CN105861336A - Nematicidal-activity P. oxalicum NBC012 and preparation method and application thereof - Google Patents
Nematicidal-activity P. oxalicum NBC012 and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to nematicidal-activity P. oxalicum NBC012 and a preparation method and application thereof. A high-activity fungus strain P. oxalicum NBC012 is isolated and screened from wheat cyst nematode group from stfold, Norway and is collected under CGMCC No. 12474. Nematicidal-activity P. oxalicum NBC012 is prepared by liquid fermentation culturing, fermentation broth of P. oxalicum NBC012 has significant poisoning action for plant parasitic nematodes. Indoor determination shows that the fermentation broth of the strain has effect on age-2 larvae and egg hatching of soybean cyst nematode. Fermentation broths with different dilution concentrations all show poisoning action for age-2 larvae and significant inhibitory action for egg hatching. Fermentation original broth may cause a decrease of 57.45% in amount of soybean cyst, and P. oxalicum NBC012 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 fungus and the preparation thereof of intoxicating plant parasitical eelworm
Methods and applications.
Background technology
Plant nematode is one of important phytopathy original, generally occurs in the whole world, and its host range is wide, causes harm serious,
The economic loss that the whole world is caused by plant nematode every year be up to 157,000,000,000 dollars (letter is permanent, 2011. plant nematologies. Beijing:
China Agricultyre University Press).Wherein, soy bean cyst roundworm (Soybean Cyst Nematode, SCN) be cause harm the most serious
One of plant nematode.
Soy bean cyst roundworm, finds the earliest (1899) in China, initial formal name used at school uses Heterodera schachtii, and this is Radix Betae
The formal name used at school of 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).Soy bean cyst roundworm is predominantly
Evil soybean root, the most at Japan (Ichinohe M.1988.Current research on the major nematode problems
, the U.S. (Wrather J.A., Koenning S.R., Anderson in Japan.Journal of Nematology.20 (2): 184-190.)
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 Heterodera
glycines 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
15 states such as the Northeast of Mato Grosso do Sul, Brazil.Tropical Plant Pathology.37 (2): 146-148.)
Family and area find report.In China, it is mainly distributed on northeast and Huang-Huai-Hai Deng Gong16Ge provinces, cities and autonomous regions.Soy bean cyst roundworm
The popular of disease causes Semen sojae atricolor underproduction 10%-20%, and severe one reaches 50%, and even (Liu X., Li J., Zhang are D.1997. in total crop failure
History and status of soybean cyst nematode in China.International Journal of Nematology.
7(1):18-25.)。
The preventing and treating of preventing and treating soy bean cyst roundworm is still based on chemical agent at present, but toxicity is high, pollute environment, to people owing to existing
Poultry health exists the problems such as potential threat, and (Zhang Keqin, the .1991. fungus such as He Shichuan, Zhou Wei and antibacterial fill the effect in biological and ecological methods to prevent plant disease, pests, and erosion online
And progress. insecticidal microorganism .3:55-66), the use reducing chemical pesticide becomes a kind of inevitable.Therefore a kind of environment is found
It is the most necessary that friendly method prevents and treats nematicide.Biological control obtains in plant pathogeny line insect is prevented and treated as the safe and effective means of one
To studying widely and applying.Paecilomyces lilacinus (Paecilomyces lilacinus), it can not only parasitic cyst roundworm, also
Can suppress nematicide activity (Jiang Peizeng, Li Hongyuan, Chen Tie protect .2006. Advance in Paecilmyces Lilacinus Research for Plant Parasitic Nematodes Control. China
Agricultural science and technology Leader, 8 (6): 38-14.).Soy bean cyst roundworm population can be made by hair spore mould (Hirsutella rhossiliensis) in Loews
Quantity reduces 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).Gen Nei probiotic bacteria India pyriform spore (Piriform
Osporaindica) density (Bajaj R., Hu W., the Huang Y..et al.2015.The of soy bean cyst roundworm ovum can be reduced
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 nematicide to live
Property, process 1h nemic death rate is up to 90%, and (Zhao Xiaohui, soy bean cyst roundworm is pressed down by the most gorgeous .2011. Fusarium spp. fermentation liquid
Make and use. Soybean Science, 30 (3): 468-470.).But, the biological prevention and control agent being developed to prevent and treat nematicide is little.Therefore, this
Bright with soy bean cyst roundworm as target, find from fungus metabolite and there is efficient nematocidal active material, new for research and development
Type GR finds new resource.
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 fermentation liquid routinely
After cultivation, remove thalline, from metabolite, extract the effective ingredient with nematocidal active material, develop wireworm-killing biologic agriculture
Medicine.
It is Penicillium oxalicum NBC012 that the strain that the present invention screens has the fungal bacterial strain of eelworm-killing activity, and preservation is compiled
Number it is CGMCC No.12474.
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 3.5-3.8.
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 fermentation
In culture 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 cyst roundworm (Heterodera spp.).
The present invention is found by research, and the most bacterial strains separated from the Semen Tritici aestivi cyst roundworm colony of Norway Dong Fuer prefecture all have
Certain eelworm-killing activity, filters out a strain higher fungal bacterial strain P.oxalicum NBC012 of activity from these bacterial strains and carries out
Further research.
The invention still further relates to the fungal fermented filtrate for preventing and treating plant nematode, it is by the fungal bacterial strain P. of the present invention
Oxalicum NBC012 is prepared after conventional liq fermentation culture.
The bacterial strain P.oxalicum NBC012 of the present invention be cultivated by flat board and prepare after, then prepare through expanding fermentation culture.
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 Rhizoma Solani tuber osi
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), and 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.Cut-off from culture dish
Footpath be the fungus pure culture biscuits involvng inoculation of 1cm in 250ml conical flask (built-in PDB 100ml), 150rpm/min under 26 DEG C of dark conditions
Shaken cultivation 120h.
Bacterial strain P.oxalicum NBC012 fermentation liquid involved in the present invention has nematocidal active material, can be used for phytotrophy
The preventing and treating of nematicide, particularly cyst roundworm.
Biological deposits information:
Penicillium oxalicum NBC012, entitled penicillium oxalicum of classifying (penicillium oxalicum)
Preserving number: CGMCC No.12474.
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 the following example.
The present invention is found by research, and the P.oxalicum NBC008 separated from Semen Tritici aestivi cyst roundworm colony has and preferably kills
Nematicide activity, after PDA culture medium 26 DEG C is cultivated one week, bacterium colony ovalize, major axis 45-50 rice is slow, short axle 35-40mm,
Early growth period bacterium colony surface is pink colour, and the later stage becomes green.Basis of microscopic observation, conidiophore length, column, the raw row in top
Arrange into branch between broom shape, conidium oval, green.PDB fermentation culture, fermentation liquid is acidity, and pH is at 3.5-3.8
Between.
Biological deposits information: be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is
CGMCC No.12474。
First bacterial strain P.oxalicum NBC012 is carried out fermentation culture, after removing thalline, fermentation liquid is carried out eelworm-killing activity
Test, measures its effect to nematicide.
Embodiment 1: the preparation of bacterial strain P.oxalicum NBC012 fermentation liquid
Bacterial strain P.oxalicum NBC012 mycelium is inoculated on PDA plate, cultivates 10 days, use sterilizing card punch for 26 DEG C
The pure culture biscuits involvng inoculation of a diameter of 1cm is taken to 250ml conical flask (built-in 100ml PDB), under 26 DEG C of dark conditions from flat board
150rpm/min, shaken cultivation 120h.0.22 μm biofilter is crossed degerming after removing mycelium with 4 layers of sterile gauze,
To fermenation raw liquid.Fermenation raw liquid is diluted different multiples and carries out the nematicide evaluation of pesticide effectiveness.
Embodiment 2: the eelworm-killing activity of bacterial strain P.oxalicum NBC012
1 is diluted successively on the basis of fermentation liquid stock solution ×, 5 ×, 10 ×, soy bean cyst roundworm 2 instar larvae is measured.
1 preparation test medicament
The fungal bacterial strain P.oxalicum NBC012 fermentation liquid prepared by aforementioned liquids fermentation process, with sending out of different extension rates
Ferment liquid carries out Nematicidal Activity.
Nematicide is used in 2 preparation tests
The collection of 2.1 soy bean cyst roundworm sporangiocysts
The sick soil endangered from the soy bean cyst roundworm of Langfang, academy of agricultural sciences Pilot Base collection is poured in a clean plastics keg, with strong
After sick soil is broken up by current, cross 20 mesh/80 mesh mesh screens, collect the deposit on 80 mesh mesh screens, picking sporangiocyst under anatomical lens.
The preparation of 2.2 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, by net
Sieve is placed in a sterilizing culture dish, adds 3mmol/L zinc chloride to not having sporangiocyst in ware, hatches under 26 DEG C of dark conditions.3 days
After start to collect 2 instar larvaes, and make the 2 instar larvae suspensions that concentration is 200/ml.
The preparation of 2.3 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
3. test method
The eelworm-killing activity of 3.1 bacterial strain P.oxalicum NBC012
In 24 orifice plates, add the fermentation liquid 0.5ml of different extension rate, dilute the PDB of same multiple as comparison, add
0.5ml 2 instar larvae suspension, then final diluted concentration be respectively 2 ×, 10 ×, 20 ×.It is dead that room temperature adds up nematicide after placing 24h
Die number, 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%
The 3.2 bacterial strain P.oxalicum NBC012 effect to soy bean cyst roundworm 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 suspension (about 50), hatches under 26 DEG C of dark conditions, adds up the nematicide number of hatching, and calculate incubation rate after 12 days.Each
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%
4 result of the tests
(1) the P.oxalicum NBC012 metabolite nematicide effect to soy bean cyst roundworm 2 instar larvae
Result shows, bacterial strain P.oxalicum NBC012 difference extension rate fermentation liquid has higher activity.Its 2 × diluent
Corrected mortality can reach more than 90%, 10 × diluent, about 50%, has higher eelworm-killing activity.
The impact on Semen Tritici aestivi cyst roundworm 2 instar larvae of the table 1 penicillium oxalicum fermentation 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 P.oxalicum NBC012 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, NBC012 fermentation liquid is described
Soy bean cyst roundworm egg hatching is all had inhibitory action, 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. penicillium oxalicum NBC012 variable concentrations fermentation liquid
Note: CK is aquesterilisa comparison, and letter is significance of difference analysis result.
Embodiment 3: bacterial strain P.oxalicum NBC012 fermentation liquid indoor control effect measuring
Utilizing fermenation raw liquid preventing and treating soy bean cyst roundworm and Semen Tritici aestivi 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 bacterial strain P.oxalicum NBC012 fermentation liquid indoor control soy bean cyst roundworm
1% hypochlorite disinfectant is used on soybean seed surface, after aquesterilisa cleans three times, is placed in batch cultur ware germination.2 days
After by the seed that germinates equipped with in the plastic cup of sterilized soil, every glass of 3 seeds.After one week, in plastic cup, add fungus
Fermenation raw liquid 20ml, PDB is as comparison.After fermented stock solution processes 24h, near Soybean Root, inoculate SCN second instar larvae,
Article 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: bacterial strain P.oxalicum NBC012 fermenation raw liquid inoculates nematicide after processing, and the soybean cyst quantity of formation shows
Writing less than matched group, sporangiocyst slip is 57.45%, has application prospect.
The effect measuring of table 3. bacterial strain P.oxalicum NBC012 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 Penicillium oxalicum NBC012, deposit number is
CGMCC No.12474。
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 is 3.5-3.8.
Fungal fermented filtrate the most according to claim 1, the culture medium used by described liquid fermentation is the Rhizoma Solani tuber osi containing chloromycetin
Liquid fermentation medium, liquid fermentation medium pH 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: with dividend right by a diameter of 1cm
Profit require the pure culture biscuits involvng inoculation of fungal bacterial strain described in 1 in Rhizoma Solani tuber osi liquid fermentation medium, 150rpm/min under 26 DEG C of dark conditions
Cultivate 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 μm
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 cyst roundworm (Heterodera spp.).
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| CN110964644A (en) * | 2019-10-21 | 2020-04-07 | 西北农林科技大学 | Penicillium, and fermentation method and application thereof |
| CN114424775A (en) * | 2017-05-04 | 2022-05-03 | 青岛益佰农肥业有限公司 | Compound microorganism wettable powder for preventing and treating nematodes and preparation method and application thereof |
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