity, the nutrient solution of most of substratum all has bacteriostatic action behind the 3d, the bacteriostatic activity of nutrient solution that wherein is numbered 1,4,9,13,17,30,36,38,40,47 substratum is stronger, therefore selected its for for the examination substratum.Further carry out postsearch screening, the postsearch screening test-results shows, the nutrient solution of No. 36 substratum fungistatic effect when 3d is better, the bacteriostatic activity of nutrient solution all is better than other for the examination substratum 4,5,6, during 7d, so draft No. 36 substratum produce antimicrobial substance for the most suitable bacterial strain YL04 substratum.
No. 36 the substratum compound method is: yeast extract paste 5g, and bitter salt 0.05g, lime carbonate 0.2g, sodium-chlor 0.4g, starch 10g, soyflour 10g, peptone 5g is settled to 1000ml with distilled water.
The screening of three, bottling amount and pH value
With No. 36 substratum serves as for the examination substratum, 10%, 20%, 30%, 40% 4 different bottling amount is set handles, and simultaneously, in each bottling amount processing the pH value is set and is respectively 4,5,6,7,8,9,10 7 different treatment, amounts to 28 processing.
Cultivate bacterial strain YL04 down in 28 ℃, the rotating speed of 180rpm, get supernatant respectively at 3,4, behind the 5d and detect fungistatic effect.Employing is with malicious flat band method to detect fungistatic effect: 100 μ l supernatant liquors are added to abundant mixing in the 100ml PDA substratum, and dull and stereotyped, inoculating diameter in dull and stereotyped central authorities is the apple rot pathogen bacterium cake of 5mm; Simultaneously, be provided with add 100 μ l sterilized waters the PDA flat board in contrast.To train in the incubator that the sample ware is placed on 26 ± 2 ℃ and secretly cultivate, measure the radius of bacterium colony behind the 3d, calculate growth inhibition ratio.
3 repetitions, results averaged are established in test.The results are shown in Table 1.
The bacteriostasis rate of bacterial strain fermentation liquor under the different culture condition of table 1
Test-results shows that different bottling amounts and pH value are produced element to YL04 to be influenced bigger.The pH value is 5 to 10, and the nutrient solution of various bottling amounts all has fungistatic effect.Initial pH value is 7 and 8, and the bottling amount is the fungistatic effect optimum of 20% o'clock nutrient solution.
Four, the screening of rotating speed
Based on the shaker test result of bottling amount and pH value, be 8,20% bottling amounts promptly in the pH value, 1% connects bacterium further screening and culturing condition of when amount.120rpm, 160rpm, 180rpm and four different rotating speeds processing of 200rpm are set under 26 ℃.Take a sample respectively at 1,2, behind the 3d, detect fungistatic effect, the same step 3 of detection method.3 repetitions, results averaged are established in test.The results are shown in Table 2.
The bacteriostasis rate of table 2 different rotating speeds condition bacterial strain fermentation liquor
| 120rpm | 160rpm | 180rpm | 200rpm | |
| 1d | -0.79 | 0.00 | 0.36 | 2.97 |
| 2d | 80.29 | 51.41 | 59.17 | 79.04 |
| 3d | 85.33 | 82.71 | 88.10 | 75.27 |
Test-results shows, different rotating speeds has certain influence to the product element of YL04, rotating speed from low to high the time bacteriostatic action of nutrient solution also show as gradually and strengthen, after rotating speed acquires a certain degree, the bacteriostatic activity of nutrient solution but reduces along with the increase of rotating speed, and rotating speed is that 180rpm, incubation time are that the fungistatic effect performance of nutrient solution under the condition of 3d is the most obvious.
The bacteriostatic activity of embodiment 3, bacterial strain YL04 (antimicrobial spectrum mensuration)
One, bacterial strain YL04 metabolism liquid is to the inhibition effect of pathogenic fungi
This experimental selection the plant pathogenic fungi of 15 kinds in 13 genus, adopt the Oxford agar diffusion method to detect the inhibition effect of YL04 metabolism liquid to pathogenic fungi, with the antibacterial biological and ecological methods to prevent plant disease, pests, and erosion effect of clear and definite YL04 thalline and meta-bolites and control spectrum, further research and development and field are used foundation are provided for this bacterial strain.
As follows for the examination pathogenic fungi: melon and fruit rotten mildew fungus (Pythium aphanidermatum), cucumber phytophthora root rot bacterium (Phytophthora melonis), pimento parasitica (Phytophthora copsici), gaeumannomyces graminis (Geaumanomyces graminis), Valsa mali (Valsa mali), Rhizoctonia solani Kuhn (Rhizoctonia solani), wheat dry thread Pyrenomycetes (Rhizoctonia cerealisbv), rice blast fungus (Pyricularia grisea), verticillium dahliae (Verticillium dahliae), tomato early blight bacterium (Alternaria solani), Exserohilum turcicum (Exserohilum turcicum), southern corn leaf blight (Bipolaris maydis), fusarium graminearum (Fusarium graminearum), cotton-wilt fusarium (Fusarium oxysporum f.sp.vasinfectum), botrytis cinerea (Botrytiscinerea).Above pathogenic bacteria all can be adopted ordinary method to separate from disease plant and be obtained, and perhaps planting disease from China Agricultural University is that the seed Pathology Lab directly obtains.
Concrete detection method is as follows:
1) will be for 25 ℃ of activation of examination fungi 5-7 days (cucumber phytophthora root rot bacterium be adopted the Semen Phaseoli Vulgaris substratum, and other fungi is adopted the PDA substratum), making it form diameter is the bacterium colony of 7～8cm, it is standby to produce the bacterium cake with punch tool (diameter 5mm).
2) fermented liquid of preparation bacterial strain YL04: bacterial strain YL04 is inoculated in No. 36 liquid nutrient mediums (yeast extract paste 5g, bitter salt 0.05g, lime carbonate 0.2g, sodium-chlor 0.4g, starch 10g, soyflour 10g, peptone 5g, distilled water 1000ml), 26 ℃, 180rpm are cultivated 3d, get the centrifugal 2min of fermented liquid 10000g/min, supernatant liquor is the aseptic metabolism liquid of YL04.
3) bacteriostatic action is measured: produce the PDA flat board, in flat board be that symmetric points are placed two Oxford cups with the center, the Oxford cup is apart from dull and stereotyped center 3cm, in each Oxford cup, add step 2) the supernatant liquor 100 μ l of preparation (with the flat board that adds sterilized water CK in contrast), 3 repetitions are established in each processing, leave standstill, inserting diameter at dull and stereotyped center behind the 24h is the target bacterium bacterium cake of 5mm, be placed in 25 ± 2 ℃ the incubator and secretly cultivate, according to the growing state of difference for the examination bacterium, measure the fungal colony diameter respectively after cultivating different number of days, calculate growth inhibition ratio.The results are shown in Table 3.
Table 3YL04 metabolism liquid is to the restraining effect for the examination pathogenic fungi
Test-results shows that YL04 all has bacteriostatic activity to 15 kinds of pathogenic fungies for examination.To the inhibiting rate of Valsa mali nearly 90%, the rotten mould inhibiting rate of melon and fruit is reached 73.18%, inhibiting rate to wheat dry thread Pyrenomycetes, total eclipse germ, tomato gray mould and rice blast fungus reaches about 60%, to the inhibiting rate of verticillium dahliae, gibberella saubinetii and cotton-wilt fusarium more than 40%, tomato early blight bacterium, Rhizoctonia solani Kuhn, Exserohilum turcicum and southern corn leaf blight inhibiting rate are reached more than 30%, mould to the cucumber epidemic disease, pimento parasitica restraining effect is relatively poor.
Two, YL04 metabolism liquid is to the inhibition effect of pathogenetic bacteria
This experimental selection the plant pathogenetic bacteria of 5 kinds in 3 genus, adopt double-layer plate culture method and Oxford cup detection method to detect bacteriostatic activity and the antimicrobial spectrum of YL04 metabolism liquid to pathogenetic bacteria.
As follows for the examination pathogenetic bacteria: cloth gram Bordetella (Ralstonia) solanum solanacearum (Ralstoniasolanaecearum); Rhodopseudomonas (Pseudomonas) angular leaf spot of cucumber bacterium (Pseudomonassyringae); Xanthomonas (Xanthomonas) cotton angular leaf spot fungus (Xanthomonas matvacearum), pepper scab fungus (Xanthomonas campestris); Firmicutes clavate Bacillaceae (Clavibacter) bacterial canker of tomato (Clavibacter michiganense).Above pathogenic bacteria all can be adopted ordinary method to separate from disease plant and be obtained, and perhaps planting disease from China Agricultural University is that the seed Pathology Lab directly obtains.
1) YL04 preparation of fermentation liquid: YL04 is inoculated in No. 36 liquid nutrient mediums (yeast extract paste 5g, bitter salt 0.05g, lime carbonate 0.2g, sodium-chlor 0.4g, starch 10g, soyflour 10g, peptone 5g, distilled water 1000ml), cultivate 3d at 26 ℃, 180rpm, get fermented liquid centrifugal 2min under the speed of 10000g/min, supernatant liquor is the aseptic metabolism liquid of YL04.
2) preparation of lower floor's substratum: in diameter is the substratum of 9cm, pour the sterilized water agar of 10ml (adding the 10g agar powder in the distilled water of 1000ml) into, be lower floor's substratum, repeat for 4 times.
3) preparation of epipelagic zone bacterium culture medium: the 5ml sterilized water is joined in the pathogenic bacteria test tube, fully vibrate with vibrator, producing into concentration is 10
8The bacteria suspension of cfu/ml; The initial bacteria suspension of 40 μ l is added in the sterilized water agar of 40ml, and mixing is poured into respectively on 4 lower layer of water nutrient agars that prepared, has promptly formed germ-carrying upper strata substratum after solidifying.
3) bacteriostatic action is measured: the Oxford cup evenly is placed on water agar double-layer substratum (adding the 10g agar powder in the distilled water of the 1000ml) flat board, and every ware is put 2 points; The supernatant liquor 100 μ l that add the step 1) preparation in each Oxford cup, sterilized water contrast (CK) is set simultaneously, the supernatant liquor that replaces the step 1) preparation with sterilized water, pour step 2 Deng supernatant liquor again into after infiltrating substratum fully) the water agar that carries disease germs of preparation, each handles 3 repetitions, and 28 ℃ dark cultivates the diameter of measuring inhibition zone after a couple of days respectively.The results are shown in Table 4.
The bacterial spectrum extremely of table 4YL04 metabolism liquid
Test-results shows that YL04 metabolism liquid is to the equal unrestraint effect of 5 kinds of pathogenetic bacterias for examination.
Embodiment 4, bacterial strain YL04 and meta-bolites thereof are measured the security of plant growth
Adopt the spray inoculation method, irritate three kinds of different methods of root inoculation method and method for soaking seed and detect the influence of bacterial strain YL04, for YL04 further application aborning provides theoretical foundation to cucumber, tomato and pimento growth.Below each processing of test all is provided with three repetitions, results averaged.
One, spray inoculation method
(1) preparation of YL04 bacteria suspension: will be at No. 2 substratum (ammonium sulfate 2g, dipotassium hydrogen phosphate 1g, Zulkovsky starch 10g, bitter salt 1g, sodium-chlor 1g, lime carbonate 3g, agar powder 10g, distilled water 1000ml) YL04 that cultivates on the flat board behind the 7d scrapes with transfering loop, and it is suspended in the sterilized water, abundant vibration mixing is got a part and is surveyed its OD600 value on vibrator, makes YL04 bacteria suspension concentration reach 10
8-10Cfu/ml.
(2) preparation of test plant: the pimento seed is placed vernalization in 28 ℃ of constant incubators, after percentage of germination reaches more than 80%, be seeded in the dish of nursing young plants in hothouses, often observe, water in good time, use in order to inoculation.
(3) spray inoculation: young plant is long to 4 true leaves, the bacteria suspension of step (1) preparation evenly is sprayed at the plant leaf surface, notice that droplet is as far as possible little, be sprayed onto on the every leaf as far as possible that spray amount is stained with till the drop with blade, to spray tap water is contrast, observe once every 7d later on, the upgrowth situation of record plant, and in time water, treat to carry out the mensuration of plant strain growth index of correlation for studying one heart stage of thing two leaves.Test-results such as table 5.
The influence to two leaves, one heart stage cucumber and pimento plant strain growth is handled in the spraying of table 5YL04 bacteria suspension
The result shows, after the suspension spray of YL04 bacterial strain was handled pimento and cucumber seedling, plant strain growth was good, and long, the average stem length of its average root, single-strain fresh weight and dry weight all are higher than contrast, show that the YL04 bacterial strain studies thing growth safety to supplying, and certain promotion is arranged for the effect of examination plant strain growth.
Two, irritate the root inoculation method
(1) preparation of YL04 bacteria suspension: will be at No. 2 substratum (ammonium sulfate 2g, dipotassium hydrogen phosphate 1g, Zulkovsky starch 10g, bitter salt 1g, sodium-chlor 1g, lime carbonate 3g, agar powder 10g, distilled water 1000ml) YL04 that cultivates on the flat board behind the 7d scrapes with transfering loop, and carry with sterilized water is floating, abundant vibration mixing is got a part and is surveyed its OD600 value on vibrator, makes YL04 bacteria suspension concentration reach 10
8-10Cfu/ml.
(2) preparation of test plant: cucumber and pimento seed are placed vernalization in 28 ℃ of constant incubators, after percentage of germination reaches more than 80%, be seeded in the dish of nursing young plants in hothouses, often observe, water in good time, use in order to inoculation.
(3) irritate the root inoculation: quantitatively join in the seedling pan of step (2) with the bacteria suspension of sampler step (1) preparation, every cave 10ml, and to add tap water in contrast.Observe once every 7d, the upgrowth situation of record plant, and in time watering is treated to carry out the mensuration of plant strain growth index of correlation for studying one heart stage of thing one leaf.Test-results such as table 6.
Table 6YL04 bacteria suspension root irrigation is to the influence of pimento and cucumber plant growth
The result shows, after the suspension liquid irrigating root inoculation of YL04 bacterial strain, pimento and cucumber plant well-grown, its average root is long suitable with contrast, average stem is long, single-strain fresh weight and dry weight all are higher than contrast, show that the YL04 bacterial strain studies thing growth safety to three kinds of confessions, and certain promotion are arranged for the effect of examination plant strain growth.
Three, seed soaking is handled
(1) preparation of YL04 bacteria suspension: will be at No. 2 substratum (ammonium sulfate 2g, dipotassium hydrogen phosphate 1g, Zulkovsky starch 10g, bitter salt 1g, sodium-chlor 1g, lime carbonate 3g, agar powder 10g, distilled water 1000ml) YL04 that cultivates on the flat board behind the 7d scrapes with transfering loop, and carry with sterilized water is floating, abundant vibration mixing is got a part and is surveyed its OD600 value on vibrator, makes YL04 bacteria suspension concentration reach 10
8-10Cfu/ml.
(2) preparation of test plant: carry out seed-coat sterilization 3 minutes with 1% clorox earlier, behind aseptic water washing 3 times, bacteria suspension seed soaking with step (1) preparation, cucumber seed soaking 12 hours, tomato and pimento were all soaked seed 16 hours, establishing with the sterilized water seed soaking simultaneously is contrast, and seed soaking time is identical with the bacteria suspension seed soaking time.
(3) preparation of aseptic river sand: river sand is with 10 purpose sieve, 160 ℃ of dry sterilization 8h, and poach in the river sand, it is standard that water content holds with hand that sand do not scatter, and divides then to install in the porcelain dish, smooth back is used in order to sowing.
(4) sowing step (2) seeds treated is sealed with plastics film, reaches requirement to guarantee humidity, detects germinating energy behind the 3d, detects percentage of germination behind the 7d, and observe every day, and moisturizing in good time prevents that the river sand drying from influencing seed germination.The result is as shown in table 7.
Table 7 is soaked seed to the influence of pimento, tomato and cucumber germination with the YL04 bacteria suspension
The result shows: after the suspension of YL04 bacterial strain was handled seed, the pimento seed germination rate is compared with the clear water contrast had increased by 23.30%, and the percentage of germination of tomato and cucumber seeds is compared with the clear water contrast does not have significant difference.Show that the YL04 bacterial strain supplies examination budding safety to three kinds, does not all have pathogenic to growth of seedling.
Embodiment 5, bacterial strain YL04 are to the prevention effect of vegetable sprout term samping off, wheat hypochnus and take-all
With reference to the test requirements document of " Ministry of Agriculture's agricultural chemicals field test criterion, GB/T 17980-2004 ",, further measure the protection effect of YL04 to vegetable sprout term samping off, wheat hypochnus and take-all by the greenhouse pot culture test.For the large scale application of YL04 bacterial strain in the field provides reference.
One, bacterial strain YL04 is to the prevention effect of take-all and wheat hypochnus
Select 18 two wheat breeds of new wheat 18 and all wheats, adopt method for soaking seed and dip in the prevention effect of root method detection YL04 wheat hypochnus and take-all.Two kinds of methods are 20 of every processing, 5 repetitions.
1, will (this pathogenic bacteria can separate from the disease plant basal part of stem and obtains with the rhizoctonia cerealis (R..cerealis) of corn sand expanding propagation, perhaps planting disease from China Agricultural University is that the seed Pathology Lab directly obtains) be inoculated in the sterilization soil (inoculative proportion is 0.6%), obtain rhizoctonia cerealis soil; Will (this pathogenic bacteria can separate from the disease plant basal part of stem and obtains with the gaeumannomyces graminis (G..graminis) of corn sand expanding propagation, perhaps planting disease from China Agricultural University is that the seed Pathology Lab directly obtains) be inoculated in the sterilization soil (inoculative proportion 0.6%), obtain gaeumannomyces graminis soil.
2, adopt two kinds of methods to detect prevention effect
1) method for soaking seed
1. be 10 with wheat seed in concentration
8Be seeded in respectively in the bacterium soil that has rhizoctonia cerealis bacterium soil and gaeumannomyces graminis after soaking 24h in the cfu/ml YL04 spore suspension; Wheat seed soaked in sterilized water be seeded in rhizoctonia cerealis bacterium soil and gaeumannomyces graminis bacterium soil, (CK) in contrast behind the 24h respectively.
2. greenhouse experiment is cultivated wheat down, investigation wheat hypochnus and the sickness rate of gaeumannomyces graminis disease and disease index during 15d.
2) dip in the root method
1. 10d after wheat seed is emerged chooses stem and leaf of Wheat of uniform size, cleans the earth of root, blots the moisture that adheres on the seedling with thieving paper.
2. seedling is immersed 10
810min in the spore suspension of cfu/ml YL04 is transplanted into respectively in the bacterium soil that contains rhizoctonia cerealis bacterium soil and total eclipse germ then; Seedling is immersed 10min in the sterilized water, be transplanted in rhizoctonia cerealis bacterium soil and the total eclipse germ bacterium soil (CK) in contrast then respectively.
3. greenhouse experiment is cultivated wheat down, investigation wheat hypochnus and the sickness rate of gaeumannomyces graminis disease and disease index during 15d.
The be in a bad way grade scale (Lipps etc., 1982) of degree of wheat hypochnus is as follows:
0 grade: asymptomatic; 1 grade: leaf sheath has browning slightly; 2 grades: leaf sheath outside scab diameter＜0.5cm; 3 grades: the leaf sheath outside has 1 to a plurality of scabs, scab diameter＞0.5cm; 4 grades: scab is all arranged inside and outside the leaf sheath; 5 grades: leaf sheath, stem stalk all have scab or diseased plant withered.
The be in a bad way grade scale (Chen Huaigu etc., 2000) of degree of gaeumannomyces graminis is as follows:
0 grade: asymptomatic; 1 grade: the root area of being injured accounts for the total area below 1/4; 2 grades: the root area of being injured accounts for total area 1/4-1/2; 3 grades: the root area of being injured accounts for the 1/2-3/4 of the total area; 4 grades: the root area of being injured accounts for more than 3/4 of the total area; 5 grades: seedling stage occurs dwarfing, seedling death phenomenon.
The results are shown in Table 8.
Table 8YL04 is to the prevention effect of wheat hypochnus and take-all
Test-results shows, YL04 dips in root or the seed soaking processing all has preventive effect preferably to wheat hypochnus and take-all, wherein the preventive effect of seed soaking processing is 62.18-71.84%, dips in root processing preventive effect and reaches 72.03～84.57%, and the effect of dipping in the root processing slightly is better than seed soaking and handles.Show that YL04 is the biocontrol strain of strain ideal control soil-borne disease bacterium.
Two, YL04 is to the control of melon dish samping off
Excellent No. 4 cucumber varieties in selection Tianjin and cooperation 908 tomato varieties adopt method for soaking seed and dip in the prevention effect of root method detection YL04 to melon dish samping off.Two kinds of methods are 20 of every processing, 5 repetitions.
1, melon and fruit corruption mould (Pythium aphanidermatum) is inoculated in the sterilization soil (inoculative proportion is 0.6%), obtains melon dish da mping-off fungi soil.
2, adopt two kinds of methods to detect prevention effect
1) method for soaking seed
1. with seed 10
8Be seeded in melon dish da mping-off fungi soil after soaking 16h in the spore suspension of cfu/ml YL04; Be seeded in melon dish da mping-off fungi soil, (CK) in contrast cucumber seeds soaked 16h in sterilized water after.
2. greenhouse experiment is cultivated down, and 10d writes down the number of emerging, each sickness rate of handling of 20d record.
2) dip in the root method
1. 10d behind the seed sprouting chooses seedling of uniform size, cleans the earth of root, blots the moisture that adheres on the seedling with thieving paper.
2. seedling is immersed 10
810min in the spore suspension of cfu/ml YL04 is transplanted in the melon dish da mping-off fungi soil then; Seedling is immersed 10min in the sterilized water, be transplanted in the melon dish da mping-off fungi soil (CK) in contrast then.
3. greenhouse experiment is cultivated down, investigation sickness rate during 25d.
The results are shown in Table 9.
Table 9YL04 is to the prevention effect of cucumber samping off
The result shows that YL04 can obviously improve the seedling rate of cucumber and tomato after handling seed or seedling root, the seedling phase, sick seedling rate significantly reduced, the preventive effect that YL04 handles samping off can reach 70～90%, and amount of increase in production reaches 9.68～37.24%, has significant diseases prevention seedling protecting effect.
Sequence table
＜110〉China Agricultural University
＜120〉streptomyces strain and application thereof
<130>CGGNARY92169
<160>1
<210>1
<211>1519
<212>DNA
＜213〉streptomycete (Streptomyces sp.)
agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60
gatgaaccgc tttcgggcgg ggattagtgg cgaacgggtg agtaacacgt gggcaatctg 120
ccctgcactc tgggacaagc cctggaaacg gggtctaata ccggatatga ccgtctgccg 180
catggtggat ggtgtaaagc tccggcggtg caggatgagc ccgcggccta tcagcttgtt 240
ggtgaggtag tggctcacca aggcgacgac gggtagccgg cctgagaggg cgaccggcca 300
cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga atattgcaca 360
atgggcgaaa gcctgatgca gcgacgccgc gtgagggatg acggccttcg ggttgtaaac 420
ctctttcagc agggaagaag cgaaagtgac ggtacctgca gaagaagcgc cggctaacta 480
cgtgccagca gccgcggtaa tacgtagggc gcaagcgttg tccggaatta ttgggcgtaa 540
agagctcgta ggcggcttgt cacgtcggtt gtgaaagccc ggggcttaac cccgggtctg 600
cagtcgatac gggcaggcta gagttcggta ggggagatcg gaattcctgg tgtagcggtg 660
aaatgcgcag atatcaggag gaacaccggt ggcgaaggcg gatctctggg ccgatactga 720
cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780
aaacggtggg cactaggtgt gggcaacatt ccacgttgtc cgtgccgcag ctaacgcatt 840
aagtgccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt gacgggggcc 900
cgcacaagcg gcggagcatg tggcttaatt cgacgcaacg cgaagaacct taccaaggct 960
tgacatacac cggaaacgtc tggagacagg cgcccccttg tggtcggtgt acaggtggtg 1020
catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080
cttgtcccgt gttgccagca ggcccttgtg gtgctgggga ctcacgggag accgccgggg 1140
tcaactcgga ggaaggtggg gacgacgtca agtcatcatg ccccttatgt cttgggctgc 1200
acacgtgcta caatggccgg tacaatgagc tgcgataccg tgaggtggag cgaatctcaa 1260
aaagccggtc tcagttcgga ttggggtctg caactcgacc ccatgaagtc ggagtcgcta 1320
gtaatcgcag atcagcattg ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380
tcacgtcacg aaagtcggta acacccgaag ccggtggccc aaccccttgt gggagggagc 1440
tgtcgaaggt gggactggcg attgggacga agtcgtaaca aggtagccgt accggaaggt 1500
gcggctggat cacctcctt 1519
Claims (10)
1, the metabolite of streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 and/or streptomycete (Streptomycessp.) YL04 CGMCC No.2884.
2, the application of the metabolite of streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 and/or streptomycete (Streptomycessp.) YL04 CGMCC No.2884 in suppressing fungi.
3, application as claimed in claim 2 is characterized in that: the described application that is applied as in plant fungal disease resistance.
4, application as claimed in claim 3 is characterized in that: described fungal diseases of plants is vegetable sprout term samping off, wheat hypochnus or take-all.
5, the cultural method of streptomycete (Streptomyces sp.) YL04 CGMCC No.2884, be with following culture medium culturing streptomycete (Streptomyces sp.) YL04 CGMCC No.2884: ammonium sulfate 2g, dipotassium hydrogen phosphate 1g, Zulkovsky starch 10g, bitter salt 1g, sodium-chlor 1g, lime carbonate 3g, agar powder 10g, water is settled to 1000ml.
6, a kind of method for preparing streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 metabolite, be with following culture medium culturing streptomycete (Streptomyces sp.) YL04 CGMCC No.2884: yeast extract paste 5g, bitter salt 0.05g, lime carbonate 0.2g, sodium-chlor 0.4g, starch 10g, soyflour 10g, peptone 5g, water is settled to 1000ml.
7, method as claimed in claim 6 is characterized in that: the culture condition of described cultivation streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 is: pH 5.0-8.0, rotating speed 120-200rpm.
8, a kind of fungistat, its activeconstituents are streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 and/or cultivate the metabolite that streptomycete (Streptomyces sp.) YL04 CGMCC No.2884 obtains.
9, fungistat as claimed in claim 8 is characterized in that: described fungistat is the biological prevention and control agent of plant fungal disease resistance.
10, fungistat as claimed in claim 8 or 9, it is characterized in that: described fungi is melon and fruit rotten mildew fungus (Pythium aphanidermatum), gaeumannomyces graminis (Geaumanomyces graminis), Valsa mali (Valsa mali), wheat dry thread Pyrenomycetes (Rhizoctonia cerealisbv), rice blast fungus (Pyricularia grisea), verticillium dahliae (Verticillium dahliae), cotton-wilt fusarium (Fusarium oxysporum f.sp.vasinfectum), fusarium graminearum (Fusarium graminearum), tomato early blight bacterium (Alternaria solani), Exserohilum turcicum (Exserohilum turcicum), southern corn leaf blight (Bipolaris maydis), Rhizoctonia solani Kuhn (Rhizoctonia solani) or botrytis cinerea (Botrytis cinerea).
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