CN101225368A - Biocontrol actinomyces-streptomyces diastatochromogenes D - Google Patents

Abstract

The invention discloses a preparation method of microbial pesticide for controlling vegetable fungal diseases, belonging to the microbial technical field, which utilizes a streptomyces diastatochromogenes D isolated and screened from a soil sample to prepare various formulation pesticides with the steps of the activation cultivation of strain, fermentation cultivation, leavening extraction and distribution of pesticide auxiliary material to the purified active composite, wherein the soil sample is acquired from Lin' an Tianmu mountain in Hangzhou, Zhejiang. The preparation method of microbial pesticide for controlling vegetable fungal diseases is safe and can be used for controlling cucumber fusarium wilt, tomato gray mold, and other vegetable fungal diseases; and the controlling effect is a little better than normal chemical agents.

Description

One strain biological and ecological methods to prevent plant disease, pests, and erosion actinomycetes-streptomyces diastatochromogenes D

Technical field

The present invention relates to microbial technology field, particularly relate to the biological and ecological methods to prevent plant disease, pests, and erosion actinomycetes---the cultivation of streptomyces diastatochromogenes D and the separation of meta-bolites and application.

Background technology

Actinomycetes (Actinomycetes) are very close with human relation.According to statistics, in the microbiotic of having found, have 61.7% to be produced by actinomycetes.Microbiotic commonly used is except that penicillin and cephamycin class, and the overwhelming majority is actinomycetic product.Soil is the important place of actinomycetes perch, and obtaining to have the active actinomycetes generation of high-efficiency antimicrobial microbiotic from soil is to develop new pharmaceutical and the requisite element task of novel pesticide in the present world wide.

At present, mainly adopt chemical pesticide to kill pathogenic bacteria, but chemical pesticide still is difficult to effective solution so far to people and animals' toxic side effect and residue problem to the control of Plant diseases.Utilize the active substance of the disease-resistant fungal pathogens that actinomycetes produce to carry out the biological control of plant pathogenic fungi disease, then have the cycle short, be easy to research, advantage such as be convenient to produce, nontoxic.

The present invention is separated to strain biological and ecological methods to prevent plant disease, pests, and erosion actinomycetes from the soil in Hangzhou, and the antimicrobial component in this biological and ecological methods to prevent plant disease, pests, and erosion actinomycete fermentation meta-bolites is separated, and has obtained main active ingredient, through four big spectroscopic analysis, and clear and definite its structural formula.This invention provides good starting strain and bioactive compounds for the further development and the exploitation of microbial pesticide.

Summary of the invention

The objective of the invention is the dangerous and less deficiency of microbial pesticide kind, a kind of actinomycetes that separate from the soil of Hangzhou-streptomyces diastatochromogenes D is provided at chemical pesticide; Another object of the present invention provides utilizes this bacterial strain to be prepared into microbial pesticide of preventing and treating the part fungal diseases of plants and preparation method thereof by its meta-bolites of fermented extracted.

The separation of streptomyces diastatochromogenes of the present invention, screening and evaluation:

Be in August, 2006 in the soil sample that gather in the Tian Mu Shan Mountain, Zhejiang Hangzhou Linan, obtain and preserve through separation, cultivation, fermentation, determination of activity and to the steps such as screening that suppress fungal diseases of plants pathogenic bacteria effective strain; Be accredited as streptomyces diastatochromogenes (Streptomycesdiastatochromogenes) through microbial taxonomy, name and be streptomyces diastatochromogenes (Streptomycesdiastatochromogenes) D.This bacterial strain oneself in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date on May 25th, 2007, preservation registration number CGMCC No.2060.

Streptomyces diastatochromogenes D microscopic morphology feature: straight, hook-shaped, the loose volution of fibrillae of spores, spore is oval, ellipse or oval.

Streptomyces diastatochromogenes D of the present invention, can obtain the inhibited active compound [2 of part fungal diseases of plants pathogenic bacteria through fermented extracted, 4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid.

The object of the invention is achieved through the following technical solutions:

A kind of preparation method who prevents and treats fungal diseases of plants microbial pesticides such as graw mold of tomato and cucumber fusarium axysporum, carry out according to the following steps:

(1) activation culture of bacterial classification: will separate the streptomyces diastatochromogenes D bacterial classification of preserving and move in vitro on the Gause I inclined-plane, and under 28 ℃ ± 1 ℃ condition, be cultured to the test tube slant and cover with spore;

(2) fermentation culture: fermentation culture is to contain soybean cake powder 20g among every 1000mL, Semen Maydis powder 15g, wheat bran 10g, NH ₄NO ₃3g, (NH ₄) ₂SO ₄5g, CaCO ₃5g, MgSO ₄1g, sterilization, to be cooled to 40 ℃, inoculation streptomyces diastatochromogenes D under 28 ℃ ± 1 ℃ condition, cultivated 5 days to fermentation culture under aseptic condition;

(3) fermented product extracts: fermentation finishes, and collects fermented liquid, chemical process extraction routinely, chromatography, and separation and purification obtains active compound

[2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, this compound molecule formula is C ₁₃H ₁₉O ₉N ₃, the structural formula See Figure;

(4) this active compound is equipped with the agricultural drugs auxiliary material, is prepared into the agricultural drugs of multiple formulation.

Beneficial effect of the present invention:

The one, the present invention separates metabolite output height and part fungal diseases of plants pathogenic bacteria is had stronger inhibiting streptomyces diastatochromogenes D from Hangzhou soil, and this provides a kind of microorganism strains for control fungal diseases of plants pathogenic bacteria on the agricultural;

The 2nd, the invention provides and from the fermented product of streptomyces diastatochromogenes D, extract its active metabolite [2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, and and then be prepared into the preparation method of the microbial pesticide that can prevent and treat fungal diseases of plants; This agricultural chemicals is applied to the control of fungal diseases of plants, and its effect is than conventional chemical medicament slightly excellent (seeing the routine table 1 of test), for the exploitation of disinfectant use in agriculture has increased new approach.

Embodiment

Separation and the screening of embodiment 1:(streptomyces diastatochromogenes D)

The present invention in August, 2006 in the soil sample that gather in the Tian Mu Shan Mountain, Linan, Zhejiang, through separation, cultivation, fermentation, determination of activity and to suppressing the steps such as screening of fungal diseases of plants pathogenic bacteria effective strain, therefrom obtain streptomyces diastatochromogenes (Streptomyces diastatochromogenes) D, preserve.

Streptomyces diastatochromogenes (Streptomyces diastatochromogenes) D bacteriostatic activity is measured: get its fermented supernatant fluid 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA substratum with 9mL, cucumber fusarium axysporum that it is 4mm that the cooling back is put 1 diameter respectively on each substratum plane or graw mold of tomato etc. are for examination bacterium bacterium cake, the bacterium cake is connected to culture dish central authorities (the culture dish diameter is 9cm), put 28 ℃ of cultivation 36h in the incubator, handle in contrast with sterilized water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:

Measurement result shows: streptomyces diastatochromogenes (Streptomyces diastatochromogenes) D meta-bolites is respectively 83.8% and 78% to the inhibiting rate of cucumber fusarium axysporum and botrytis cinerea.

The preparation method of embodiment 2:(streptomyces diastatochromogenes D fermentating metabolism product)

Carry out according to the following steps:

(1) bacterial classification test tube slant activation culture: slant medium is the Gause I substratum, and its component and content are: K ₂HPO ₄0.5g, KNO ₃1g, MgSO ₄0.5g, Zulkovsky starch 20g, NaCl 0.5g, FeSO ₄0.01g, agar 20g, supply water to 1L after dissolving; With the spore of a little streptomyces diastatochromogenes D of transfering loop picking, (in 15 * 150mm), put interior 28 ℃ ± 1 ℃ of incubator, activation culture 96h covers with spore to the test tube slant to sterilized Gause I substratum test tube under aseptic condition;

(2) fermentation culture: fermentation culture is to contain soybean cake powder 20g among every 1000mL, Semen Maydis powder 15g, wheat bran 10g, NH ₄NO ₃3g, (NH ₄) ₂8O ₄5g, CaCO ₃5g, MgSO ₄1g, surplus is a water; The bottled 40mL fermentation culture of every 300mL triangle, prepare back 121 ℃ the sterilization 20min, to be cooled to 40 ℃ under aseptic condition, picking one platinum ring streptomyces diastatochromogenes D spore inoculating, 28 ℃ ± 1 ℃, fermentation culture 5 days;

(3) fermented product extracts: collect fermented liquid after fermentation finishes, under vacuum condition, be concentrated into medicinal extract, dissolve with Virahol again, the centrifuging and taking supernatant liquor, be concentrated into medicinal extract, then go up silica gel column chromatography, with Virahol and water (volume ratio 10%～90%) wash-out, separation and purification gets active compound, and [2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid.

Streptomyces diastatochromogenes D meta-bolites described in following examples 3,4 is embodiment 2 products made therebies; The percentage composition of described product is mass percent.

Embodiment 3:(utilizes streptomyces diastatochromogenes D fermentating metabolism product to be prepared into wettable powder)

Wherein:

White carbon black: (the rich strange chemical industry company limited in Shanghai produces)

Blue star auxiliary reagent factory, farming newborn 0203-B:(Xingtai produces)

Sodium lignosulfonate: (the Shanghai prestige is chemical industry company limited and produces)

Light calcium carbonate: (Shanghai DaYu biochemistry Co., Ltd's production)

Product contains 1.5% active compound [2,4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, the white carbon black of adding 2% is as sorbent material, and add 1% agricultural newborn 0203-B and 3% sodium lignosulfonate auxiliary material, supply 100% with light calcium carbonate at last, behind the mixing, make wettable powder (claiming " product A " in the following content) through comminution by gas stream.

Embodiment 4:(utilizes streptomyces diastatochromogenes D fermentating metabolism product to be prepared into missible oil)

Wherein:

Ethanol (production of Shanghai Shen Xing pharmaceutical factory)

0204 (production of Xingtai blue star auxiliary reagent factory) of farming breast

Dimethylbenzene (production of Shanghai Shen Xing pharmaceutical factory)

The active compound [2 of product 2%, 4-Dioxo-1-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-1,2,3,4-tetrahydro-pyrimidin-5-yl]-methylamino-acetic acid, the dissolve with ethanol with 4% adds quality 5% agricultural newborn 0204-C as emulsifying agent, supply 100% with dimethylbenzene at last, stirring and evenly mixing is made missible oil (claiming " product B " in the following content).

Test example: (product of the present invention and conventional pesticide prevention effect simultaneous test)

" product A, B " prevented and treated test to Plant diseasess such as cucumber fusarium axysporum or graw mold of tomato, is contrast with conventional chemical agricultural chemicals, clear water, and shown in table 1, table 2, the disinfectant use in agriculture of trial-production " product A, B " is slightly more excellent than conventional chemical pesticide control effect.

Table 1 product A is to graw mold of tomato preventive effect cartogram

(test of pesticide effectiveness is all carried out in test farmland, suburbs, Hangzhou in July, 2007-September)

Product and contrast medicine	Extension rate	Using method	Proofread and correct preventive effect (%)
				Graw mold of tomato
			Product A		1000	Spraying	90.9
1500	Spraying	88.5
				2000	Spraying	85.1
But 70% thiophanate methyl wet-milling	1000	Spraying					90.7
			Clear water	-	Spraying	4.3

Table 2 product B is to cucumber fusarium axysporum preventive effect cartogram

(test of pesticide effectiveness is all carried out in test farmland, suburbs, Hangzhou in July, 2007-September)

Product and contrast medicine	Extension rate	Using method	Proofread and correct preventive effect (%)
				Cucumber fusarium axysporum
			Product B		800	Spraying	93.8
1200	Spraying	85.3
				1600	Spraying	79.0
But 50% derosal wet-milling	500	Spraying					84.4
			Clear water	-	Spraying	-10.2

Claims (1)

1. streptomyces diastatochromogenes bacterial strain, its preservation registration number is CGMCC No.2060.