CN102217656B - Application of alternaria alternata metabolite to controlling Botrytis cirerea - Google Patents

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 alternaria alternata 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 the 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 owing to endogenetic fungus Growth and Reproduction in plant corpus, 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 broad interest in recent years, becomes gradually the focus of domestic and international research.The at present research and development of microbial source biochemical pesticides is very active in China, but microbial resources and application potential thereof still do not fully played, and plant endogenesis epiphyte namely is the untapped emerging microbial resources of such class.

At present to the control of plant disease, 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.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 the 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 further development and the exploitation of microbial pesticide.

Summary of the invention

The objective of the invention is provides a kind of endogenetic fungus-alternaric bacteria that separates (Alternaria alternata) for the deficiency that chemical pesticide is dangerous and the microbial pesticide kind is less from camphor tree; Another object of the present invention provides the method for utilizing this 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 the Zhejiang Hangzhou Tian Mu Shan Mountain gathers, 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 sprawl growth, middle grey black, the edge canescence, reverse side black, the mycelia tool is every, branch.Micro-morphology: conidiophore Dan Sheng; Conidium is oval, circle.

Alternaric bacteria 31 of the present invention can obtain the inhibited metabolite of Activities of Some Plants fungal disease pathogen through the fermentation extraction.

The object of the invention is achieved through the following technical solutions:

A kind of preparation method who prevents and treats the 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, under 28 ℃ ± 1 ℃ condition, cultivate, covering whole culture dish to mycelia, is that the punching of 4mm card punch obtains the bacterium cake with diameter, for inoculating;

(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 rear 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: it is complete ferment, centrifugal collection fermented supernatant fluid, and learning from else's experience with conventional organic solvent extracting obtains medicinal extract behind the Vacuum Concentration, for Antibacterial Activity.

Beneficial effect of the present invention:

The one, the present invention separates metabolite Activities of Some Plants fungal disease 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 isolation and 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 Antibacterial Activities: 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 cooling is put respectively 1 diameter on each medium plane afterwards 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, process 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, under 28 ℃ ± 1 ℃ condition, cultivate, covering whole culture dish to mycelia, is that the punching of 4mm card punch obtains the bacterium cake with diameter, for inoculating;

(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 rear 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: the complete rear centrifugal collection zymotic fluid that ferments, add the equal-volume ethyl acetate extraction, and take off layer, use again the equal-volume extracting n-butyl alcohol, get the upper strata, under vacuum condition, be concentrated into medicinal extract, get n-butyl alcohol extract, for Antibacterial 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) Antibacterial Activity: get above-mentioned mother liquor and be diluted to respectively the solution that concentration is 1800mg/L and 900mg/L, 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 cooling is put respectively 1 diameter on each medium plane afterwards, the bacterium cake is connected to culture dish central authorities (the culture dish diameter is 9cm), put 28 ℃ of cultivation 36h in the incubator, process 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, alternaric bacteria 31 metabolite inhibitions are slightly excellent than the conventional chemical medicament, and safety.

Table 1 alternaric bacteria 31 metabolites are to the inhibition for the examination disease fungus

"-" expression not test (N.T.).

Claims (1)

1. the application of alternaric bacteria (Alternaria alternata) 31 metabolites control botrytis cinereas (Botrytis cinerea), described alternaric bacteria 31 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation registration number is CGMCC No.4508, and described metabolite prepares by the following method:

(1) activation culture of bacterial classification: will separate alternaric bacteria 31 bacterial classifications of preserving and move on the potato glucose PDA solid plate, under 28 ℃ ± 1 ℃ condition, cultivate, covering whole culture dish to mycelia, is that the punching of 4mm card punch obtains the bacterium cake with diameter, for inoculating;

(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 rear 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: the complete rear centrifugal collection zymotic fluid that ferments, add the equal-volume ethyl acetate extraction, and take off layer, use again the equal-volume extracting n-butyl alcohol, get the upper strata, under vacuum condition, be concentrated into medicinal extract, get n-butyl alcohol extract.