CN103039495B - Jiang chuan mycin for preventing and controlling plant diseases - Google Patents
Jiang chuan mycin for preventing and controlling plant diseases Download PDFInfo
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Abstract
The invention provides a Jiang chuan mycin for preventing and controlling plant diseases and a pesticide compound of the Jiang chuan mycin, which is used for disinfecting and/or resisting crop diseases. The pesticide compound has excellent effects for resisting plant pathogens such as phoma asparagi, pellicularia gramineum, vanturia pirina, saccharomycete, botrytis cinerea, helmintosporium maydis, pyricuraria oryzae, colletotrichum lagenarium and/or alternaria mali Roberts.
Description
Technical field
The invention belongs to pesticide technology and fermentation technical field, particularly, the present invention relates to fermentation preparation and river and mountain mycin, for controlling plant diseases.
Background technology
Blasticidin S (Blasticidin S) be a kind of initial separation from streptomyces griseus (
streptomyces griseochromogenes) farm antibiotics, be mainly used in killing fungus.In the fermentation process of blasticidin S, can produce the metabolic by-product of being permitted lot of trace, but to their research seldom, the compound being especially shown below for structure (being called in this article, " river and mountain mycin ").
At present and do not know this compound whether can aspect crop pest management, there is effect, one of add is because its structure participates in the process that the antibiotic activity of blasticidin S is lost, thereby generally assert that it does not possess antibiotic activity, the research thereby nobody can spend a lot of time and energy; The 2nd, because its fermentation yield is low, also limited people and gone research, nobody reports that its agricultural chemicals function is (referring to Canadian Journal of Chemistry (1994), 72 (1): 6-11 so far; Phytochemistry (1993), 32 (3): 535-541; With, Journal of Antibiotics (1987), 40 (12): 1791-1793).
Yet, the inventor is through long-term research and practice, and some fortune have been relied on, in inventor's strain bacterial strain serendipitous, find that it can ferment and accumulate more river and mountain mycin, in conjunction with the inventor's purification process, for research, it provides the foundation a large amount of river and mountain mycins of acquisition; And, the inventor has overcome the obstruction that its antibiotic activity that relates to blasticidin S of formerly reporting is lost process, still having dropped into a large amount of man power and materials is studied river and mountain mycin, found that it still has antibiotic activity to some specific germs, thereby can be used for preventing and treating corresponding plant disease, more surprisingly, its resistance of rainwater washing against and light durability are strong, safety is good, without off-odor and chafe not, thereby control efficiency is in practice good and be easy to promote.
Summary of the invention
The composition pesticide that relates to river and mountain mycin that the problem that will solve of the present invention is to provide new, preparation method and application's method etc.
Particularly, in first aspect, the invention provides composition pesticide, it comprises acceptable auxiliary material on the river and mountain mycin that is shown below and agricultural chemicals
。
In this article, composition pesticide can be tangible (agricultural chemicals) preparation, for example composition pesticide can be aqua, missible oil, aqueous emulsion, suspension emulsion, microemulsion, wetting powder, water dispersible granules, or other preparations of being convenient to active component performance drug effect or being convenient to the applications of pesticide.Wherein, " acceptable auxiliary material on agricultural chemicals " refer to can make an addition to pesticide activity component (as, river and mountain mycin) thus preparation forms the auxiliary material of composition pesticide, it does not generally possess independent pesticide activity.Conventional auxiliary material but be not limited to carrier (as water), emulsifier, stabilizing agent, filler, wetting agent, antifreezing agent, synergist and/or surfactant etc.In the specific embodiment of the present invention, river and mountain mycin is soluble in water using directly.Preferably, in the composition pesticide of first aspect present invention, the percentage by weight that river and mountain mycin accounts for composition pesticide is 0.0025-99.5%.
The composition pesticide of first aspect present invention can be for anti-plant pathogen.In this article, plant is the term that the present invention knows, and crop preferably, as crops.Described phytopathogen, for can cause the germ of plant injury arbitrarily, comprises bacterium, fungi and/or the actinomycetes that can cause plant injury.Through the inventor, study discovery, the composition pesticide of first aspect present invention is preferred for anti-asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, corn bottom class germ, rice blast fungus, melon anthrax bacteria and/or Alternaria mali.
Correspondingly, for due to above-mentioned germ disease, the composition pesticide of first aspect present invention also can be for controlling plant diseases.Through the inventor, study discovery, the composition pesticide of first aspect present invention is preferred for the plant disease that control is caused by asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, corn bottom class germ, rice blast fungus, melon anthrax bacteria and/or Alternaria mali.In this article, if not contrary or specific description, term " control " refers to and prevents and/or treats, i.e. remedial after preventative processing before disease occurs and/or disease generation is processed.
Preferably the composition pesticide of first aspect present invention is prepared by fermentation preparation, and described fermentation preparation comprises uses the bacterial classification CGMCC No. 6829 that is preserved in CGMCC to ferment.The inventor, through studying for a long period of time, has just fortunately obtained the bacterial classification CGMCC No. 6829 of the product river and mountain mycin with practical value, makes the technology based on river and mountain mycin be able to realize in reality.This river and mountain mycin not only can controlling plant diseases, and its resistance of rainwater washing against and light durability strong, safety is good, produces, in addition without off-odor and chafe not without remarkable poisoning.Especially with respect to its analogue, without remarkable poisoning, produce, after medication, do not make plant growth undesired or occur that physiology hinders, thereby can use to larger dose, coordinate the positive correlation characteristic of its dosage-effect, thereby can realize better control efficiency.
Fermentation preparation can carry out with reference to the fermentation preparation of conventional ash product look streptomycete (Streptomyces griseochromogenes), and just bacterial classification CGMCC No. 6829 can produce with the expression having practical value river and mountain mycin.Preferred described fermentation preparation comprises,
(1) with the bacterial classification CGMCC No. 6829 that is preserved in CGMCC, ferment, obtain zymotic fluid;
(2) concentrated broth, with enrichment river and mountain mycin, obtains concentrate;
(3) concentrate is passed through successively to cation-exchange chromatography, anion-exchange chromatography and macroporous adsorption resin chromatography purifying; With,
(4) optionally carry out crystallization purifying.
More preferably in described fermentation preparation, in step (1), the medium that fermentation is used comprises organic carbon source, organic nitrogen source, quick-acting carbon source, inorganic nitrogen-sourced and mineral salt.Wherein, organic carbon source comprises corn flour, dextrin, starch and/or wheat flour; Organic nitrogen source comprises analysis for soybean powder, soybean cake powder, groundnut meal, cottonseed meal, coarse colza meal, dried silkworm chrysalis meal, dusty yeast (cream), fish meal, peptone, rice bran, rice bran, Mai Pi and/or corn steep liquor; Quick-acting carbon sources comprise glucose, mannose, fructose, arabinose and/or maltose; Inorganic nitrogen-sourced ammonium nitrate, ammonium sulfate, ammonium chloride, sodium nitrate and/or the potassium nitrate of comprising; And/or mineral salt comprise magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate and/or sodium chloride.In the specific embodiment of the present invention, bacterial classification CGMCC No. 6829 first cultivates and obtains cultivating seed liquor in seed culture medium, is then inoculated into fermentation in fermentation medium and obtains zymotic fluid.
Also more preferably in described fermentation preparation, in step (2), enrichment can be processed and be concentrated by filter membrane.Conventional filter membrane has milipore filter and/or NF membrane.In the specific embodiment of the present invention, zymotic fluid, after reconciling pH, first passes through ultrafiltration membrane treatment, then passes through nanofiltration membrane treatment, obtains concentrate.
Research according to the inventor to the separating property of river and mountain mycin, also more preferably in described fermentation preparation, in step (3), cation-exchange chromatography can be Subacidity cation displacement chromatography, anion-exchange chromatography can be weakly-basic anion displacement chromatography, pass through again macroporous adsorption resin chromatography, can obtain highly purified river and mountain mycin.
Therefore, of the present invention one independent aspect, the invention provides fermentation and preparing the method for river and mountain mycin, its process as mentioned above.Particularly, the method comprises fermenting prepares the method for river and mountain mycin, and it comprises uses the bacterial classification CGMCC No. 6829 that is preserved in CGMCC to ferment.
Preferably, the method comprises,
(1) with the bacterial classification CGMCC No. 6829 that is preserved in CGMCC, ferment, obtain zymotic fluid;
(2) concentrated broth, with enrichment river and mountain mycin, obtains concentrate;
(3) concentrate is passed through successively to cation-exchange chromatography, anion-exchange chromatography and macroporous adsorption resin chromatography purifying; With,
(4) optionally carry out crystallization purifying.
More preferably, in the method,
In step (1), the medium that fermentation is used comprises organic carbon source, organic nitrogen source, quick-acting carbon source, inorganic nitrogen-sourced and mineral salt;
In step (2), concentrated broth is processed and is concentrated by filter membrane; And/or
In step (3), cation-exchange chromatography is Subacidity cation displacement chromatography, and anion-exchange chromatography is weakly-basic anion displacement chromatography, then passes through macroporous adsorption resin chromatography.
In second aspect, the invention provides the method for the composition pesticide of preparation first aspect present invention, it comprises:
(1) method of preparing river and mountain mycin by the fermentation of above-mentioned independent aspect is prepared river and mountain mycin; With
(2) acceptable auxiliary material on river and mountain mycin and agricultural chemicals is mixed with to the composition pesticide that obtains first aspect present invention.
Also the process that prepared by preferred described fermentation can comprise the step of the fermentation preparation of mentioning in the composition pesticide of first aspect present invention.
Correspondingly, in the third aspect, the invention provides the river and mountain mycin that the is shown below application method in the composition pesticide for the preparation of anti-plant pathogen or controlling plant diseases
In fourth aspect, the invention provides the method for anti-plant pathogen or controlling plant diseases, it comprises composition pesticide from first aspect present invention to plant that use river and mountain mycin or.The inventor has found the performance for Exposure to Sunlight and water pouring of river and mountain mycin excellence, therefore preferred in the method for fourth aspect present invention, the process that also comprises Exposure to Sunlight, waters and/or drench with rain.Like this, in the time of can avoiding using, (for example to consider the factors such as Exposure to Sunlight, rain condition and irrigation, be not necessarily limited in booth and use, also needn't be in order to irrigate be dragged to an opportunity of using water after), also can avoid using anti-illumination poisonous or that cost is high and/or the auxiliary material of waterproof, the composition pesticide that can use more without restrictions first aspect present invention, makes to use better adaptability.
In this article, it is 25-150mg/L that the application concentration of preferred described composition pesticide be take the cubage of river and mountain mycin, and 50-125mg/L more preferably, as 100mg/L.
Aspect the 5th, the invention provides the river and mountain mycin that is shown below for the application method of the method for anti-plant pathogen or controlling plant diseases
Further, in preferred method aspect the present invention the 3rd, the 4th and/or the 5th, phytopathogen is asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, corn bottom class germ, rice blast fungus, melon anthrax bacteria and/or Alternaria mali.Correspondingly, also in the further preferably method aspect the present invention the 3rd, the 4th and/or the 5th, plant disease is the plant disease being caused by asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, corn bottom class germ, rice blast fungus, melon anthrax bacteria and/or Alternaria mali.
In addition, also, in the further preferably method aspect the present invention the 3rd, the 4th and/or the 5th, river and mountain mycin is the river and mountain mycin that ferments and prepare with the bacterial classification CGMCC No. 6829 that is preserved in CGMCC.
Aspect the 6th, the ash the invention provides for the preparation of river and mountain mycin produces look streptomycete (Streptomyces griseochromogenes) bacterial strain, described bacterial strain is the bacterial strain CGMCC No. 6829 of China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and its preservation day is on November 15th, 2012.
Beneficial effect of the present invention and unforeseeable effect are: the manufacturing of the practical scale of industry of river and mountain mycin is achieved, and production equipment etc. can to produce the production equipment of look streptomycete general with the existing ash for other fermentation purposes, saved cost; Research finds that river and mountain mycin is to people and animals' nonhazardous, without off-odor, and chafe not; The composition pesticide that river and mountain mycin is found in research, thereby can heavy dose be used without poisoning crop; Research finds that river and mountain mycin itself is pretty good to some specific control of plant disease effects, excellent safety and can heavy dose using in addition, so can produce the control efficiency that is significantly higher than its analogue (blasticidin S); Research finds that resistance of rainwater washing against and the light durability of river and mountain mycin is fine, more can avoid using some poisonous or cost is high auxiliary materials, and the adaptability of using is good, has facilitated actual control operation.
For the ease of understanding, below the accompanying drawing by concrete, embodiment are described in detail the present invention.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification, many variations of the present invention, change are all apparent concerning one of ordinary skill in the art.In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included in and carried out reference herein, just looks like that repeated description is excessively the same in this article for their full text.
Accompanying drawing explanation
Fig. 1 has shown the control efficiency block diagram of exemplary resistance of rainwater washing against test, the concentration of the river and mountain mycin that wherein numeral of abscissa is sprayed (unit: mg/L), " water spray "/" not spraying water " represents respectively whether wash away through rainwater in test; Percentage (the unit: %) of the numeral control efficiency of ordinate; Legend represents that respectively the Time interval that sprays river and mountain mycin is from the infected number of days of plant.
Embodiment
Below will invention be described by specific embodiment herein.As do not specialize part, can implement according to listed method in the familiar microorganism fermentology of those skilled in the art, pesticidal preparations and agronomic laboratory manual, textbook and the list of references of quoting herein.Wherein reagent used, instrument all can be bought acquisition by commercial channel.
fermentation preparation and the evaluation of embodiment 1 river and mountain mycin
The ash that the applicant is preserved in to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on November 15th, 2012 produces look streptomycete (Streptomyces griseochromogenes) bacterial classification CGMCC No. 6829, (formula is by weight percentage: glucose 2% to be inoculated in seed culture medium, starch 5%, groundnut meal 2%, soybean cake powder 1%, peptone 0.1%, yeast extract 0.4%, sodium chloride 0.2%, magnesium sulfate 0.5%, ammonium nitrate 0.2%, sodium hydrogen phosphate 0.05%, pH7.2, surplus is water) in, in 28 ℃ of 300rpm shaking table concussions, cultivate 64 hours, must cultivate seed liquor.
Seed liquor be take to 10% inoculum concentration (V/V) to be inoculated in fermentation medium in fermentation tank (formula is by weight percentage glucose 0.4%, starch 7%, groundnut meal 1.5%, soybean cake powder 1.5%, peptone 0.1%, yeast extract 0.4%, sodium chloride 0.2%, magnesium sulfate 0.5%, ammonium nitrate 0.2%, sodium hydrogen phosphate 0.05%, pH7.2, surplus is water) in, with tank pressure 0.08MP, the condition that ventilating ratio 1:1.5 and mixing speed are 240 revs/min was in 30 ℃ of fermented and cultured 96 hours, obtain fermentation culture, the fermentation cultures of a plurality of batches are carried out to HPLC detection, wherein the content of river and mountain mycin is 3300mg/L.
Fermentation culture is added to sulphur acid for adjusting pH to 2.5, after standing one hour, with sodium hydroxide, regulate pH to 5.0, then milipore filter (the F801-4040FM that is 20kDa by the culture fluid that has regulated pH by molecular cut off, can be purchased from Hangzhou Wo Tengmo Engineering Co., Ltd) ultrafiltration, milipore filter (the F202-4040FM that to get filtrate be 2.5kDa by molecular cut off, the same) ultrafiltration, NF membrane (the D201-4040FM that to get filtrate be 300-400Da by molecular cut off, the same) concentrated, when in the liquid that NF membrane is held back, the content of river and mountain mycin reaches 10000mg/L, get NF membrane trapped fluid as concentrate.
By 1mol/L salt acid for adjusting pH to 2.5 for concentrate; be splined on the chromatographic column with HZD-2 weak-acid cation-exchange resin (can purchased from Shanghai Huazhen Science and Technology Co., Ltd.) dress post; discard percolation liquid; use again the water elution of 3 times of column volumes; discard eluent; then use 1mol/L hydrochloric acid wash-out, now start to collect eluent, until eluent pH reaches 2.5.
The eluent of collection is splined on to the chromatographic column with D315 macroporous type weak-base anion-exchange resin (can purchased from Xingtai people's livelihood resin Science and Technology Ltd.) dress post, collect percolation liquid and be splined on the chromatographic column with HZ818 macroporous absorbent resin (can purchased from Zhengzhou Qin Shi Science and Technology Ltd.) dress post, discard percolation liquid and use aqueous acetone solution (acetone: water (V:V)=1:1) wash-out, collection eluent.
To after in the pasty state, add warm water to be dissolved into saturated solution eluent decompression distillation, add absolute ethyl alcohol to carry out crystallization after being cooled to room temperature, obtain white needle-like crystals.After testing, this crystal purity is more than 98%, and fusing point is 253 ~ 255 ℃, specific rotatory power+108.4 °, elementary analysis consist of C
17h
26n
8o
5, soluble in water, acetic acid, is insoluble to such as organic solvents such as ethyl acetate; Through NMR and molecular weight identification, its NMR data:
1h NMR (TFA-
dl) δ: 6.24-6.28 (m, 1H), 7.84 (d, 1H; J=7.9), 6.58 (br s, 1H), 6.24-6.28 (m; 1H), 5.96 (d, 1H; J=10.3), 4.33 (d, 1H; J=8.2), 2.69 (dd, 1H; J=15.4,8.5), 2.58 (dd; 1H, J=15.4,8.5); 4.03 (m, 1H), 2.06 (m; 2H), 3.15 (t, 2H; J=8.1), 2.75 (s, 3H);
13c NMR (TFA-dl) δ: 161.0,150.1,97.0,147.0,80.7,135.1,125.9,46.2,77.1,174.0,173.5,41.9,48.8,31.9,47.1,156.2,34.4.Molecular weight data is: anion FAB-MS (thioglycerin-glycerine)
m/z(ref. int.): 422 [M-H]
+(65), 113 (100); HR anion FAB-MS (thioglycerin-glycerine), accurate mass calculates C
17h
26n
8o
5[M-H]
+be 422.4387, finally determine that molecular weight is 421.44, found in person that it has antibiotic activity in addition, therefore by this white needle-like crystals called after river and mountain mycin, its definite structural formula is shown below:
the mensuration of the antimicrobial spectrum of embodiment 2 river and mountain mycins
On the medium that is mixed with the river and mountain mycin of variable concentrations (100,50,25mg/L), cultivate plant disease microorganism, measure the colony growth diameter of each disease bacterium, recently calculate mutually inhibiting rate with blank (medium is containing river and mountain mycin) separately.The disease bacterium of measuring except powdery mildew of cucumber bacterium (
sphaerotheca fuliginea, below study in great detail) outside, also comprise:
xanthomonas oryzae(Xanthomonas oryzae),
erwinia carotovora(soft rot of cabbage bacterium),
bacillus subtilis(hay bacillus),
eshchcrichia coli(Escherichia coli),
helmintosporium maydis(corn bottom class germ),
pyricuraria oryzaecaV(Pyricularia oryzae),
colletotrichum lagenarium(melon anthrax bacteria),
phoma asparagi saccardo(Asparagus Stem Blight),
helminthosporium oryzae(paddy rice flax class germ),
alternaria solani(tomato early blight bacterium),
fusarium oxysporiumf.sp.lycopersici (Sacc) Snyder (tomato wilt),
pellicularia gramineumikata et Matsmura (Wheat sheath blight bacterium),
gibbrella zeae(fusarium graminearum),
helminthosporium turcicumthe Pass(corn top class in a kindergarten is sick),
fusarium moniliforme(cotton boll rot pathogens),
fusarium oxysporum, f.Vasinfectum(cotton-wilt fusarium),
physalospora piricoranose(black rot of pear bacterium),
phytophthora capsici(phytophthora capsici),
cladosporium fulvumcooke(Fulvia fulva),
vanturia pirina(pear cucumerinum), Saccharomycete(saccharomyces sake bacterium),
alternaria bassicae(BerK) Sacc (Alternaria brassicae),
alternaria maliroberts(alternaria leaf spot of apple),
fusarium oxysporium(withered germ of water-melon),
candida albicans(Candida albicans),
elsinoe ampelina(de Baly) Shear(anthracnose of grape),
rhizoctonia solani(Rhizoctonia solani Kuhn) and
botrytis cinerea(botrytis cinerea pers).
The measurement result of table 1 river and mountain mycin antimicrobial spectrum
Result as shown in Table 1, the antimicrobial spectrum of river and mountain mycin and the antimicrobial spectrum of blasticidin S make a big difference, river and mountain mycin is to asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium and botrytis cinerea pers have excellent fungistatic effect, to corn bottom class germ, rice blast fungus, melon anthrax bacteria and Alternaria mali etc. have good fungistatic effect, and to other test microorganisms bacteriostatic activity all a little less than, even do not have, estimate that it is as the inactivation intermediate of blasticidin S, really scope and the antibacterial properties at antibioticogram has larger shortcoming with respect to blasticidin S.
the control efficiency test of embodiment 3 river and mountain mycins to melon anthracnose
On a leaf period cucumber seedling, spray in advance the river and mountain mycin of isopyknic variable concentrations, after 24 hours, then artificial infection anthrax bacteria conidial suspension (spore concentration is 6.0 * 10
5individual/ml), then cucumber seedling being put to the moisturizing of shading in thermostatic chamber (24 ℃ of left and right) humidistat brings out, after 42 hours, melon seedling is taken out and put thermostatic chamber light from humidistat, to the 7th day blank (not spraying in advance river and mountain mycin) stable disease, anthracnose scab number on counting blade, compares with blank, calculates control efficiency and further by the method for linear regression, calculates the EC to melon anthracnose
50value.
The control efficiency of table 2 river and mountain mycin to melon anthracnose
Result is as shown in table 2, and river and mountain mycin is to the control efficiency of melon anthracnose and its consumption correlation, its effective concentration 50 EC
50=20.45mg/L.This has proved that river and mountain mycin can be applied in actual control of plant disease.
the control efficiency test of embodiment 4 river and mountain mycins to rice blast
On 2 leaf phase pot rices (kind is Tai Hu Nuo) seedling, spray in advance river and mountain mycin or the blasticidin S of isopyknic variable concentrations, after 24 hours, then artificial infection rice blast fungus conidial suspension (spore concentration is 5.0 * 10
5individual/ml), then rice seedling being put to moisturizing in thermostatic chamber humidistat brings out, after 36 hours, rice seedling is put under humidistat light again, about 6 days, after blank (not spraying in advance river and mountain mycin or blasticidin S) stable disease, by occurring degree classification investigation, whether naked-eye observation there is poisoning phenomenon to paddy rice seedling, the calculating control efficiency of simultaneously comparing with blank with disease index, more further by the method for linear regression, calculate the EC to rice blast
50value.
The control efficiency of table 3 river and mountain mycin to rice blast
Result is as shown in table 3, and river and mountain mycin is to the control efficiency of rice blast and its consumption correlation, its effective concentration 50 EC
50=29.88mg/L, is much higher than the EC of blasticidin S
50(0.82mg/L), this result of comparing with the corresponding germ research of both antimicrobial spectrums conforms to.Yet, in test, though working concentration high to 50,100mg/L, river and mountain mycin only observes slight poisoning phenomenon to paddy rice seedling and occurs, still can use safely; And blasticidin S just has later significant symptom of chemical damage to occur from 12.5 mg/L, be difficult in practice the more use of high dose.That is to say, thereby river and mountain mycin can be used the dosage that can reach approximately 90% control efficiency to reach corresponding control efficiency, even and if the limit of blasticidin S use safe dose also only can reach approximately 80% control efficiency.
the control efficiency test of embodiment 5 river and mountain mycins to gray mold of cucumber
On a leaf period cucumber seedling, spray in advance river and mountain mycin or the blasticidin S of isopyknic variable concentrations, after 24 hours, then artificial infection ash arrhizus bacteria conidial suspension (spore concentration is 5.0 * 10
5individual/ml), then cucumber seedling being put to moisturizing in thermostatic chamber (20 ℃ of left and right) humidistat brings out, about 5 days, after blank (not spraying in advance river and mountain mycin or blasticidin S) stable disease, press occurring degree classification investigation, the calculating control efficiency of comparing with blank with disease index.
The control efficiency of table 4 river and mountain mycin to gray mold of cucumber
Result is as shown in table 4, river and mountain mycin is to the control efficiency of gray mold of cucumber and its consumption correlation, and it is 12.5 with substantially reach the control efficiency of blasticidin S 50, during 100mg/L during the consumption of 25.0mg/L, and its 50.0 and control efficiency during the consumption of 100mg/L reach especially more than 90%; Yet, this does not conform to the result that the corresponding germ research of both antimicrobial spectrums is compared, this prompting in person, the antibiotic properties of river and mountain mycin is not only subject to the impact of its antimicrobial spectrum, also may have relation with its some characteristic of in use reflecting, therefore I need the further more concrete test of design to grope.
the detailed test of embodiment 6 river and mountain mycins to powdery mildew of cucumber
In order to further investigate river and mountain mycin, I except the test of conventional control efficiency, also further designed reality such as considering illumination, water punching with time the factor that can make a difference multiple test grope.
1, to the preventive effect of powdery mildew of cucumber and effect protracted test
At leaf period and have on the potted plant cucumber seedling of obvious growth tendency, spray in advance the river and mountain mycin of isopyknic variable concentrations, put respectively thermostatic chamber light after lower 4 days, 3 days, 1 day, artificial infection Powdery Mildew conidial suspension again, then putting powdery mildew brings out under greenhouse light, after 6 days, count powdery mildew scab number on blade, with blank (not spraying in advance river and mountain mycin) the calculating control efficiency of comparing, more further by the method for linear regression, calculate EC
50value.
Table 5 river and mountain mycin is to the preventive effect of powdery mildew of cucumber and continuation thereof
?result is as shown in table 5, river and mountain mycin is to the preventive effect of powdery mildew of cucumber and its consumption correlation, and under the concentration separately of 25-100mg/L, no matter spray in advance 1 day, 3 days, or 4 days, mutual preventive effect does not have significant difference, shows that the effect continuation of river and mountain mycin is good.This is likely that the actual control efficiency of river and mountain mycin is better than one of reason of its effect showing in antimicrobial spectrum research.
2, to the result for the treatment of test of powdery mildew of cucumber and light durability test
On a potted plant cucumber seedling of leaf period, artificial infection Powdery Mildew conidial suspension in advance, putting powdery mildew brings out under greenhouse light, after 68 hours, the river and mountain mycin that sprays again isopyknic variable concentrations on melon seedling, then becomes two parts by each component: a part is put powdery mildew by melon seedling and brought out under greenhouse light; Another part was put melon seedling in 10 o'clock every mornings under greenhouse outdoor sunlight in the south according to shining 3.5 hours, and afternoon, 1:30 puts powdery mildew by melon seedling and brings out under greenhouse light.Powdery mildew scab number on the 9th day counting blade, with blank (not spraying in advance river and mountain mycin) the calculating control efficiency of comparing, more further calculates EC by the method for linear regression
50value.
The impact of table 6 river and mountain mycin on the result for the treatment of of powdery mildew of cucumber and illumination
Result is as shown in table 6, and river and mountain mycin is to the result for the treatment of of powdery mildew of cucumber and its consumption correlation, and it carries out the effective concentration 50 EC of the treatment of more approaching reality (getting sun) to powdery mildew of cucumber
50=1.72mg/L, the EC higher than it under the condition of not getting sun
50value (2.32mg/L).This shows, river and mountain mycin has good light durability, and this physical condition of getting sun also can strengthen its antibacterial effect.This is likely that the actual control efficiency of river and mountain mycin is better than one of reason of its effect showing in antimicrobial spectrum research.
3, the resistance of rainwater washing against test to powdery mildew of cucumber
Select the true leaf consistent potted plant cucumber seedling grouping of growing, respectively at river and mountain mycin, process first 5 days, 4 days, 3 days, artificial infection Powdery Mildew conidial suspension on melon seedling, juxtaposition powdery mildew is brought out under greenhouse light, within after inoculation the 3rd day, (before now 5 days, on the melon seedling of inoculation, indistinctly can see there is powdery mildew scab the last time, also cannot naked eyes differentiating of other two inoculations batch), now on melon seedling, spray the river and mountain mycin of isopyknic variable concentrations, after 6 hours, on melon seedling, with the precipitation that watering pot imitates medium rainfall, carry out sprinkle running water, then will melon seedling putting powdery mildew brings out under greenhouse light, powdery mildew scab number on the 10th day counting blade, with blank (not spraying in advance river and mountain mycin) the calculating control efficiency of comparing.
Result as shown in Figure 1, under each concentration, is inoculated latter 5 days, 4 days and 3 days and is sprayed after river and mountain mycin water spray again, all powdery mildew is had to excellence or good control efficiency, and no matter sprays water and do not spray water, and effect is there was no significant difference all.This shows, river and mountain mycin has good resistance of rainwater washing against.This is likely that the actual control efficiency of river and mountain mycin is better than one of reason of its effect showing in antimicrobial spectrum research.
4, the interior suction effect test to powdery mildew of cucumber
The river and mountain mycin of variable concentrations is evenly injected on the table soil of the cucumber seedling of planting, then artificial infection Powdery Mildew conidial suspension, juxtaposition powdery mildew is brought out under greenhouse light, yet control efficiency is undesirable, the control efficiency of the river and mountain mycin of 100mg/L concentration also with blank (not injecting river and mountain mycin) there was no significant difference.This shows, the systemic action of river and mountain mycin on plant a little less than, but one of this reason that to be likely the poisoning effect of river and mountain mycin (even under heavy dose) slight.
5, prevent and treat the field plot trial of powdery mildew of cucumber
On the loam of cultivate in outdoor cucumber, between 9～October, carry out herbal sprinkling during 5 true leaves of cucumber, artificial infection Powdery Mildew conidial suspension on melon seedling now, the every 4m of knapsack sprayer for second day
2spray respectively the different pharmaceutical 800ml(agricultural chemicals of variable concentrations and concentration thereof in Table 7), same sprinkling once again after a week, after 10 days when blank (not spraying any medicine) stable disease, the calculating control efficiency of comparing with blank.
The field test effect of table 7 river and mountain mycin to powdery mildew of cucumber
Result is as shown in table 7, and river and mountain mycin is as broad as long with at present conventional agricultural chemicals to the control efficiency of powdery mildew of cucumber in field, can substitute current agricultural chemicals completely and use.
Claims (10)
1. the method for preparing composition pesticide, described composition pesticide comprises acceptable auxiliary material on the river and mountain mycin that is shown below and agricultural chemicals, wherein to account for the percentage by weight of composition pesticide be 0.0005-99.5% to river and mountain mycin
，
Described method comprises:
(1) with the bacterial classification CGMCC No. 6829 that is preserved in CGMCC, ferment, obtain zymotic fluid;
(2) concentrated broth, with enrichment river and mountain mycin, obtains concentrate;
(3) concentrate is passed through successively to cation-exchange chromatography, anion-exchange chromatography and macroporous adsorption resin chromatography purifying;
(4) optionally carry out crystallization purifying; With
(5) acceptable auxiliary material on river and mountain mycin and agricultural chemicals is mixed with and obtains described composition pesticide.
2. method claimed in claim 1, wherein
In step (1), the medium that fermentation is used comprises organic carbon source, organic nitrogen source, quick-acting carbon source, inorganic nitrogen-sourced and mineral salt;
In step (2), concentrated broth is processed and is concentrated by filter membrane; And/or,
In step (3), cation-exchange chromatography is Subacidity cation displacement chromatography, and anion-exchange chromatography is weakly-basic anion displacement chromatography, then passes through macroporous adsorption resin chromatography.
3. the application of the river and mountain mycin being shown below in the composition pesticide for the preparation of anti-plant pathogen or controlling plant diseases
4. application claimed in claim 3, wherein said composition pesticide comprises acceptable auxiliary material on the river and mountain mycin that is shown below and agricultural chemicals, wherein to account for the percentage by weight of composition pesticide be 0.0005-99.5% to river and mountain mycin
5. application claimed in claim 3, wherein phytopathogen is asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, southern corn leaf blight, rice blast fungus, melon anthrax bacteria and/or Alternaria mali; Or plant disease is the plant disease being caused by asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, southern corn leaf blight, rice blast fungus, melon anthrax bacteria and/or Alternaria mali.
6. the method for anti-plant pathogen or controlling plant diseases, it comprises to plant uses the river and mountain mycin that is shown below or to plant, uses the composition pesticide that comprises acceptable auxiliary material on the river and mountain mycin that is shown below and agricultural chemicals
7. method claimed in claim 6, it is 25-150mg/L that the application concentration of wherein said composition pesticide be take the cubage of river and mountain mycin.
8. method claimed in claim 7, it is 50-125mg/L that the application concentration of wherein said composition pesticide be take the cubage of river and mountain mycin.
9. method claimed in claim 8, it is 100mg/L that the application concentration of wherein said composition pesticide be take the cubage of river and mountain mycin.
10. method claimed in claim 6, wherein phytopathogen is asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, southern corn leaf blight, rice blast fungus, melon anthrax bacteria and/or Alternaria mali; Or plant disease is the plant disease being caused by asparagus stem wilt bacteria, Wheat sheath blight bacterium, pear cucumerinum, saccharomyces sake bacterium, botrytis cinerea pers, southern corn leaf blight, rice blast fungus, melon anthrax bacteria and/or Alternaria mali.
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| Cytosine glycosides from Streptomyces Griseochromogenes;Jincan Guo等;《Phytochemistry》;19931231;第32卷(第3期);第539页左栏化合物20，右栏倒数第1-4段，第540页左栏第3段 * |
| Jincan Guo等.Cytosine glycosides from Streptomyces Griseochromogenes.《Phytochemistry》.1993,第32卷(第3期),第539页左栏化合物20，右栏倒数第1-4段，第540页左栏第3段. |
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