CN102191185A - Biological control endophytic fungi - Aspergillus restrictus - Google Patents
Biological control endophytic fungi - Aspergillus restrictus Download PDFInfo
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- CN102191185A CN102191185A CN 201110088205 CN201110088205A CN102191185A CN 102191185 A CN102191185 A CN 102191185A CN 201110088205 CN201110088205 CN 201110088205 CN 201110088205 A CN201110088205 A CN 201110088205A CN 102191185 A CN102191185 A CN 102191185A
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Abstract
The invention relates to the microbial technology field. The garlic endophytic fungi are obtained from a garlic living body with endophytic fungi microbe separation technology, wherein the garlic living body is obtained at a vegetable garden in the suburb of Hangzhou city Zhejiang province. Through the microbe taxology identification, the garlic endophytic fungi are named as Aspergillus restrictus 28. The bacterial strain is preserved at China General Microbiological Culture Collection Center with a preservation date December 31st 2010 and a preservation registry number CGMCC No. 4507. The most substantial characteristic of the endophytic fungi is that through liquid fermentation the bacterial strain can produce an active metabolite which has an inhibition effect on pathogen of plant fungous disease such as Rhizoctonia solani, Thanatephorus cucumeris and the like. The bacterial strain is an important microbial resource.
Description
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 endogenetic fungus---the cultivation of Aspergillus restrictus bacterium (Aspergillus restrictus) and the application of meta-bolites thereof.
Background technology
All have a large amount of endogenetic fungus in the plant materials, endogenetic fungus is found in herbage the earliest, is found in plants such as xylophyta, herbaceous plant, gramineae plant, liver moss, lichens, marine alga again afterwards.Studies show that so far, plant endogenesis epiphyte can strengthen the host disease resistance, improve the productivity, degeneration-resistant pest-resistant etc. of plant.Bringing into play these effects is because endogenetic fungus is bred in plant materials and grown, and generation has bioactive secondary metabolite in vivo.Therefore, endogenetic fungus may become potential biocontrol agent in the biological control, has important purposes aspect the ecological agriculture and the biological pesticide development.Plant endogenesis epiphyte has caused microbiologist, ecologist, plant protection scholar's extensive interest in recent years, becomes the focus of domestic and international research gradually.The research and development of microbial source biological chemistry agricultural chemicals at present is very active in China, but Microbial resources and application potential thereof still do not fully played, and plant endogenesis epiphyte promptly is the untapped emerging Microbial resources of such class.
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 of endogenetic fungus generation to carry out the biological control of plant pathogenic fungi disease, then have the cycle weak point, be easy to research, be convenient to advantages such as production and low toxicity.
The present invention is separated to a strain biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus from garlic, and this strain fermentation meta-bolites has been carried out bacteriostatic action research.This invention provides good starting strain 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 endogenetic fungus that separates from garlic-Aspergillus restrictus bacterium (Aspergillus restrictus) is provided at chemical pesticide; Another object of the present invention provides the method for utilizing this bacterial strain meta-bolites control fungal diseases of plants.
The separation of Aspergillus restrictus bacterium of the present invention, screening and evaluation:
Be in vegetable garden, Zhejiang Hangzhou countryside, to gather garlic, obtain and preservation through steps such as separation, purifying, cultivation, fermentation and determinations of activity in April, 2009; Be accredited as Aspergillus restrictus bacterium (Aspergillus restrictus) through microbial taxonomy, name and be Aspergillus restrictus bacterium (Aspergillus restrictus) 28.This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date on December 31st, 2010, preservation registration number CGMCC No.4507.
Aspergillus restrictus bacterium 28, its solid culture is characterized as: bacterium colony on the PDA flat board 28 ℃ cultivate and to extend to diameter 9cm in 5-7 days, initial stage white is sparse, mycelia is along the media surface growth of crawling, the back forms grey, reverse side is colourless, the mycelia tool is every, branch.The microscopic morphology feature: the conidiophore ultimate swelling is head; The conidium light gray, subsphaeroidal.
Aspergillus restrictus bacterium 28 of the present invention can obtain the inhibited meta-bolites of part fungal diseases of plants pathogenic bacteria through fermented extracted.
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 metabolites such as rice sheath blight disease or cucumber rhizoctonia rot, carry out according to the following steps:
(1) activation culture of bacterial classification: will separate Aspergillus restrictus 28 bacterial classifications of preserving and move on the potato glucose PDA solid plate, and under 28 ℃ ± 1 ℃ condition, cultivate, and cover whole culture dish to mycelia, and obtain the bacterium cake with diameter for the punching of 4mm punch tool, for inoculation usefulness;
(2) fermentation culture: fermentation culture is to contain soybean cake powder 20g among every 1000mL, Semen Maydis powder 15g, Zulkovsky starch 20g, wheat bran 10g, peptone 8g, NH
4NO
33g, (NH
4)
2SO
45g, CaCO
35g, MgSO
41g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare back 121 ℃ the sterilization 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 4mm bacterium cake, under 28 ℃ ± 1 ℃ condition, 180 rev/mins of shaking speed, fermentation culture 7 days;
(3) fermented product extracts: fermentation finishes, and centrifugal collection fermented supernatant fluid is used conventional organic solvent extraction, obtains medicinal extract behind vacuum concentration, is used for bacteriostatic activity and measures.
Beneficial effect of the present invention:
The one, the present invention separates metabolite part fungal diseases of plants pathogenic bacteria is had stronger inhibiting Aspergillus restrictus 28 from the plant garlic, and this provides a kind of microorganism strains for control fungal diseases of plants pathogenic bacteria on the agricultural;
The 2nd, the invention provides the method for utilizing Aspergillus restrictus 28 fermentations to obtain active metabolite; This meta-bolites can be applicable to the control of fungal diseases of plants, for the exploitation of disinfectant use in agriculture has increased new approach.
Embodiment
The separation and the screening of embodiment 1:(Aspergillus restrictus 28)
The present invention gathers garlic in April, 2009 in vegetable garden, Zhejiang Hangzhou countryside, through surface sterilization, endogenetic fungus 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 Aspergillus restrictus bacterium (Aspergillus restrictus) 28, preserve.
Aspergillus restrictus bacterium (Aspergillus restrictus) 28 bacteriostatic activities are measured: the fermented supernatant fluid 1mL of the 0.22 μ m membrane filtration of learning from else's experience is in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA substratum with 9mL, cucumber rhizoctonia rot bacterium (Rhizoctonia solani) that it is 4mm that the cooling back is put 1 diameter respectively on each substratum plane or Rhizoctonia solani Kuhn (Thanatephoruscucumeris) 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: Aspergillus restrictus bacterium (Aspergillus restrictus) 28 meta-bolitess are respectively 85% and 88% to the inhibiting rate of cucumber rhizoctonia rot bacterium and Rhizoctonia solani Kuhn.
The preparation method of embodiment 2:(Aspergillus restrictus 28 fermentating metabolism products)
Carry out according to the following steps:
(1) activation culture of bacterial classification: will separate Aspergillus restrictus 28 bacterial classifications of preserving and move on the potato glucose PDA solid plate, and under 28 ℃ ± 1 ℃ condition, cultivate, and cover whole culture dish to mycelia, and obtain the bacterium cake with diameter for the punching of 4mm punch tool, for inoculation usefulness;
(2) fermentation culture: fermentation culture is to contain soybean cake powder 20g among every 1000mL, Semen Maydis powder 15g, Zulkovsky starch 20g, wheat bran 10g, peptone 8g, NH
4NO
33g, (NH
4)
2SO
45g, CaCO
35g, MgSO
41g, the bottled fermentation culture 60mL of every 300mL triangle; Prepare back 121 ℃ the sterilization 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 4mm bacterium cake, under 28 ℃ ± 1 ℃ condition, 180 rev/mins of shaking speed, fermentation culture 7 days;
(3) fermented product extracts: centrifugal collection fermented liquid after fermentation finishes, adopt the equal-volume ethyl acetate extraction, and get the upper strata, and under vacuum condition, be concentrated into medicinal extract, get acetic acid ethyl ester extract, measure for bacteriostatic activity.
Aspergillus restrictus 28 meta-bolitess described in following examples 3,4 are embodiment 2 products made therebies; The percentage composition of described product is mass/volume per-cent.
Embodiment 3:(Aspergillus restrictus 28 meta-bolitess are to the restraining effect of Rhizoctonia solani Kuhn and cucumber rhizoctonia rot bacterium)
(1) accurately take by weighing Aspergillus restrictus 28 meta-bolites 30mg, and add the sterilized water of 10mL volume, ultrasonic oscillation fully dissolves, and finally is configured to the mother liquor that concentration is 3000mg/L;
(2) bacteriostatic activity is measured: getting above-mentioned mother liquor, to be diluted to concentration respectively be 1250mg/L, the solution of 1000mg/L and 500mg/L, get each concentration solution 1mL in sterile petri dish, (Aspergillus restrictus 28 meta-bolites final concentrations are 125mg/L to be cooled to 50 ℃ the rapid mixing of PDA substratum with 9mL, 100mg/L and 50mg/L), Rhizoctonia solani Kuhn or cucumber rhizoctonia rot bacterium bacterium cake that it is 4mm that the cooling back is put 1 diameter respectively on each substratum plane, 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 jingganmycin medicament and sterilized water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
Test example: (product of the present invention and conventional pesticide prevention effect simultaneous test)
Test-results sees Table 1.As seen from Table 1, it is slightly excellent than the conventional chemical medicament that Aspergillus restrictus 28 meta-bolitess suppress effect, and safety.
Table 1 Aspergillus restrictus 28 meta-bolitess are to the inhibition effect of Rhizoctonia solani Kuhn and cucumber rhizoctonia rot bacterium
"-" expression not test (N.T.).
Claims (1)
1. Aspergillus restrictus bacteria strain, it is characterized in that called after Aspergillus restrictus bacterium (Aspergillus restrictus) 28, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation day: on December 31st, 2010, preservation registration number is CGMCC No.4507.
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|---|---|---|---|
| CN201110088205A CN102191185B (en) | 2011-04-08 | 2011-04-08 | Biological control endophytic fungi - Aspergillus restrictus |
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| CN201110088205A CN102191185B (en) | 2011-04-08 | 2011-04-08 | Biological control endophytic fungi - Aspergillus restrictus |
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| CN102191185B CN102191185B (en) | 2012-09-05 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504610A (en) * | 2018-12-12 | 2019-03-22 | 安徽中医药大学 | A kind of plant endogenesis epiphyte Aspergillus sp.MBL1612 extract and application thereof |
| CN109706083A (en) * | 2018-12-10 | 2019-05-03 | 贵州省中国科学院天然产物化学重点实验室 | A kind of Eucommia Ulmoieds that the antibacterial activity after metabolic regulation dramatically increases |
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| CN1279581A (en) * | 1997-10-23 | 2001-01-10 | 阿克佐诺贝尔英国有限公司 | Method of controlling house dust mites and bedmites |
| WO2009061950A2 (en) * | 2007-11-09 | 2009-05-14 | Montana State University | Endophytic fungi from pteromischum sp. plant, compounds and methods of use |
| US7901927B1 (en) * | 2005-08-09 | 2011-03-08 | The United States Of America As Represented By The Secretary Of Agriculture | Transfer and incorporation of heritable symbiotic fungi into non-host plants |
-
2011
- 2011-04-08 CN CN201110088205A patent/CN102191185B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1279581A (en) * | 1997-10-23 | 2001-01-10 | 阿克佐诺贝尔英国有限公司 | Method of controlling house dust mites and bedmites |
| US7901927B1 (en) * | 2005-08-09 | 2011-03-08 | The United States Of America As Represented By The Secretary Of Agriculture | Transfer and incorporation of heritable symbiotic fungi into non-host plants |
| WO2009061950A2 (en) * | 2007-11-09 | 2009-05-14 | Montana State University | Endophytic fungi from pteromischum sp. plant, compounds and methods of use |
Non-Patent Citations (2)
| Title |
|---|
| 《浙江农业学报》 20061231 俞晓平 植物内生菌及其代谢产物在生物农药创制中的应用 289-293 1 第18卷, 第5期 * |
| 《浙江农业学报》 20061231 申屠旭萍 雷公藤内生真菌的分离即活性菌株的筛选 308-312 1 第18卷, 第5期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706083A (en) * | 2018-12-10 | 2019-05-03 | 贵州省中国科学院天然产物化学重点实验室 | A kind of Eucommia Ulmoieds that the antibacterial activity after metabolic regulation dramatically increases |
| CN109706083B (en) * | 2018-12-10 | 2023-05-30 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Eucommia ulmoides endophytic fungus with remarkably increased antibacterial activity after metabolic regulation |
| CN109504610A (en) * | 2018-12-12 | 2019-03-22 | 安徽中医药大学 | A kind of plant endogenesis epiphyte Aspergillus sp.MBL1612 extract and application thereof |
| CN109504610B (en) * | 2018-12-12 | 2021-11-19 | 安徽中医药大学 | Endophytic fungus Aspergillus sp.MBL1612 extract and application thereof |
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| CN102191185B (en) | 2012-09-05 |
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