CN102191184B - Biocontrol endophytic fungi-Alternaria alternata - Google Patents
Biocontrol endophytic fungi-Alternaria alternata Download PDFInfo
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- CN102191184B CN102191184B CN201110088193A CN201110088193A CN102191184B CN 102191184 B CN102191184 B CN 102191184B CN 201110088193 A CN201110088193 A CN 201110088193A CN 201110088193 A CN201110088193 A CN 201110088193A CN 102191184 B CN102191184 B CN 102191184B
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Abstract
The invention provides biocontrol endophytic fungi- Alternaria alternata and relates to the technical field of microorganisms. The endophytic fungi of camphor trees are separated from camphor tree living bodies collected from Tianmu Mountain in Hangzhou, Zhejiang by an endophytic fungi separation technique and are named as Alternaria alternata 31 according to microbial taxonomy identification. The biocontrol endophytic fungi-Alternaria alternata is collected in the General Microbiology Center of China Microorganism Strain Collection Management Committee, wherein the collection date is 2010.12.31 and the collection registered number is CGMCC No.4508. The biocontrol endophytic fungi-Alternaria alternata has the most remarkable characteristic that: the strain liquid is fermented to generate active metabolites which can inhibit plant fungi disease pathogenic bacteria such as rhizoctonia solani, fusarium oxysporium, botrytis cinerea and the like and is important microbial resources agriculturally.
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 alternaric bacteria (Alternariaalternata) 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, in plants such as xylophyta, herbage, gramineae plant, liver moss, lichens, marine alga, comes to light again afterwards.Research so far shows, plant endogenesis epiphyte can strengthen the host disease resistance, improve the yield-power, 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 possibly 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 such one type of untapped emerging Microbial resources.
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 camphor tree, 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-alternaric bacteria that from camphor tree, separates (Alternaria alternata) is provided to 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 alternaric bacteria of the present invention, screening and evaluation:
Be in June, 2008 in the camphor tree stem that gather in the Zhejiang Hangzhou Tian Mu Shan Mountain, obtain and preserve through steps such as separation, purifying, cultivation, fermentation and determinations of activity; Be accredited as alternaric bacteria (Alternaria alternata) through microbial taxonomy, name and be alternaric bacteria (Alternaria alternata) 31.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.4508.
Alternaric bacteria 31, its solid culture is characterized as: it is 9cm that bacterium colony 28 ℃ of cultivations on the PDA flat board extended to diameter in 6-7 days, and the initial stage light gray is sparse, and mycelia crawls to grow along media surface, middle grey black, the edge pearl, reverse side black, the mycelia tool is separated, branch.Microscopic morphology characteristic: conidiophore Dan Sheng; Conidium is oval, circle.
Alternaric bacteria 31 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 realizes through following technical scheme:
A kind of preparation method who prevents and treats fungal diseases of plants microbial metabolites such as cucumber rhizoctonia rot, graw mold of tomato or watermelon blight, carry out according to the following steps:
(1) activation culture of bacterial classification: will separate alternaric bacteria 31 bacterial classifications of preserving and move on the potato glucose PDA solid plate, and under 28 ℃ ± 1 ℃ condition, cultivate, and cover whole petridish to mycelia, using diameter is that the punching of 4mm punch tool obtains the bacterium cake, supplies 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 126h;
(3) fermented product extracts: fermentation finishes, centrifugal collection fermented supernatant fluid, and learning from else's experience with conventional organic solvent extracting obtains medicinal extract behind the vacuum concentration, is used for bacteriostatic activity mensuration.
Beneficial effect of the present invention:
The one, the present invention separates metabolite part fungal diseases of plants pathogenic bacteria is had stronger inhibiting alternaric bacteria 31 from plant camphor tree stem, and this provides a kind of microorganism strains for agricultural goes up control fungal diseases of plants pathogenic bacteria;
The 2nd, the invention provides the method for utilizing alternaric bacteria 31 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
Embodiment 1: (separation of alternaric bacteria 31 and screening)
The present invention gathers the camphor tree stem in the Zhejiang Hangzhou Tian Mu Shan Mountain in June, 2008; 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 active bacterial strain; Therefrom obtain alternaric bacteria 31, preserve.
Alternaric bacteria 31 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; The cucumber rhizoctonia rot bacterium (Rhizoctonia solani) that it is 4mm that the cooling back is put 1 diameter respectively on each substratum plane or withered germ of water-melon (Fusarium oxysporium) or botrytis cinerea (Botrytis cinerea) etc. supply examination bacterium bacterium cake; The bacterium cake is connected to petridish central authorities (the petridish diameter is 9cm); Put 28 ℃ of cultivation 72h in the incubator, handle as contrast, repeat 3 times with sterilized water; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with formula:
Mensuration result shows: alternaric bacteria 31 meta-bolitess are respectively 90.9%, 87.5% and 84.6% to the inhibiting rate of cucumber rhizoctonia rot bacterium, withered germ of water-melon and botrytis cinerea.
Embodiment 2: (preparing methods of alternaric bacteria 31 fermentating metabolism products)
Carry out according to the following steps:
(1) activation culture of bacterial classification: will separate alternaric bacteria 31 bacterial classifications of preserving and move on the potato glucose PDA solid plate, and under 28 ℃ ± 1 ℃ condition, cultivate, and cover whole petridish to mycelia, using diameter is that the punching of 4mm punch tool obtains the bacterium cake, supplies 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 126h;
(3) fermented product extracts: centrifugal collection fermented liquid after fermentation finishes, add the equal-volume ethyl acetate extraction, and take off layer, use the equal-volume n-butanol extraction again, get the upper strata, under vacuum condition, be concentrated into medicinal extract, get n-butyl alcohol extract, supply bacteriostatic activity to measure.
Following examples 3,31 meta-bolitess of the alternaric bacteria described in 4 are embodiment 2 products made therebies; The percentage composition of said product is mass/volume per-cent.
Embodiment 3: (alternaric bacteria 31 meta-bolitess are to the restraining effect of cucumber rhizoctonia rot bacterium, withered germ of water-melon and botrytis cinerea)
(1) accurately take by weighing alternaric bacteria 31 meta-bolites 0.18g, and add the sterilized water of 10mL volume, ultrasonic oscillation fully dissolves, and finally is configured to the mother liquor that concentration is 18g/L;
(2) bacteriostatic activity is measured: get above-mentioned mother liquor and be diluted to the solution that concentration is 1800mg/L and 900mg/L respectively; Get each concentration solution 1mL in sterile petri dish; Be cooled to 50 ℃ the rapid mixing of PDA substratum (alternaric bacteria 31 meta-bolites final concentrations are 180mg/L and 90mg/L) with 9mL; Withered germ of water-melon or cucumber rhizoctonia rot bacterium or botrytis cinerea 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 petridish central authorities (the petridish diameter is 9cm), puts 28 ℃ of cultivation 36h in the incubator; Handle as contrast with conventional chemical medicament and sterilized water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with formula:
Test Example: (product of the present invention and conventional pesticide control effect simultaneous test)
Test-results is seen table 1.Find out that from table 1 it is slightly excellent than the conventional chemical medicament that alternaric bacteria 31 meta-bolitess suppress effect, and safety.
Table 1 alternaric bacteria 31 meta-bolitess are to supplying the inhibition effect of examination pathogenic fungi
"-" expression not test (N.T.).
Claims (1)
1. alternaric bacteria bacterial strain; It is characterized in that called after alternaric bacteria (Alternaria alternata) 31, 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.4508.
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| CN201110088193A CN102191184B (en) | 2011-04-08 | 2011-04-08 | Biocontrol endophytic fungi-Alternaria alternata |
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| CN201110088193A CN102191184B (en) | 2011-04-08 | 2011-04-08 | Biocontrol endophytic fungi-Alternaria alternata |
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| CN102191184B true CN102191184B (en) | 2012-09-12 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719362A (en) * | 2011-11-23 | 2012-10-10 | 西北农林科技大学 | Alternaria. sp. xnsp-MG1 and use thereof |
| CN102952756B (en) * | 2012-08-23 | 2015-06-03 | 浙江工业大学 | Marine fungi altemaria MNP801 and application thereof |
| CN109280624B (en) * | 2018-10-30 | 2021-05-28 | 宁夏医科大学 | Alternaria alternata and application thereof in antagonizing pathogenic bacteria of potato ring rot |
| CN111676143A (en) * | 2020-06-29 | 2020-09-18 | 新疆农业科学院植物保护研究所 | Application of Alternaria alternata (Alternaria alternata) in biocontrol of cucurbitaceae |
| CN112646733B (en) * | 2020-12-28 | 2023-01-17 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Tamarix chinensis endophytic antagonistic fungus as well as separation method and application thereof |
| CN113773967B (en) * | 2021-08-19 | 2023-04-28 | 杨凌未来中科环保科技有限公司 | Alternaria tenuissima and application thereof |
| CN113980816B (en) * | 2021-08-19 | 2023-03-07 | 杨凌未来中科环保科技有限公司 | Alternaria brassicae and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1880309A (en) * | 2004-09-29 | 2006-12-20 | 汕头市双骏生物科技有限公司 | Novel compound, bacteria strain and method for producing novel compound using the strain |
| CN101484010A (en) * | 2006-06-16 | 2009-07-15 | 乌迪内大学 | Antifungal compositions containing the endophyte fungus alternaria alternata and/or its metabolites, as antagonist agents of plasmopara viticola |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1128210C (en) * | 2000-09-20 | 2003-11-19 | 南京农业大学 | Method for using crude metabolite of alternaric bacteria in biologic herbiciding |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1880309A (en) * | 2004-09-29 | 2006-12-20 | 汕头市双骏生物科技有限公司 | Novel compound, bacteria strain and method for producing novel compound using the strain |
| CN101484010A (en) * | 2006-06-16 | 2009-07-15 | 乌迪内大学 | Antifungal compositions containing the endophyte fungus alternaria alternata and/or its metabolites, as antagonist agents of plasmopara viticola |
Non-Patent Citations (4)
| Title |
|---|
| M.R.V.Fernanders et al.Biological activities of the fermentation extract of the endophytic fungus alternaria alternata isolated from coffea arabica L..《BJPS》.2009,第45卷(第4期), * |
| 万佐玺 等.链格孢菌寄主选择性毒素的研究现状.《湖北民族学院学报(自然科学版)》.2001,第19卷(第4期), * |
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