CN1260351C - Streptomyces antifungus - Google Patents

Abstract

The present invention relates to the microbial field, particularly to a microbe which has the function of resisting pyricularia oryzae, botrytis sp., etc. The present invention is characterized in that the microbe is named as streptomyces antifungus YIM 35551 which is preserved in a preservation organization designated by the State Intellectual Property Bureau; the preservation date is Feb. 14th, 2003, the name of the preservation organization is the China Center for Type Culture Collection, and the preservation number is CCTCCNO: M203015. The fermentation liquor of the bacterial strain of the present invention has the function of resisting pyriculariaoryzae and botrytis sp., and the effect is durable and stable; the present invention has a large potentiality for developing novel, highly effective and non-poisonous natural agricultural chemical from the microbe.

Description

Antimycotic streptomycete

Technical field

The present invention relates to microbial technology field, particularly relate to a kind of microorganism with effects such as blast resisting Pyricularia oryzae, gray mold Botrytis sp..

Background technology

Rice blast is the rice disease that is caused by Pyricularia oryzae; Gray mold is the disease that is caused by Botrytis sp., and a lot of farm crop all have generation.The pathogenic bacterium that cause this two classes corps diseases all are fungies.

Summary of the invention

The purpose of this invention is to provide microorganism-antimycotic streptomycete that a kind of its fermented liquid has antifungic action.

The antimycotic streptomycete Streptomycesantifungus of antimycotic streptomycete called after of the present invention YIM 35551 now is deposited in specified depositary institution of State Intellectual Property Office, and deposit number is CCTCC NO:M203015.

Antimycotic strepto-fungus strain of the present invention separates from the pedotheque of Yunnan Province of China Tengchong collection and obtains.Now be deposited in the preservation of depositary institution of Patent Office of State Intellectual Property Office, depositary institution's title: Chinese typical culture collection center, preservation date are on February 14th, 2003, and deposit number is M203015.

Streptomycete of the present invention is well-grown on most of substratum, and aerial hyphae is white in color, and substrate mycelium is yellowish-white to yellow.Spore chain is dredged spirrillum.The spore rod-short, spore surface is smooth.Gelatine liquefication, starch hydrolysis, nitrate reductase are positive, and milk solidifies, peptonizes and be negative, and does not produce hydrogen sulfide, does not form melanochrome.Can utilize glucose, seminose, wood sugar, ribose, N.F,USP MANNITOL, glycine, Histidine, methionine(Met).Cell walls contains L-diaminopimelic acid and glycine, and full cell hydrolyzate contains semi-lactosi and wood sugar.

Culture condition:

(1) slant culture condition: substratum: glucose 4g; Yeast extract paste 4g; Malt extract 5g; Vitamin complex 3.75mg/L; Agar 15g; Distilled water water 1000ml, pH7.2; Culture temperature is 28 ℃, and incubation time was 1 week.

(2) seed culture condition: insert the seed liquor shake-flask culture 36 hours from inclined-plane picking part mycelium.Seed culture medium: dextrin 120g; Soyflour 40g; Yeast extract paste 2g; Tryptophane 0.5g; Beta-alanine 5g; Sal epsom 0.5g; Ammonium phosphate 0.2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.

(3) fermentation culture conditions: receive in the seed culture medium shake-flask culture 6 days by 10% inoculum size.Substratum: soyflour 10g; Glucose 10g; Peptone 3g; Sodium-chlor 2.5g; Lime carbonate 2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.Promptly obtain fermented liquid.

This fermented liquid is as follows to the restraining effect test of fungi:

(1), the potted plant bioassay method of gray mold (the Shanghai pesticide research assists to measure)

A leaf period (spore or the inoculation of bacterium piece with) or cotyledon period (inoculation of bacterium piece with) the consistent potted plant seedling of growing way is selected in the test medication the day before yesterday.With spray method medicament (alcohol extractive of this fermented liquid) is sprayed on the melon seedling leaf.Sprayed and to have tried material behind the medicine and in spray medicine corridor or greenhouse, dry in the air about three hours.Select suitable inoculation time, inoculation is brought out.The inoculation of mycelia piece method, in long good ash arrhizus bacteria plate,, cut the bacterium colony nahlock at colony edge with diameter 5mm punch tool, be attached to the melon seedling leaf central authorities that chemicals treatment is crossed then, put in 20 ℃ of left and right sides thermostatic chamber humidistats and to look the blank incidence in heat and moisture preserving 4-5 days and investigate.The spore inoculating method, spore is cultivated referring to " botrytis cinerea pers cultivation administrative standard ".In covering with the substratum ware of spore, add pure water, scrape gently with the flat spoon of stainless steel and to get surperficial spore, with four layers of filtered through gauze, make spore suspension, use blood counting chamber, microscopy is observed spore quantity, the control spore concentration adds 5% glucose and 1% yeast decoction then between ten thousand/ml of 50-60; Use mist hand lance and WM-4 type oil-free gas compressor control pressure 0.1MP (1kgf/cm2) then, even spraying inoculating spores suspension on cucumber seedling, about every deep bid (80-90 basin seedling) spray bacterium liquid 40ml, move to carefully immediately that heat and moisture preserving brings out in thermostatic chamber (20 ℃) humidistat, look the blank incidence about 4-5 days and carry out the classification investigation.Carry out data processing and analysis by related standards.

(2), the potted plant bioassay method of rice blast (the Shanghai pesticide research assists to measure)

The test medication select the day before yesterday two leaves wholeheartedly or three leaves wholeheartedly, the consistent potted plant rice seedling of growing way, discharging is for experiment according to the order of sequence.Spray referring to " medicament sprinkling standard ", sprayed and to have tried material behind the medicine (alcohol extractive of this fermented liquid) and in spray medicine corridor or greenhouse, dry (3 hours (about).Perhaps use root-pouring method, press every mu, according to the used potted plant basin size of using, evenly impose on according to quantity in each basin rice seedling of shoaling layer respectively with significant quantity 50g, 100g, three kinds of dosage of 200g.

Inoculation is brought out: select suitable inoculation time inoculation to bring out.Spore is cultivated referring to " Pyricularia oryzae cultivation administrative standard ".In covering with the substratum ware of spore, add pure water, scrape gently with the flat spoon of stainless steel and to get surperficial spore, note not breaking surperficial subiculum into pieces as far as possible, filter with double gauze, make spore suspension, get the 0.1ml spore suspension, add the tally, in the microscopically rolling counters forward from blood counting chamber cover glass edge, the control spore concentration is at ten thousand/ml of 20-30, and is standby.With mist hand lance and WM-4 type oil-free gas compressor control pressure 0.1MP (1kgf/cm2), spray about bacterium liquid 40ml at the every dish of even spraying inoculating spores suspension (80-90 basin seedling) on the rice seedling, carefully move to immediately in the thermostatic chamber (26-28 ℃) and preserve moisture in lower floor's humidistat, preserve moisture (RH100%) bring out, after 24 hours again with rice seedling therefrom lower floor move to top layer and have in the humidistat of light-illuminating (2000 Lux intensity of illumination), open the humidistat ventilating pit gradually, make the interior humidity of case keep 80-90%, do not make rice seedling cross wet jaundice, and every day observe rice growth and have no abnormally, can how much carry out the classification investigation according to scab after five days left and right sides blank scabs are stable.

Be allowed for access below 2 grades during general sieve and rescreen.Carry out data processing and analysis by relevant regulations.

(3) The selection result of bacterial strain YIM 35551 is seen Fig. 7.

This bacterial strain has antifungic action, its fermented liquid is strong to the anti-microbial effect of rice blast (Pyricularia oryzae), gray mold (Botrytis sp.) etc., and lasting medicine, stable, nontoxic, have the great potential of therefrom developing novel, efficient, nontoxic natural agricultural chemicals.

The antimycotic streptomycete of streptomycete called after of the present invention, on February 14th, 2003 in China's typical culture collection center preservation, depositary institution is called for short: CCTCC, deposit number is M203015, the depositary institution address: Wuhan, Luo Jiashan, in the Wuhan University, postcode 430072.

Description of drawings

Fig. 1 is the electromicroscopic photograph of Nanning of the present invention streptomycete on yeast extract paste-malt extract substratum.

Fig. 2 is the cultural characteristic of antimycotic streptomycete of the present invention.

Fig. 3 is the physiological and biochemical property of antimycotic streptomycete of the present invention.

Fig. 4 utilizes situation for the carbon nitrogen source of antimycotic streptomycete of the present invention.

Fig. 5 is that antimycotic streptomycete of the present invention and part correlation bacterium are in the 16S of GenBank database rDNA sequence and accession number.

Fig. 6 is the The selection result of bacterial strain YIM35551 and the part correlation bacterial strain phylogenetic tree according to 16S rDNA sequence construct.

Fig. 7 is the The selection result of antimycotic streptomycete bacterial strain YIM35551 of the present invention.

Embodiment

Embodiment:

Economize the pedotheque that Tengchong is gathered from Yunnan Province of China, in the laboratory, cultivate, can obtain antimycotic streptomycete of the present invention by following condition.

(1) strain culturing condition:

1. slant culture condition: substratum: glucose 4g; Yeast extract paste 4g; Malt extract 5g; Vitamin complex 3.75mg/L; Agar 15g; Distilled water water 1000ml, pH7.2; Culture temperature is 28 ℃, and incubation time was 1 week.

2. seed culture condition: insert the seed liquor shake-flask culture 36 hours from inclined-plane picking part mycelium.Seed culture medium: dextrin 120g; Soyflour 40g; Yeast extract paste 2g; Tryptophane 0.5g; Beta-alanine 5g; Sal epsom 0.5g; Ammonium phosphate 0.2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.

Cultivate by following fermentation culture conditions, can obtain fermented liquid: receive in the seed culture medium shake-flask culture 6 days by 10% inoculum size.Substratum: soyflour 10g; Glucose 10g; Peptone 3g; Sodium-chlor 2.5g; Lime carbonate 2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.

The 16S rDNA partial sequence of streptomycete of the present invention is analyzed as follows:

GCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCTTCGGGTGGATTA

GTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGAC

AAGCCCTGGAAACGGGGTCTAATACCGGATAATACTGTGCCCCTCATGGGG

GACGGTTGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTT

GTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGA

GGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGA

GGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACG

CCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGA

AGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGC

CAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGC

GTAAAGAGCTCGTAGGCGGTCTGTCACGTCGGGTGTGAAAGCCCGGGGCT

TAACCCCGGGTCTGCATTCGATACGGGCAGACTAGAGTGTGGTAGGGGAGA

TCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCG

GTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCG

TGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGG

GAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATT

AAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATT

GACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACG

CGAAGAACCTTACCAAGGCTTGACATACACCGGAAAGCATCAGAGATGGT

GCCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTG

TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGT

TGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACCGCCGGGGT

CAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCT

TGGGCTGCACACGTGNTACAATGGCCGGTACAATGAGCTGCGATACCGCAA

GGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAA

CTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGC

GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGT

CGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCG

AAGGTGGGACTGGCGATTGGGACGAAGTCGTAACAAGGTAGC

Claims (1)

1. antimycotic streptomycete, according to the antimycotic streptomycete Streptomyces of its feature called after antifungus YIM 35551, now be kept at the preservation of specified depositary institution of State Intellectual Property Office, preservation date is on February 14th, 2003, depositary institution's title: Chinese typical culture collection center, preserving number CCTCC NO:M203015.

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