CN100489083C - Penicillium fungus with nematocidal activity and its preparing method and use - Google Patents
Penicillium fungus with nematocidal activity and its preparing method and use Download PDFInfo
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Abstract
本发明涉及一株具毒杀植物寄生线虫作用的真菌的培养方法及其代谢物制备方法和应用，属于微生物农药技术领域。本发明涉及的真菌菌株Penicilliumpinophilum snf44-1，已于2006年10月27日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为：CGMCC No.1852。snf44-1的分类地位是半知菌亚门，丝孢纲，丛梗孢目，丛梗孢科，青霉属，嗜松青霉。通过液体发酵培养制备，其代谢物对植物寄生线虫，特别是对根结线虫具有毒力高，杀线虫效果明显的突出特点，室内测定本菌株发酵液对北方根结线虫(Meloidogyne hapla)2龄幼虫和卵囊孵化的影响。不同稀释浓度发酵液的处理和无菌水处理有显著差异，发酵液的1倍液以上浓度与10μg/ml阿维菌素的防效相当。具良好的应用开发前景。The invention relates to a method for cultivating a fungus capable of poisoning and killing plant parasitic nematodes and a method for preparing and using metabolites thereof, belonging to the technical field of microbial pesticides. The fungal strain Penicillium pinophilum snf44-1 involved in the present invention has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on October 27, 2006, and the preservation number is: CGMCC No.1852. The taxonomic status of snf44-1 is Deuteromycotina, Hyphomycetes, Polygonales, Polygonalaceae, Penicillium, and Penicillium pinophilum. Prepared by liquid fermentation culture, its metabolites have high toxicity to plant parasitic nematodes, especially root-knot nematodes, and prominent characteristics of nematicidal effect. Indoor measurement of the fermentation liquid of this strain against the 2nd instar of northern root-knot nematodes (Meloidogyne hapla) Effects on larval and oocyst hatching. There are significant differences between the treatment of different dilution concentrations of fermentation broth and the treatment of sterile water, and the control effect of 10 μg/ml avermectin is equivalent to the concentration of fermentation broth above 1 times. It has a good application development prospect.
Description
Technical field:
The invention belongs to the microbial pesticide technical field, be specifically related to fungi of a kind of intoxicating plant parasitical eelworm and its production and application.
Background technology:
The many in the world countries and regions of plant nematode take place and cause harm, and kind is varied.In the world, the generation of plant nematode and harm are along with the variation of the development of agroforestry and tillage and cultivation system is serious day by day, add up according to Esser, the whole world has been reported and has been found that plant nematode 207 belongs to 4832 kinds of (Liu Weizhi till the nineteen ninety, Duan Yuxi .2000. plant pathogeny line insect is learned. Beijing: and Chinese agriculture press, p1-2).According to estimates, it is 12.3% that plant nematode is caused the year rate of loss of world staple crops, above 1,000 hundred million dollars, and actual loss is considerably beyond estimation, reason be the harm that causes of many nematodes because of do not have visible symptom in field or specialization feature do not arouse attention (Wang Shouhua. the fruit tree nematology. Beijing: Chinese agriculture science and technology press, 1994.P.1-5), in addition, the new discovery of in the world wide nematode being caused harm is also in continuous increase, and the agricultural cultivation system of some renewals makes the nematode problem more outstanding, moreover nematode and other biological interaction and direct or indirect influence that plant is produced are also in rapid increase.The harm that nematode is caused is more hidden than other biology in this sense! At present the deleterious nematode of plant there is kind more than 3000 approximately, mainly contains root knot nematode, Cyst nematode, pine wood nematode, sweet potato stem nematode etc. in China.Root knot nematode is the maximum class nematode of causing harm on the agricultural, after disease takes place, the general underproduction about 10%, serious in more than 75%, even total crop failure (A.G.Whitehead, 1998.Plant Nematode Control.ISBN0851991882 CABInternational).At present, the control of plant nematode is still based on chemical prevention, but in recent years, the application waste of chemistry nematode killing agent is big, contaminate environment, poor selectivity, the natural disposition of going out is strong, drawbacks such as destruction soil organisms fauna are paid attention to (Zhang Keqin by people day by day, He Shichuan .1991. fungi and effect and the progress thereof of bacterium in the nematode biological and ecological methods to prevent plant disease, pests, and erosion such as Zhou Wei. insecticidal microorganism .3:55-66), 21 century is called environmental protection century, the application of some effective chemical nematode killing agents progressively is restricted, and therefore the biological control research nature to plant nematode becomes hot issue.The present invention just is being based on above-mentioned theory, is target with the plant nematode, seeks to have efficient nematocidal active material from fungus metabolite, intends seeking new resource into research and development new bio nematocides.
Summary of the invention:
The objective of the invention is to select fungal bacterial strain to the high insecticidal activity of root knot nematode tool, bacterial strain routinely after the liquid fermentation and culture, is extracted the effective constituent of tool nematocidal active material, exploitation wireworm-killing biologic agricultural chemicals from meta-bolites, wherein, the preferably northern root knot nematode of described root knot nematode.
The fungal bacterial strain that the strain that the present invention screens has eelworm-killing activity is Penicillium pinophilum snf44-1, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on October 27th, 2006; The numbering that preservation is registered on the books: CGMCC No.1852.P.pinophilum snf44-1 is the strain that filters out from a large amount of soil fungi bacterial strains has good eelworm-killing activity to northern root knot nematode a fungal bacterial strain.
The present invention is by discovering, soil fungi has a lot of fungal strains all to have higher eelworm-killing activity, filters out the best relatively fungal bacterial strain P.pinophilum snf44-1 of a strain proterties and carried out further research from these fungies.
The invention still further relates to the fungal metabolite that is used to prevent and treat plant nematode, it is to be prepared by extraction after the liquid fermentation and culture of fungal bacterial strain P.pinophilum snf44-1 process routine of the present invention.
Fungal bacterial strain P.pinophilum snf44-1 of the present invention after cultivating by the test tube kind, prepares through enlarging fermentation culture again, and its concrete grammar is as follows:
1. the test tube kind is cultivated
Culture medium prescription is potato agar (PDA) substratum, i.e. potato 200g; Glucose 20g; Agar 17g; Distilled water 1000mL.
2. liquid fermentation and culture
Wherein the liquid fermentation medium prescription is: by weight percentage, contain glucose 2%-4%, NH
4Cl0.1%-0.2%, K
2HPO
40.1%-0.5%, MgSO
47H
2O 0.05%-0.1%, KCl 0.02%-0.2%, FeSO
40.002-0.04%, remainder is a distilled water, and pH is 6-7, and preferred pH is 6.5.
The test tube kind is inoculated in 250mL triangular flask (the every bottled 100mL) liquid nutrient medium, and under 25 ℃～28 ℃, shaking speed is with 150rmin
-1～200rmin
-1, the fermentation 240h.
The invention still further relates to a kind of mematocide, it comprises the fungal bacterial strain P.pinophilumsnf44-1 of the present invention of significant quantity itself or its metabolite as the effective active composition.
Fungal bacterial strain P.pinophilum snf44-1 of the present invention or its metabolite have nematocidal active material, can be used for the biological control of plant nematode, particularly root knot nematode (Meloidogyne spp.).
Embodiment:
In order further to illustrate in more detail rather than, to have provided the following example in order to limit the present invention.
At first P.pinophilum snf44-1 bacterial strain is carried out fermentation culture, then the meta-bolites after the fermentation culture is carried out the eelworm-killing activity test, determine its effect nematode.
Embodiment 1: the cultivation of bacterial strain P.pinophilum snf44-1
The mycelium of P.pinophilum snf44-1 is inoculated on the test tube nutrient agar, and culture medium prescription is the PDA substratum, i.e. potato 200g; Glucose 20g; Agar 17g; Distilled water 1000mL.Cultivate 10d for 25 ℃, obtain the test tube kind.
The test tube kind is inoculated in 250mL triangular flask (the every bottled 100mL) liquid nutrient medium, culture medium prescription is (weight percent): glucose (2.83%), NH again
4Cl (0.15%), K
2HPO
4(0.12%), MgSO
47H
2O (0.08%), KCl (0.02%), FeSO
4(0.02%), remainder is a distilled water, pH6.5.Under 25 ℃～28 ℃, shaking speed is with 150rmin
-1～200rmin
-1, the fermentation 240h.Fermented liquid is made into different multiples carries out the test of nematicide drug effect.
Embodiment 2: the cultivation of bacterial strain P.pinophilum snf44-1
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is:
The liquid culture based formulas is: glucose (2%), NH
4Cl (0.1%), K
2HPO
4(0.08%), MgSO
47H
2O (0.04%), KCl (0.02%), FeSO
4(0.002%), pH7.
Embodiment 3: the cultivation of bacterial strain P.pinophilum snf44-1
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is:
The liquid culture based formulas is: glucose (3%), NH
4Cl (0.15%), K
2HPO
4(2%), MgSO
47H
2O (0.1%), KCl (0.2%), FeSO
4(0.004%), pH6.
Embodiment 4: the cultivation of bacterial strain P.pinophilum snf44-1
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is:
The liquid culture based formulas is: glucose (4%), NH
4Cl (2%), K
2HPO
4(1.5%), MgSO
47H
2O (0.08%), KCl (0.15%), FeSO
4(0.01%), pH6.5.
Embodiment 5: the eelworm-killing activity of bacterial strain P.pinophilum snf44-1:
Fermented liquid is concentrated on the basis of 10 times of liquid dilution successively for-5 * (concentrating 5 times of liquid), 1 *, 5 *, 10 *, handle northern root knot nematode 2 instar larvaes respectively.
1. preparation is tested and is used preparation
By the fungal bacterial strain P.pinophilum snf44-1 of aforementioned liquids fermentation culture method preparation, carry out eelworm-killing activity with the fermented liquid of different multiples and test.
2. nematode is used in the preparation test
(1) northern root knot nematode egg capsule of preparation and 2 instar larvaes
The northern root knot nematode of breeding on the potted plant susceptible tomato variety L402 in the greenhouse.The tomato seeds sowing in the bed of growing seedlings, is treated tomato
Seedling length is to about the 20cm, and it is potted plant showing sturdy time shift slightly.Inoculate northern root knot nematode, after about one month, root can produce a large amount of egg capsules.Root is taken out and flush away earth, place culture dish with tweezers picking egg capsule, the clorox with 0.5% is handled 3min, uses aseptic water washing again three times, places 25 ℃ of incubators to hatch.Will hatch a lot of second instar larvaes about 3 days, be drawn onto in the triangular flask of sterilization, place the refrigerator of 4C standby.
3. test method:
3.1 P.pinophilum snf44-1 metabolite is to the effect of nematode: the test that adds different concns in the crown capsule of sterilization is with fermented liquid 100 μ L respectively, to add 100 μ L sterilized waters in contrast, then respectively to handle and contrast in respectively add 100 μ L nematode suspension (contain approximately nematode 48.11. ± 10.66), put into 25 ℃ of incubators, write down nemic death rate behind the 48h, the calculation correction mortality ratio is the nematicide drug effect.Every processing repeats 3 times.
3.2 the effect that P.pinophilum snf44-1 metabolite is hatched northern root knot nematode egg capsule:
At each consistent egg capsule of growth of in the crown capsule of high-temperature sterilization, putting into 1 surface sterilization, add the test fermented liquid of 100 μ L different concns respectively, to add 100 μ L sterilized waters in contrast.Test is carried out under 25 ℃, each is handled culture dish obturage with sealing film, pollutes and liquid evaporation to reduce.Microscopy is respectively handled egg capsule hatching situation behind 2d and 4d respectively.Calculate the relative inhibition of egg capsule hatching nematode number and egg capsule hatching.Every processing repeats 3 times.
The operation of test 3.1 and 3.2 is all carried out under gnotobasis, avoids the influence to test-results of environment and human factor as far as possible.
4. test-results
(1) P.pinophilum snf44-1 metabolite is to the nematicide effect of northern root knot nematode 2 instar larvaes
The different extension rates of table 1 Penicillium notatum 44-1 fermented liquid are to the influence of northern root knot nematode 2 instar larvaes
Annotate: " 5 * ": concentrate five times of liquid; CK1:10 μ g/ml Avrmectin is handled; CK2: sterilized water is handled;
The result shows, significant difference between the fermented liquid of each weaker concn and the sterilized water contrast, and about 90%, difference is not remarkable between the two to the relative lethality rate of nematode for 1 times of liquid and 10 μ g/ml Avrmectins.
(2) P.pinophilum snf44-1 metabolite is to the effect of northern root knot nematode egg capsule hatching
The different extension rates of table 2 Penicillium notatum 44-1 fermented liquid are to the influence of northern root knot nematode egg capsule hatching
The result shows, between the egg capsule of the fermented liquid of each weaker concn and sterilized water control treatment, the nematode hatching is influenced significant difference, and particularly 1 times of liquid almost can not be hatched, and is suitable with the inhibiting rate of 10 μ g/ml Avrmectins, and its relative inhibition is about 95%.
Show by above two kinds of restraining effect test-results nematode vigor and egg capsule hatching, P.pinophilum snf44-1 is the fungi that a strain has using value, particularly, demonstrate its good application development prospect to nematocidal effect of northern root knot nematode and the restraining effect that its egg capsule is hatched.
Embodiment 6: the field eelworm-killing activity of bacterial strain P.pinophilum snf44-1
The fermented liquid that adopts liquid fermenting to obtain concentrates 20 times of preservations after 6 months through vacuum decompression, and at field control tomato root-knot eelworm (Meloidogyne hapla), effect is as follows:
For trying material and method:
Tomato: kind is L402
Nematode: northern root knot nematode Meloidogyne hapla
5000 ovum of the 5th day inoculation root knot nematode after the field planting of every strain tomato, root irrigation was carried out with fermented liquid, every strain perfusion liquid 200ml on the 7th day in the inoculation back.Normal field management, 8 all " Invest, Then Investigate " root knot indexes.Fresh water pouring is contrast CK.
Result such as following table:
The result shows, this fermented liquid under the condition of dosing without any auxiliary agent, 200 * can reach 60%-70% to the effect of 500 * diluent control root knot nematode, this weaker concn has reached the concentration requirement that industrialization development utilizes.
Claims (8)
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| CN101182468B (en) * | 2007-11-08 | 2010-06-02 | 沈阳农业大学 | Trichoderma fungus with nematicidal activity and its preparation method and application |
| CN105368715B (en) * | 2015-09-09 | 2019-04-23 | 中国热带农业科学院热带生物技术研究所 | A cellulose-degrading Penicillium pinophilum and its application in straw fermentation |
| CN106085911A (en) * | 2016-05-19 | 2016-11-09 | 中国农业科学院植物保护研究所 | One strain has penicillium oxalicum NBC008, its preparation method and the application of eelworm-killing activity |
| CN105861336A (en) * | 2016-05-19 | 2016-08-17 | 中国农业科学院植物保护研究所 | Nematicidal-activity P. oxalicum NBC012 and preparation method and application thereof |
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Non-Patent Citations (4)
| Title |
|---|
| 大豆胞囊线虫胞囊内寄生真菌研究. 范圣长，段玉玺，陈立杰.大豆科学，第23卷第1期. 2004 |
| 大豆胞囊线虫胞囊内寄生真菌研究. 范圣长，段玉玺，陈立杰.大豆科学，第23卷第1期. 2004 * |
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