CN102936570A - Azadirachtaindica endogeny xylaria - Google Patents

Abstract

The present invention relates to the technical field of microorganism, particularly to a strain of microorganism bacterial having a broad-spectrum antibacterial activity. According to the present invention, a name of the azadirachtaindica endogeny xylaria is Xylariasp.YM311647; the azadirachtaindica endogeny xylaria is preserved in a preservation institution designated by state intellectual property office, and a preservation number is CCTCCNo:M2012365; the strain is firstly isolated from the azadirachtaindica, and provides significant growth inhibition effects for two human pathogenic fungi, four plant pathogenic fungi and three bacteria; and the plant endogeny fungi resource is utilized, and fermentation is adopted to obtain novel agricultural or medical natural antibacterial drug active ingredients.

Description

Give birth to charcoal angle bacterium in the one strain seal chinaberry

Technical field

The present invention relates to microbial technology field, particularly relate to a kind of microorganism strains with broad spectrum antibiotic activity.

Background technology

The seal chinaberry ( Azadirachta indicaA. Juss) belong to the Meliaceae plant, be tropical tree species, have higher biological pesticide and pharmaceutical value.The India researchist once from the seal chinaberry plant that is grown in India, separated and identified endogenetic fungus (Rajagopal K et al, Current Science, 2000, 78, 1375; Mahesh B et al, Current Science, 2005, 88, 218; Verma VC et al, Microbial Ecology, 2007, 54, 119).We separate from the seal chinaberry plant that is grown in the Yuanjiang county and have obtained endogenetic fungus, and studied its species composition and anti-microbial activity screening (Shao Shicheng, Wu Shaohua etc., Research and development of natural products, 2007, 19, 761; Shao Shicheng, Wu Shaohua etc., Species diversity, 2008, 16, 63).

Summary of the invention

The object of the present invention is to provide a kind of separation from Chinese yunnan economize Salvador in Yuanjiang County of Yunnan Province seal chinaberry ( Azadirachta indicaA. Juss) have in the plant living body in the microorganism strains of broad spectrum antibiotic activity-seal chinaberry and give birth to charcoal angle bacterium.

Give birth to charcoal angle fungus strain in the seal chinaberry of the present invention and from Chinese Yuanjiang county seal chinaberry plant living body, separate acquisition.Depositary institution's preservation of now State Intellectual Property Office's approval, depositary institution's title: Chinese Typical Representative culture collection center, be called for short: CCTCC, depositary institution address: China, Wuhan University, postcode: 430072.Preservation date is on September 19th, 2012, and deposit number is CCTCC No:M 2012365.

Give birth to charcoal angle bacterium called after charcoal angle bacterium YM311647(in the seal chinaberry of the present invention XylariaSp. YM311647), now be deposited in the depositary institution of State Intellectual Property Office's approval, deposit number is CCTCC No:M 2012365.

The solid culture feature of bacterial strain YM311647:

1,28 ℃ of cultivations of PDA substratum 5d, colony diameter reaches 5.2cm, and bacterium colony is open and flat, just is white, the short flannel shape, aerial hyphae grows radially, and the edge begins blackening gradually.Black club-like stromatic tissue occurs at the colony edge place behind the 15d, form one and significantly take turns line, it is circular-arc gently that the edge is.

2,28 ℃ of cultivations of malt extract medium 5d, colony diameter reaches 6.4cm, and mycelia grows radially, and middle body white has projection, and the gradually ageing of edge mycelia is the fine hair shape.

3,28 ℃ of cultivations of Cha Shi substratum, colony growthing slow, bacterium colony just are white, the short flannel shape, and 7d is cultivated in the radial growth of mycelia, and colony diameter reaches 4.2cm, and white is kept at the bacterium colony center, and the edge begins slowly blackening.

The stroma morphological specificity (see figure 1) of bacterial strain YM311647:

The stroma ring is given birth to, long 1.5cm-4.5cm, and thick 1.5 mm-2mm, a large amount of branches, handle is about 1cm.Adularescent infertile little point in top is about 2mm.The stroma exterior carbon turns to black, and surface black scab matter has aperture.Inside still is white.Along with the prolongation of incubation time, stroma is elongated to attenuate, and white is most advanced and sophisticated to disappear.Having no thecaspore in the process of growth produces.

The molecular biological characteristics of bacterial strain YM311647:

Adopt the molecular biology round pcr, the determined dna sequence analysis, YM 311647 bacterial strain ITS rDNA genomes are comprised of 539 bases (bp).Take ITS rDNA sequence as the fundamental construction phylogenetic tree, this bacterium and Xylaria have higher sibship, with known kind Xylaria venosulaITS rDNA sequence homology reach 98.7%, the difference (Fig. 2) of 7 bases is arranged.

The fermentation culture feature of bacterial strain YM311647:

1, substratum: PDB substratum.

2, culture temperature: 28 ± 2 ℃.

3, fermentation time: 7 days.

4, fermentation culture feature: cultivated the 1st day, and had fragmentary white hypha point to occur; Cultivated the 3rd day, and occurred a small amount of white hypha in the fermented liquid; Cultivated the 5th day, white hypha increases gradually, and a small amount of mycelia note wall is arranged; Cultivated the 7th day, and covered with the canescence mycelium, the fermented liquid thickness.

The zymotechnique of bacterial strain YM311647 is as follows:

Bacterial classification ---Activation---first order seed (PDA substratum) ---Cultivate (28 ± 2 ℃, 5-7 days) ---Inoculation ---Secondary seed (PDB substratum, shaking table 200 r/min, 28 ± 2 ℃, 3-4 days) ---Inoculation (by 5% inoculum size) ---Fermentation (PDB substratum, shaking table 200 r/min, 28 ± 2 ℃, 7 days) ---Fermented product.

Fermentation condition in the above-mentioned zymotechnique is as follows:

1, fermentation mode: liquid fermenting.

2, seed culture medium: potato glucose liquid nutrient medium (PDB).

The preparation of potato glucose liquid nutrient medium: get fresh potato 200 grams, peeling is cut into small pieces, and adds 1 liter in water and boils 30 minutes, filters, and filtrate adds water and complements to 1 liter, adds glucose 20 grams, the pH nature.

Add 15 gram agar in the above-mentioned substratum, be the PDA substratum.

3, fermention medium: potato glucose liquid nutrient medium (PDB).

4, incubation time: strain activation and culture 5-7 days, the seed shaking table was cultivated 3-4 days, and the fermentation shaking table was cultivated 7 days.

5, culture temperature: seed and fermentation culture temperature are 28 ± 2 ℃.

6, fermentation is complete, adds equal-volume 95% alcohol steep 48 hours, filters, and filtrate is concentrated into dried, adds sterilized water and makes antibiotic fermented liquid goods.

The anti-microbial activity test of bacterial strain YM311647 of the present invention:

1, cause of disease indicator totally 9 strains: the human body cause illness fungi comprises: Candida albicans ( Candida albicans), the star trichophyton gypseum ( Trichophyton gypseum); Plant pathogenic fungi comprises: Pyricularia oryzae ( Pyricularia oryzae), Botrytis cinerea ( Botrytis cinerea), Fusarium graminearum ( Fusarium graminearum), oat sickle spore ( Fusarium avenaceum); Bacterium comprises: streptococcus aureus ( Staphylococcus aureus), intestinal bacteria ( Escherichia coli), pseudomonas aeruginosa ( Pseudomonas aeruginosa).

2. cause of disease indicator substratum: bacterium class indicator is adopted beef-protein medium; Skin pathomycete class indicator is adopted sabouraud culture medium; Plant pathogenic fungi class indicator is adopted the PDA substratum.

3. the cultivation of endogenetic fungus:

The triangular flask that charcoal angle bacteria strain YM311647 access contains 7 mL PDA liquid nutrient mediums will be given birth in the seal chinaberry, place 28 ± 2 ° of C shaking tables (200 r/ min) to cultivate 7d, added equal-volume 95 % alcohol steeps 48 hours, filter, filtrate is concentrated into dried, gets the broth extraction matter sample for subsequent use.

4. the mensuration of anti-microbial activity:

Add the 1mL sterilized water in growing the solid slant culture base that indicator is arranged, the indicator suspension is made in concussion for a moment.The broth extraction matter sample is dissolved with sterilized water, be mixed with the solution that concentration is 1mg/mL, with the PDB liquid nutrient medium with sample respectively doubling dilution be 6 concentration: 1mg/mL, 500mg/mL, 250mg/mL, 125mg/mL, 62.5mg/mL, 31.25mg/mL.By every hole 50 μ L sample is joined respectively in 96 orifice plates, add again the active indicator bacteria suspension of 50 μ L in every hole, take the hole that do not add bacterium liquid as blank, 3 parallel processing are set.After placing 28 ± 1 ℃ (fungies) or 37 ± 1 ℃ of (bacterium) incubators to cultivate 24 h 96 orifice plates, the upgrowth situation of the active indicator of naked-eye observation is take the sample minimum concentration that do not have the indicator growth as minimal inhibitory concentration (MIC).The result shows, the broth extraction matter sample of bacterial strain YM311647 to the MIC value of 9 kinds of cause of disease indicator Candida albicanss, star trichophyton gypseum, Pyricularia oryzae, Botrytis cinerea, Fusarium graminearum, oat sickle spore, streptococcus aureus, intestinal bacteria, pseudomonas aeruginosa be respectively 62.5,125,250,125,125,250,62.5,31.25,250mg/mL.

The present invention first from seal chinaberry plant ( Azadirachta indica) in separate to obtain endogenetic fungal bacterial strain YM311647, be defined as charcoal angle bacterium ( XylariaSp.), find this bacterium to 2 kinds of human body cause illness fungies, 4 kind of plant pathogenic fungies and 3 kinds of bacteriums, totally 9 kinds of pathogenic micro-organisms have apparent in view inhibition growth.The present invention utilizes endophytic fungus resources of plants, obtains agricultural or Novel medical natural antibacterial active constituents of medicine by fermentation.

Description of drawings

Fig. 1 is the stroma morphological specificity of charcoal of the present invention angle bacterium YM311647.

Fig. 2 is the phylogeny tree graph of charcoal of the present invention angle bacterium YM311647 homology.

Embodiment

Embodiment one:

Charcoal angle bacterium YM311647 bacterial strain is the microorganism medicine source that obtains agricultural or Novel medical natural antibacterial active constituents of medicine by fermentation.

Get the YM311647 bacterial classification, under aseptic condition, with inoculating needle picking a small amount of mycelia, be inoculated in and be equipped with in the sterilized PDA substratum test tube, put in the incubator in 28 ± 2 ° of C activation culture 5-7 days, for subsequent use.Under aseptic condition, the bacterial classification that activation is good accesses in the sterilized first order seed PDB substratum in the same way, cultivates 3-4 days at 28 ± 2 ° of C, is the first order seed of YM311647 bacterial strain.

The PDB substratum 100ml for preparing is packed in the 500ml triangular flask, and 121 ℃ of sterilizations 30 minutes are for subsequent use.Get the first order seed of YM311647 bacterial strain, under aseptic condition, be inoculated in the 500ml triangular flask that 100ml PDB substratum is housed, inoculum size is by 7-10%(v/v), place 28 ± 2 ° of C shaking tables (200 r/min) to cultivate 7 days, during note checking and have or not the microbiological contamination phenomenon.

Ferment complete, added equal-volume 95 % alcohol steeps 48 hours in the fermented liquid, filter, filtrate is concentrated into dried, add sterilized water and namely make antibiotic fermentation broth sample.

The ITS rDNA genome of charcoal of the present invention angle bacterium YM311647 is comprised of 539 bases (bp), and its sequence is as follows:

1?taaccctgtg?aacctacctt?tgttgcctcg?gcaggtctgc?aacctaccct?gagagtccct

61?accctgtagg?gaccttaccc?ggtagttgcg?ggcataacct?gccggtggtc?taccaaactc

121?tgtttactat?gttattctga?ataatataac?taaataagtt?aaaactttca?acaacggatc

181?tcttggttct?ggcatcgatg?aagaacgcag?cgaaatgcga?taagtaatgt?gaattgcaga

241?attcagtgaa?tcatcgaatc?tttgaacgca?cattgcgccc?attagtattc?tagtgggcat

301?gcctgttcga?gcgtcatttc?aacccttaag?ccctgttgct?tagcgttggg?agcctacaga

361?tacactctgt?agttccttaa?agttagtggc?ggagtcggtt?tcacactcta?gacgtagtaa

421?attttatctc?gcctatagat?gagccggtcc?cttgccgtaa?aaccccctaa?tttctaaagg

481?ttgacctcgg?atcaggtagg?aatacccgct?gaacttaagc?atatcaataa?gcggaggaa

 

Claims (3)

1. give birth to charcoal angle bacterium in a seal chinaberry, it is characterized in that called after charcoal angle bacterium YM311647( XylariaSp. YM311647), now be deposited in the depositary institution of State Intellectual Property Office's appointment, deposit number is CCTCC No:M 2012365.

2. according to giving birth to charcoal angle bacterium in the seal chinaberry claimed in claim 1, it is characterized in that its ITS rDNA genome by 539 based compositions, sequence is as follows:

1?taaccctgtg?aacctacctt?tgttgcctcg?gcaggtctgc?aacctaccct?gagagtccct

61?accctgtagg?gaccttaccc?ggtagttgcg?ggcataacct?gccggtggtc?taccaaactc

121?tgtttactat?gttattctga?ataatataac?taaataagtt?aaaactttca?acaacggatc

181?tcttggttct?ggcatcgatg?aagaacgcag?cgaaatgcga?taagtaatgt?gaattgcaga

241?attcagtgaa?tcatcgaatc?tttgaacgca?cattgcgccc?attagtattc?tagtgggcat

301?gcctgttcga?gcgtcatttc?aacccttaag?ccctgttgct?tagcgttggg?agcctacaga

361?tacactctgt?agttccttaa?agttagtggc?ggagtcggtt?tcacactcta?gacgtagtaa

421?attttatctc?gcctatagat?gagccggtcc?cttgccgtaa?aaccccctaa?tttctaaagg

481?ttgacctcgg?atcaggtagg?aatacccgct?gaacttaagc?atatcaataa?gcggaggaa。

3. one kind is printed the purposes of giving birth to charcoal angle bacterium in the chinaberry, it is characterized in that: for the preparation of antibacterials.

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