CN102217656A - Application of alternaria alternata metabolite to controlling Botrytis cirerea - Google Patents

Application of alternaria alternata metabolite to controlling Botrytis cirerea Download PDF

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Publication number
CN102217656A
CN102217656A CN2011100882037A CN201110088203A CN102217656A CN 102217656 A CN102217656 A CN 102217656A CN 2011100882037 A CN2011100882037 A CN 2011100882037A CN 201110088203 A CN201110088203 A CN 201110088203A CN 102217656 A CN102217656 A CN 102217656A
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bacterial strain
alternaric bacteria
metabolite
plant
application
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CN102217656B (en
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申屠旭萍
俞晓平
董胜张
郝培应
边亚琳
马正
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China Jiliang University
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China Jiliang University
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Abstract

The invention relates to the technical field of microorganism. The Endophytic Fungi in a camphor tree is separated and obtained from the living plant of the camphor tree collected in Tianmu Mountain Hangzhou, Zhejiang, by the separating technology of Endophytic Fungi. Verified by microbial taxonomy, the Endophytic Fungi is named alternaria alternata, and the bacterial strain is preserved in China General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms, with the preservation data being 2010-12-31 and the preservation registration number being CGMCC No.4508. The most remarkable characteristic of the bacterial strain is that the liquid of the bacterial strain can ferment to produce active metabolite having inhibitory action on plant fungi disease pathogenic bacteria such as Rhizoctonia solani, Fusarium oxysporium and Botrytis cinerea, and the bacterial strain is a significant microorganism resource in agriculture.

Description

The application of alternaric bacteria metabolite in the control botrytis cinerea
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 (Alternaria alternata) and the application of metabolite thereof.
Background technology
All have a large amount of endogenetic fungus in the plant corpus, endogenetic fungus is found in herbage the earliest, is found in plants such as woody plant, 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 corpus 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 biopesticide 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 biochemistry 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 pathogen, but chemical pesticide still is difficult to effective solution so far to people and animals' toxic and side effect and residue problem to the control of plant disease.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 metabolite has been carried out bacteriostasis 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 separates from camphor tree (Alternaria alternata) is provided at chemical pesticide; Another object of the present invention provides the method for utilizing this bacterial strain metabolite 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: bacterium colony on the PDA flat board 28 ℃ cultivate that to extend to diameter in 6-7 days be 9cm, the initial stage light gray is sparse, mycelia is along the media surface growth of crawling, middle grey black, the edge canescence, reverse side black, the mycelia tool is every, branch.Microscopic morphology feature: conidiophore Dan Sheng; The conidium ellipse, circle.
Alternaric bacteria 31 of the present invention can obtain the inhibited metabolite of part fungal diseases of plants pathogen 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 metabolic products 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 culture dish to mycelia, and obtain the bacterium cake with diameter for the punching of 4mm card punch, for inoculation usefulness;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, soluble 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, fermented and cultured 126h;
(3) fermentation 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 pathogen is had stronger inhibiting alternaric bacteria 31 from plant camphor tree stem, and this provides a kind of microbial strains for control fungal diseases of plants pathogen on the agricultural;
The 2nd, the invention provides the method for utilizing alternaric bacteria 31 fermentations to obtain active metabolite; This metabolite 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:(alternaric bacteria 31)
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 pathogen 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 medium with 9mL, the cucumber rhizoctonia rot bacterium (Rhizoctonia solani) that it is 4mm that the cooling back is put 1 diameter respectively on each medium plane or withered germ of water-melon (Fusarium oxysporium) or botrytis cinerea (Botrytis cinerea) 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 72h in the incubator, handle in contrast with sterile water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
Measurement result shows: alternaric bacteria 31 metabolites 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.
The preparation method of embodiment 2:(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 culture dish to mycelia, and obtain the bacterium cake with diameter for the punching of 4mm card punch, for inoculation usefulness;
(2) fermented and cultured: fermentation culture is to contain soybean cake powder 20g among every 1000mL, corn flour 15g, soluble 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, fermented and cultured 126h;
(3) fermentation product extracts: centrifugal collection zymotic fluid after fermentation finishes, add the equal-volume ethyl acetate extraction, and take off layer, use the equal-volume extracting n-butyl alcohol again, get the upper strata, under vacuum condition, be concentrated into medicinal extract, get n-butyl alcohol extract, measure for bacteriostatic activity.
Alternaric bacteria 31 metabolites described in following examples 3,4 are embodiment 2 products made therebies; The percentage composition of described product is mass/volume percentage.
Embodiment 3:(alternaric bacteria 31 metabolites are to the inhibitory action of cucumber rhizoctonia rot bacterium, withered germ of water-melon and botrytis cinerea)
(1) accurately take by weighing alternaric bacteria 31 metabolite 0.18g, and add the sterile 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 medium (alternaric bacteria 31 metabolite 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 medium 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 conventional chemical medicament and sterile 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 control efficiency comparative trial)
Result of the test sees Table 1.As seen from Table 1, it is slightly excellent than the conventional chemical medicament that alternaric bacteria 31 metabolites suppress effect, and safety.
Table 1 alternaric bacteria 31 metabolites are to the inhibition effect for the examination disease fungus
"-" expression not test (N.T.).

Claims (2)

1. the application of alternaric bacteria (Alternaria alternata) 31 metabolites control botrytis cinereas (Botrytis cinerea).
2. the described application of claim 1, wherein alternaric bacteria 31 metabolites are obtained by following method: alternaric bacteria 31 obtains zymotic fluid through slant activation cultivation, fermented and cultured, add the equal-volume ethyl acetate extraction, take off layer, use the equal-volume extracting n-butyl alcohol again, get the upper strata, under vacuum condition, be concentrated into medicinal extract, get n-butyl alcohol extract, described alternaric bacteria 31 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preservation registration number is CGMCC No.4508.
CN 201110088203 2011-04-08 2011-04-08 Application of alternaria alternata metabolite to controlling Botrytis cirerea Expired - Fee Related CN102217656B (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103070198A (en) * 2013-01-07 2013-05-01 中国计量学院 Preparation method of fungal disease prevention metabolite
CN103232942A (en) * 2013-04-22 2013-08-07 安徽农业大学 Preparation method of anti-phytopathogen fermentation liquid of spirea endophytic fungus
CN103952327A (en) * 2013-08-03 2014-07-30 山西省农业科学院农产品贮藏保鲜研究所 Biological antagonistic bacterial strain for controlling postharvest disease of fruit and vegetable, and preparation method and application thereof
CN108085259A (en) * 2017-12-14 2018-05-29 浙江农林大学 Biological and ecological methods to prevent plant disease, pests, and erosion plant endogenesis epiphyte and its application in industrial crops gray mold is prevented
CN109055238A (en) * 2018-08-29 2018-12-21 青岛农业大学 One plant of aspergillus flavipes bacteria strain TR32 and its application for having inhibiting effect to phytopathogen
CN110373331A (en) * 2019-06-21 2019-10-25 华中农业大学 The Huperzia serrata endogenetic epiphyte of one plant of anti-botrytis cinerea and its application
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

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WO1994028723A1 (en) * 1993-06-03 1994-12-22 Japan Tobacco Inc. Colletotrichum and use thereof
CN101223879A (en) * 2007-11-19 2008-07-23 中国计量学院 Method of preparing microorganisms pesticide for preventing vegetable fungi disease
CN101961014A (en) * 2010-03-19 2011-02-02 中国计量学院 Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables

Patent Citations (3)

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WO1994028723A1 (en) * 1993-06-03 1994-12-22 Japan Tobacco Inc. Colletotrichum and use thereof
CN101223879A (en) * 2007-11-19 2008-07-23 中国计量学院 Method of preparing microorganisms pesticide for preventing vegetable fungi disease
CN101961014A (en) * 2010-03-19 2011-02-02 中国计量学院 Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103070198A (en) * 2013-01-07 2013-05-01 中国计量学院 Preparation method of fungal disease prevention metabolite
CN103070198B (en) * 2013-01-07 2015-01-28 中国计量学院 Preparation method of fungal disease prevention metabolite
CN103232942A (en) * 2013-04-22 2013-08-07 安徽农业大学 Preparation method of anti-phytopathogen fermentation liquid of spirea endophytic fungus
CN103232942B (en) * 2013-04-22 2014-05-14 安徽农业大学 Preparation method of anti-phytopathogen fermentation liquid of spirea endophytic fungus
CN103952327A (en) * 2013-08-03 2014-07-30 山西省农业科学院农产品贮藏保鲜研究所 Biological antagonistic bacterial strain for controlling postharvest disease of fruit and vegetable, and preparation method and application thereof
CN103952327B (en) * 2013-08-03 2016-04-27 山西省农业科学院农产品贮藏保鲜研究所 Prevent and treat the biological antagonist bacteria strain of postharvest disease of fruits and vegetables, its preparation method and application thereof
CN108085259A (en) * 2017-12-14 2018-05-29 浙江农林大学 Biological and ecological methods to prevent plant disease, pests, and erosion plant endogenesis epiphyte and its application in industrial crops gray mold is prevented
CN109055238A (en) * 2018-08-29 2018-12-21 青岛农业大学 One plant of aspergillus flavipes bacteria strain TR32 and its application for having inhibiting effect to phytopathogen
CN110373331A (en) * 2019-06-21 2019-10-25 华中农业大学 The Huperzia serrata endogenetic epiphyte of one plant of anti-botrytis cinerea and its application
CN110373331B (en) * 2019-06-21 2021-09-10 华中农业大学 Huperzia serrata endophytic fungus for resisting botrytis cinerea and application thereof
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

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