CN105861337A - Nematicidal-activity A. niger NBC001 and preparation method and application thereof - Google Patents
Nematicidal-activity A. niger NBC001 and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to nematicidal-activity A. niger NBC001 and a preparation method and application thereof. High-activity fungus strain A. niger NBC001 is isolated from wheat cyst nematode group and is collected under CGMCC No. 12472. This strain is prepared by liquid fermentation culturing, liquid broth of the strain has significant poisoning action for age-2 larvae of soybean cyst nematode and significant inhibitory action for egg hatching, fermentation original broth may cause a decrease in amount of cysts of soybean cyst nematode, and this strain has a promising development and application prospect.
Description
Technical field
The invention belongs to microbial pesticide technical field, be specifically related to a strain and there is aspergillus niger and the system thereof of eelworm-killing activity
Preparation Method and application.
Background technology
Plant nematode is one of important phytopathy original, generally occurs in the whole world, and its host range is wide, causes harm
Seriously, the economic loss that the whole world is caused by plant nematode every year is up to 157,000,000,000 dollars of (letter perseverance, 2011. Plant nematodes
Learn. Beijing: China Agricultyre University Press).Wherein, (Heterodera glycines, is commonly called as Soybean to soy bean cyst roundworm
Cyst Nematode, SCN) it is one of the most serious plant nematode of causing harm.
Soy bean cyst roundworm, finds the earliest (1899) in China, initial formal name used at school uses Heterodera schachtii,
This is the formal name used at school of Radix Betae sporangiocyst class nematicide.Nineteen fifty-two Ichinohe, by careful observation, individually sets up soy bean cyst roundworm
Heterodera this kind of glycines (Liu Wei will .2004. Plant nematode will. Beijing: China Agricultyre University Press).Greatly
Bean cyst roundworm is mainly caused harm soybean root, the most at Japan (Ichinohe M.1988.Current research on
, beautiful the major nematode problems in Japan.Journal of Nematology.20 (2): 184-190.)
State (Wrather J.A., Koenning S.R., Anderson T.R.2003.Effect of diseases on soybean
yields in the United States and Ontario(1999to 2002).Plant Health Progress
(March) .0-16.), Italy (Manachini D.2000.First report of Heteroderaglycines
Ichinohe on soybean in Italy.Bolletino di Zoologia Agraria e Bachicoltura,
Serie II.32:261-267.), Brazil (Asmus G.L., Teles T.S., Anselmo J.et al.2012.Races
of Heterodera glycines in the Northeast of Mato Grosso do Sul,Brazil.Tropical
Etc. 15 countries and regions occur Plant Pathology.37 (2): 146-148.).In China, soy bean cyst roundworm mainly divides
Cloth northeastward with Huang-Huai-Hai Deng Gong16Ge provinces, cities and autonomous regions.The popular of soybean cyst nematode causes Semen sojae atricolor underproduction 10%-
20%, severe one reaches 50%, even total crop failure (Liu X., Li J., Zhang D.1997.History and status of
soybean cyst nematode in China.International Journal of Nematology.7(1):18-
25.)。
The preventing and treating of soy bean cyst roundworm is based on chemical prevention, but toxicity is high, pollute environment owing to chemical agent exists, right
Human and livestock health exists the problems such as potential threat, and (Zhang Keqin, the .1991. fungus such as He Shichuan, Zhou Wei and antibacterial are filled in biological and ecological methods to prevent plant disease, pests, and erosion online
Effect and progress thereof. insecticidal microorganism .3:55-66), reduce the use of chemical pesticide, find eco-friendly method
Preventing and treating soy bean cyst roundworm has become the focus studied in recent years.Biological control is to utilize beneficial organism or other biological to suppress
Or a kind of prevention and controls of killing harmful living, obtain studying widely and applying, at present in plant pathogeny line insect is prevented and treated
Study more biocontrol microorganisms to have.In Paecilomyces lilacinus (Paecilomyces lilacinus), Loews mould by hair spore
(Hirsutella rhossiliensis), India's pyriform spore (Piriform osporaindica), Fusarium spp. (Fusarium
Spp.) etc..Paecilomyces lilacinus can not only parasitic cyst roundworm, additionally it is possible to and the activity of suppression nematicide (Jiang Peizeng, Li Hongyuan, old
.2006. Advance in Paecilmyces Lilacinus Research for Plant Parasitic Nematodes Control protected by ferrum. Chinese agriculture science and technology Leader, and 8 (6): 38-14.).In Loews
By hair spore mould can make soy bean cyst roundworm population quantity reduce by 79% (Zhang L., Yang E., Xiang M.et
al.2008.Population dynamics and biocontrol efficacy of the nematophagous
fungus Hirsutella rhossiliensisas affected by stage of the soybean cyst
nematode.Biological Control,47(2):244-249).India's pyriform spore can reduce soy bean cyst roundworm ovum
Density (Bajaj R., Hu W., Huang Y..et al.2015.The beneficial root endophyte
Piriform osporaindica reduces egg density of the soybean cyst
nematode.Biological Control,90:193-199.).Fusarium spp. fermenation raw liquid can suppress cysts hatch and have
Extremely strong eelworm-killing activity, process 1h nemic death rate is up to 90%, and (Zhao Xiaohui, the most gorgeous .2011. Fusarium spp. fermentation liquid is to greatly
The inhibitory action of bean cyst roundworm. Soybean Science, 30 (3): 468-470.).But, these biocontrol microorganisms are developed to biological and ecological methods to prevent plant disease, pests, and erosion system
Agent, produce on be used for prevent and treat the little of soy bean cyst roundworm.Therefore, the present invention is with soy bean cyst roundworm as target, from fungus
Metabolite is found there is efficient nematocidal active material, find new resource for research and development new bio nematicide.
Summary of the invention
It is an object of the invention to the fungal bacterial strain selecting that cyst roundworm is had high eelworm-killing activity, by bacterial strain liquid routinely
After fermentation liquid is cultivated, remove thalline, from metabolite, extract the effective ingredient with nematocidal active material, big in order to prevent and treat
Bean cyst roundworm.
It is Aspergillus niger NBC001 that the strain that the present invention screens has the fungal bacterial strain of eelworm-killing activity,
Deposit number is CGMCC No.12472.
A kind of fungal fermented filtrate, it is characterised in that: above-mentioned fungal bacterial strain is made through liquid fermentation and culture, and its pH value is 3.0-
3.4。
Culture medium used by described liquid fermentation is the Rhizoma Solani tuber osi liquid fermentation medium containing chloromycetin, and its pH is 5.4-
5.8。
The preparation method of fungal fermented filtrate, the method is particularly as follows: by a diameter of 1cm fungus pure culture biscuits involvng inoculation to Rhizoma Solani tuber osi liquid
In fermentation medium, under 26 DEG C of dark conditions, 150rpm/min cultivates 120h, obtains after removing thalline.
Described removing thalline uses 4 layers of sterilizing yarn to filter, and filtrate, again through 0.22 μm biofilter, collects filtered solution.
The application in plant nematode Biological control of the above-mentioned fungal bacterial strain.
Described application is fungal bacterial strain to be prepared as fungal fermented filtrate spray or the crop of pouring infection plant parasitic nematode.
Described plant nematode is soy bean cyst roundworm (Heterodera glycines.).
The present invention relates to the fungal fermented filtrate for preventing and treating plant nematode, be by fungal bacterial strain A.niger NBC001
Preparation after conventional liq fermentation culture.
The strains A .niger NBC001 of the present invention be by flat board cultivate preparation after, then through expand fermentation culture system
Standby.
Its concrete grammar is as follows:
1. flat board is cultivated
Culture medium is potato agar culture medium (PDA), and purchased from Qingdao Hai Bo Bioisystech Co., Ltd, its formula is horse
Bell potato leaching powder 6.0g, glucose 20.0g, agar 20.0g, water 1000ml, pH value 5.4-5.8.
2. liquid fermentation and culture
Wherein fluid medium is Rhizoma Solani tuber osi fluid medium (PDB), purchased from Qingdao Hai Bo Bioisystech Co., Ltd, its
Formula is: Rhizoma Solani tuber osi leaches powder 6.0g, glucose 20.0g, chloromycetin 0.1g, and water 1000ml, pH are 5.4-5.8.From culture dish
On take the fungus pure culture biscuits involvng inoculation of a diameter of 1cm in 250ml conical flask (built-in PDB 100ml), under 26 DEG C of dark conditions
150rpm/min shaken cultivation 120h.
Strains A .niger NBC001 fermentation liquid involved in the present invention has nematocidal active material, can be used for Semen sojae atricolor spore
The preventing and treating of capsule nematicide.
3. biological deposits information:
Aspergillus niger NBC001, entitled aspergillus niger of classifying (Aspergillus niger)
Preserving number: CGMCC No.12472.
Preservation date: on May 17th, 2016
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode
100101
Detailed description of the invention
In order to illustrate in more detail rather than limit the present invention, give following embodiment.
The present invention is found by research, and the A.niger NBC001 separated from Semen Tritici aestivi cyst roundworm colony has preferably
Eelworm-killing activity, after PDA culture medium 26 DEG C is cultivated one week, bacterium colony is rounded, diameter 50-55mm, and rat is fine hair shape, raw
Long initial stage mycelia is white, after become foresythia until black.Basis of microscopic observation, conidiophore length, column, top raw
Ball-type top capsule, conidium is circular, in black.PDB fermentation culture, fermentation liquid is acidity, and pH is between 3.0-3.4.
Preserving number: CGMCC No.12472.
Preservation date: on May 17th, 2016
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Embodiment:
Strains A .niger NBC001 is carried out fermentation culture, after removing thalline, carries out eelworm-killing activity examination with fermentation liquid
Test, measure its effect to nematicide.
Embodiment 1: the preparation of strains A .niger NBC001 fermentation liquid
Strains A .niger NBC001 mycelium is inoculated on PDA plate, 26 DEG C cultivate 10 days, with sterilizing card punch from
The pure culture biscuits involvng inoculation of a diameter of 1cm is taken to 250ml conical flask (built-in 100ml PDB), 150rpm/ under 26 DEG C of dark conditions on flat board
Min, shaken cultivation 120h.Cross 0.22 μm biofilter after removing mycelium with 4 layers of sterile gauze degerming, obtain proferment
Liquid.Fermenation raw liquid is diluted different multiples and carries out the nematicide evaluation of pesticide effectiveness.
Embodiment 2: the eelworm-killing activity of strains A .niger NBC001
The preparation of 1 fermentation liquid
On the basis of fermentation liquid stock solution successively dilute with water 1 ×, 5 ×, 10 ×, for soy bean cyst roundworm 2 children in age
Worm carries out toxic biological mensuration.
The collection of 2 soy bean cyst roundworm sporangiocysts
Pour the sick soil endangered from the soy bean cyst roundworm of Langfang, academy of agricultural sciences Pilot Base collection into a clean plastics keg
In, after sick soil being broken up with strong current, cross 20 mesh/80 mesh mesh screens, collect the deposit on 80 mesh mesh screens, picking under anatomical lens
Sporangiocyst.
The preparation of 3 soy bean cyst roundworm 2 instar larvaes
Sporangiocyst, through 0.5% hypochlorite disinfectant 3min, after sterilizing distilled water is cleaned, is placed on 100 clean mesh mesh screens,
Mesh screen is placed in a sterilizing culture dish, in ware, adds 3mmol/L zinc chloride to not having sporangiocyst, hatch under 26 DEG C of dark conditions.3
Start to collect 2 instar larvaes after it, and make the 2 instar larvae suspensions that concentration is 200/ml.
The preparation of 4 soy bean cyst roundworm ovum suspensions
Sporangiocyst, through 0.5% hypochlorite disinfectant 3min, after sterilizing distilled water is cleaned, galls sporangiocyst with cork, makes ovum
Suspension
The toxic biological of larva is measured by 5 fermentation liquids
In 24 orifice plates, add the fermentation liquid 0.5ml of different extension rate, dilute the PDB of same multiple as comparison, then
Add 0.5ml 2 instar larvae suspension, then final diluted concentration be respectively 2 ×, 10 ×, 20 ×.Room temperature adds up line after placing 24h
Worm death toll, and calculate mortality rate and corrected mortality.Each process is repeated 4 times, and this test is repeated 2 times.
Mortality rate (%)=death nematicide/confession examination nematicide × 100%
Corrected mortality (%)=(processing nemic death rate-comparison nemic death rate)/(1-compares nemic death rate) ×
100%
6 fermentation liquids inhibitory action to egg hatching
The fermentation liquid 100 μ l taking variable concentrations is placed in 96 orifice plates, and 100 μ l aquesterilisa are as comparison, then are added thereto to
100 μ l ovum suspensions (about 50), hatch under 26 DEG C of dark conditions, add up the ovum number of hatching, and calculate incubation rate after 12 days.Often
Individual process is repeated 4 times.
The ovum number of incubation rate (%)=hatching/total number × 100% of ovum
Hatching suppression ratio (%)=(process group incubation rate-matched group incubation rate)/matched group incubation rate × 100%
7 result of the tests
(1) the A.niger NBC001 metabolite nematicide effect to soy bean cyst roundworm 2 instar larvae
Result shows, strains A .niger NBC001 difference extension rate fermentation liquid has higher activity.Its 2 × dilution
The corrected mortality of liquid is about 90%, and 10 × diluent, more than 40%, has higher eelworm-killing activity.
The impact on Semen Tritici aestivi cyst roundworm 2 instar larvae of the table 1 fermentation of Aspergillus niger liquid difference extension rate
Note: CK1 is dilution 2 × PDB, and CK2 is dilution 10 × PDB, and CK3 is dilution 20 × PDB；Letter is the significance of difference
Analysis result.Value is meansigma methods ± standard error, and letter represents the notable analysis result of diversity, and following table is same.
(2) the A.niger NBC001 metabolite effect to soy bean cyst roundworm egg hatching
Result shows, through the egg hatching rate all substantially less than matched group of variable concentrations fermentation liquor treatment, illustrates that NBC001 sends out
Ferment liquid all has inhibitory action to soy bean cyst roundworm egg hatching, and the inhibitory action difference of variable concentrations is the most notable.
The impact on soy bean cyst roundworm egg hatching of the table 2. aspergillus niger NBC001 variable concentrations fermentation liquid
Note: CK is aquesterilisa comparison, and letter is significance of difference analysis result.
Embodiment 3: strains A .niger NBC001 fermentation liquid potted plant experiment prevention effect measures
Utilizing fermenation raw liquid to prevent and treat soy bean cyst roundworm, effect is as follows:
1 material to be tested:
Soybean varieties: middle yellow 13
Nematicide: soy bean cyst roundworm
2 test methods:
The effect measuring of strains A .niger NBC001 fermentation liquid potted plant experiment preventing and treating soy bean cyst roundworm
1% hypochlorite disinfectant is used on soybean seed surface, after aquesterilisa cleans three times, is placed in batch cultur ware and sends out
Bud.By the seed that germinates equipped with in the plastic cup of sterilized soil after 2 days, every glass of 3 seeds.After one week, add in plastic cup
Entering fungi fermentation stock solution 20ml, PDB is as comparison.After fermented stock solution processes 24h, near Soybean Root, inoculate SCN bis-children in age
Worm, 300/strain, second time inoculation 300/strain of SCN second instar larvae, after inoculating 30 days, adds up in every glass of root soil two days later
Sporangiocyst quantity.
3 result of the tests
Result shows: strains A .niger NBC001 fermenation raw liquid inoculates nematicide, the soybean cyst quantity of formation after processing
Being substantially less than matched group, sporangiocyst slip is 50.07%, has preferable application prospect.
The effect measuring of table 3. strains A .niger NBC001 fermenation raw liquid preventing and treating soy bean cyst roundworm
Note: CK is liquid fermentation culture medium PDB.
Claims (8)
1. a strain has the fungal bacterial strain of eelworm-killing activity, and named Aspergillus niger NBC001, deposit number is
CGMCC No.12472。
2. a fungal fermented filtrate, it is characterised in that: it is made up through liquid fermentation and culture of the fungal bacterial strain described in claim 1,
Fermentation liquid pH value is 3.0～3.4.
Fungal fermented filtrate the most according to claim 1, the culture medium used by described liquid fermentation is the Ma Ling containing chloromycetin
Potato liquid fermentation medium, the pH of culture medium is 5.4-5.8.
4. the preparation method of the fungal fermented filtrate described in Claims 2 or 3, it is characterised in that: wanting a diameter of 1cm containing right
Ask the pure culture biscuits involvng inoculation of fungal bacterial strain described in 1 in Rhizoma Solani tuber osi liquid fermentation medium, 150rpm/min training under 26 DEG C of dark conditions
Support 120h, obtain after removing thalline.
Preparation method the most according to claim 4, described removing thalline uses 4 layers of sterilizing yarn to filter, and filtrate is again through 0.22 μ
M biofilter, collects filtered solution.
6. the application in plant nematode Biological control of the fungal bacterial strain described in claim 1.
Application the most according to claim 6, is the fungal bacterial strain described in claim 1 to be prepared as fungal fermented filtrate spray
Or the crop of pouring infection plant parasitic nematode.
Application the most according to claim 6, described plant nematode is soy bean cyst roundworm (Heteroderaglycines).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107118978A (en) * | 2017-04-01 | 2017-09-01 | 云南大学 | One plants endogenetic fungus |
| CN108004156A (en) * | 2017-04-01 | 2018-05-08 | 云南大学 | One plant of aquatic fungi |
| CN108441426A (en) * | 2017-04-01 | 2018-08-24 | 云南大学 | One plant of endophytic bacterial controlled effect that can prevent Plant nematode |
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| CN107118978A (en) * | 2017-04-01 | 2017-09-01 | 云南大学 | One plants endogenetic fungus |
| CN108004156A (en) * | 2017-04-01 | 2018-05-08 | 云南大学 | One plant of aquatic fungi |
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