CN101724569B - Method for inducing nematode-trapping fungi to synchronously produce capturing organs - Google Patents
Method for inducing nematode-trapping fungi to synchronously produce capturing organs Download PDFInfo
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- CN101724569B CN101724569B CN2009102183129A CN200910218312A CN101724569B CN 101724569 B CN101724569 B CN 101724569B CN 2009102183129 A CN2009102183129 A CN 2009102183129A CN 200910218312 A CN200910218312 A CN 200910218312A CN 101724569 B CN101724569 B CN 101724569B
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Abstract
The invention relates to a method for inducing nematode-trapping fungi to synchronously produce trapping organs, which belongs to the field of applied microbiology. The method comprises the steps of culture of nematodes, preparation of a nematode extract, culture of nematode-trapping fungi and induction of trapping organs. The method can utilize the nematode extract to induce the nematode-trapping fungi to synchronously produce a large number of trapping organs (160 trapping organs/mg mycelium) by culturing the nematodes (Caenorhabditis elegans) and preparing the nematode extract by utilizing ultrasonic waves for crushing and centrifugalizing. The method has the advantages of simple and easy operation and good repeatability, and can obtain a large number of synchronous three-dimensional fungal nets, thereby providing good experimental materials for further research of the molecular mechanism of forming the trapping organs of the nematode-trapping fungi.
Description
Technical field:
The present invention relates to a kind of method of inducing nematode-trapping fungi to synchronously produce trapping organs, belong to applied microbiology field.
Background technology:
Plant nematode is one of important disease of plant, and the whole world is annual because the caused farm crop financial loss of plant nematode reaches 78,000,000,000 dollars (Barker, 1998).In China, nearly all cash crop such as eelworm harm tobacco, flowers, vegetables, cotton, soybean, peanut, wheat, paddy rice, forest, Chinese medicinal materials become one of limiting factor important in the agriculture production.At present to the control of nematodiasiss still based on chemical prevention, but because chemical pesticide is to the influence and the drug-fast increase of parasitic nematode of ecotope, its application progressively is restricted, therefore concern (Siddiqui and Mahmood, 1996 of adopting nemic natural enemy to carry out biological control to be subjected to the researchist day by day; Liu Xingzhong etc., 2004).Research to nematode fungi natural enemy has had the history in more than 160 year, and wherein nematode-trapping fungi (Nematode-trapping fungi) is the important fungal organism factor of a class of occurring in nature control nematode population quantity.Nematode-trapping fungi is divided into according to morphological feature that the nodal plexus spore belongs to that (Arthrobotrys), genus monacrosporium belong to (Monacrosporium) and every referring to that spore belongs to (Dactylella) (Subramanian, 1963), the trapping organs that they utilize the vegetative hyphae specialization to form, finish seizure and infection processs (Zhang Keqin etc., 2006) as shrunk ring, non-shrunk ring, viscosity mycelia, viscosity branch, viscosity net etc. to plant nematode.The morphological structure of the trapping organs of nematode-trapping fungi is very stable, and is not only significant in the function aspects of nematode-trapping, and is one of important evidence of evaluation and classification.At present, nematode-trapping fungi has been widely used in the biological control (Liu Xingzhong etc., 2004) of plant nematode as a kind of effective biological control resource.Yet people are also very limited to the understanding of the infection mechanism of nematode-trapping fungi and molecular background, limited the research and development and the application of efficient nematode biological prevention and control agent.
The trapping organs of nematode-trapping fungi is that it infects one of prerequisite of plant nematode, trapping organs can spontaneously produce on nutrition barren substratum, also can be by environmental factor (amino acid and polypeptide etc.) induce generation (Friman et al., 1985).At present the induction method of the nematode-trapping fungi trapping organs of the formal report of institute mainly comprises: starvation method, on the nutrition barren substratum as maize powder medium on the cultivation generation; Revulsion adds inductive substances such as specific amino acid or polypeptide and induces trapping organs to produce in solid or liquid nutrient medium; Cell method (digging the piece method): in solid medium (in dull and stereotyped), dig out one (0.5x 1cm), and add fresh nematode therein, induce trapping organs to produce.More than the induction method of several trapping organs the trapping organs limited amount, the trapping organs that exist to produce produce asynchronous and shortcoming such as controllability is not strong.
By literature search, find open report with content same document of the present invention.
Summary of the invention:
The objective of the invention is to overcome the prior art deficiency, a kind of method of rapid induction nematode-trapping fungi to synchronously produce trapping organs is provided, obtain a large amount of synchronous trapping organs rapidly, the molecular mechanism that forms for further research nematode-trapping fungi trapping organs provides good experiment material.
The Caenorhabditis elegans that the present invention relates to (Caenorhabditis elegans) is used nematode for the laboratory normal experiment, can buy from nematode preservation both domestic and external mechanism to obtain.
The present invention improves on the ordinary method basis and forms.The step that the present invention realizes is as follows:
1. the cultivation of substratum and nematode
1) oat medium: be used to cultivate nematode.In the 250mL triangular flask, add 10g rolled oats (supermarket purchase) adding 30mL deionized water rolled oats is soaked into, sterilized 20 minutes for 121 ℃.
2) cultivation of Caenorhabditis elegans: in the oat medium of sterilization, add the nematode suspension of 1mL, cultivated 6-7 days for 21-26 ℃.Cultured nematode preserved for 1 week at 4 ℃.
2. the preparation of nematode extract
1) oat medium that will contain Caenorhabditis elegans with 3 layers of lens wiping paper is wrapped, and spends the night with Bei Shi funnel method and collects the nematode that oat medium is cultivated;
2) nematode of collecting is used M9 damping fluid (Sodium phosphate dibasic 6g; Potassium primary phosphate 3g; Sodium-chlor 5g; Sal epsom 0.25g, water 1000mL) clean, remove residual substratum;
3) centrifugal collection nematode, 4 ℃, 8,000g, centrifugal 20 minutes;
4) with the aseptic deionized water nematode that suspends, centrifugal collection nematode, 4 ℃, 8,000g, centrifugal 20 minutes; Repeat 3 times; With the aseptic deionized water nematode that suspends, 10g (weight in wet base) nematode adds the 30mL aseptic deionized water at last;
5) place on the mixture of ice and water nematode suspension ultrasonic: 50% intensity, 6s ON, 3s OFF, 10 minutes; Place after 30 minutes, ultrasonic once more, 6s ON, 3s OFF, 5 minutes;
6) 12,000g, 30 minutes, centrifugal collection supernatant;
7) with the membrane filtration supernatant liquor of 0.22 μ m, the supernatant liquor after the filtration is the nematode extract, be stored in after the packing-80 ℃ stand-by.
3. the cultivation of nematode-trapping fungi (the few spore nodal plexus of the type strain spore A.oligospora with nematode-trapping fungi is an example)
1) substratum: the composition and the compound method of PDA substratum and CMA substratum are as follows:
CMA substratum: take by weighing the 20g Semen Maydis powder, add the 1000mL deionized water, boiled (100 ℃) 20 minutes, collect liquid, add 20g agar, sterilized 20 minutes for 121 ℃ with 6 layers of filtered through gauze.
The PDA substratum: peeling potato 200g, stripping and slicing, to boil 30 minutes, 6 layers of filtered through gauze are collected supernatant liquors, add glucose 20g, add water and replenish volume to 1000mL, sterilize 20 minutes for 121 ℃.
2) activation of few spore nodal plexus spore and spore are cultivated: the few spore nodal plexus spore of inoculation on the CMA solid medium, cultivated 10 days in 26 ℃ of constant incubators, to obtain a large amount of spores.With the few spore nodal plexus spore mycelia on the aseptic water washing CMA flat board; With pipettor the liquid on the flat board is transferred to the funnel inner filtration that aseptic lens wiping paper is housed, collects spore; Concentration with blood counting chamber counting and calculating spore.
3) liquid fermenting of few spore nodal plexus spore and the collection of fresh mycelia
Preparation PDA liquid nutrient medium is sub-packed in the triangular flask of 500mL 200mL substratum/bottle.Inoculating spores in the liquid medium within, 100 spore/mL liquid nutrient mediums, shaking table is cultivated: 26 ℃, 160rpm, 5-6 days.Collect mycelia with 6 layers of aseptic filtered through gauze, and wash mycelia 3-4 time with aseptic deionized water, remove the substratum composition, with 40mL aseptic deionized water suspension mycelia, the liquid that is used for three-dimensional bacterium net is induced.
4. the inducing of nematode-trapping fungi trapping organs (the few spore nodal plexus of the type strain spore A.oligospora with nematode-trapping fungi is an example)
After the fresh mycelia of few spore nodal plexus spore that collection is obtained suspends with aseptic deionized water, and adding nematode extract in the experimental group (three-dimensional bacterium net is induced group) (1: 10, v/v); Add the equivalent aseptic deionized water in the control group (vegetative hyphae group); Plate is statically placed in 26 ℃ of constant incubators hatches 24-48h, a large amount of trapping organs (three-dimensional bacterium net) produces.After inducing end, filter the mycelia of collecting control group and experimental group respectively, and wash mycelia 3-4 time with aseptic deionized water with 6 layers of aseptic gauze; With the moisture in the aseptic filter paper removal mycelia; Mycelia is with after the liquid nitrogen freeze-drying, be stored in-80 ℃ stand-by.
5. nematode-trapping fungi is observed (the few spore nodal plexus of the type strain spore A.oligospora with nematode-trapping fungi is an example) to the seizure of Caenorhabditis elegans
1) with 1% clorox to the Caenorhabditis elegans disinfection after, wash polypide 3-4 time repeatedly with aseptic deionized water.The three-dimensional bacterium net that the liquid attractant artificial delivery is given birth to is layered on the water agar plate, adds the Caenorhabditis elegans after 100/ware is sterilized.
2) (Olympus Japan) observes the form of the three-dimensional bacterium net that the liquid attractant artificial delivery gives birth to and to the seizure of nematode with opticmicroscope.
Compare with report in the past, the present invention has following feature: 1) the nematode extract effectively inducing nematode-trapping fungi produce a large amount of synchronous trapping organs (10
6Trapping organs/mg mycelia); 2) the nematode extract is a water-soluble substances, induce experiment to finish after, by filter, the aseptic deionized water process of washing can remove the nematode extract, to not influence of subsequent experimental; 3) nematode preparation method of extract and three-dimensional bacterium net induction method are simple, good reproducibility, and controllability is strong.4) the present invention provides good experiment material for the molecular mechanism of further research nematode-trapping fungi trapping organs formation.
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment one: the inducing of the few spore nodal plexus spore mycelia trapping organs of nematode-trapping fungi
Prepare the nematode extract according to method described above, few spore nodal plexus spore inoculation PDA liquid nutrient medium, 26 ℃, 160rpm cultivated 5 or 6 days, filtered and collected mycelia.And, remove the substratum composition with aseptic deionized water flushing mycelia 3 or 4 times, with an amount of aseptic aseptic deionized water suspension mycelia, what be used for that three-dimensional bacterium nets induces.
The fresh mycelia of few spore nodal plexus spore that collection is obtained places in the glass dish 40mL/ ware after suspending with aseptic deionized water: and adding nematode extract in the experimental group (three-dimensional bacterium net is induced group) (1: 10, v/v); Add the equivalent aseptic deionized water in the control group (vegetative hyphae group); Plate is statically placed in hatches 24 or 36 or 48h in 26 ℃ of constant incubators.The mycelia of few spore nodal plexus spore is induced and promptly can be observed three-dimensional bacterium net behind the 15h and produce, and 20 or during 24h, a large amount of trapping organs (three-dimensional bacterium net) produces.The nematode extract of different batches is less to the inducibility difference of trapping organs.
Embodiment two: the few spore nodal plexus of nematode-trapping fungi spore conidium forms inducing of trapping organs
Prepare the nematode extract according to method described above, few spore nodal plexus spore inoculation CMA solid medium was cultivated 10 days in 26 ℃ of constant incubators, to obtain a large amount of spores.Few spore nodal plexus spore mycelia with on the aseptic water washing CMA flat board is transferred to the funnel inner filtration that aseptic lens wiping paper is housed with pipettor with the liquid on the flat board, collects spore.
The few spore nodal plexus spore spore that collection is obtained places in the glass dish 20mL/ ware after suspending with aseptic deionized water: and adding nematode extract in the experimental group (three-dimensional bacterium net is induced group) (1: 10, v/v); Add the equivalent aseptic deionized water in the control group (vegetative hyphae group); Plate is statically placed in hatches 24 or 36 or 48h in 26 ℃ of constant incubators.The mycelia of few spore nodal plexus spore is induced and promptly can be observed three-dimensional bacterium net behind the 12-15h and produce, and 20 or during 24h, a large amount of trapping organs (three-dimensional bacterium net) produces.
Claims (1)
1. the method for an inducing nematode-trapping fungi to synchronously produce trapping organs comprises culture of nematodes, the preparation of nematode extract, and nematode-trapping fungi is cultivated and trapping organs is induced step, it is characterized in that present method is specific as follows:
(1). the cultivation of substratum and nematode
A. in the 250mL triangular flask, add 10g rolled oats, add the 30mL deionized water rolled oats is soaked into, sterilized 20 minutes for 121 ℃;
B. the cultivation of Caenorhabditis elegans: add the nematode suspension of 1mL in the oat medium of sterilization, cultivated 6-7 days for 21-26 ℃, cultured nematode preserved for 1 week at 4 ℃;
(2). the preparation of nematode extract
A. the oat medium that will contain Caenorhabditis elegans with 3 layers of lens wiping paper is wrapped, and spends the night with Bei Shi funnel method and collects the nematode that oat medium is cultivated;
B. the nematode of collecting is used the M9 buffer solution for cleaning, remove residual substratum;
C. centrifugal collection nematode, 4 ℃, 8,000g, centrifugal 20 minutes;
D. with the aseptic deionized water nematode that suspends, 4 ℃, 8,000g collected nematode in centrifugal 20 minutes; Repeat 3 times; With the aseptic deionized water nematode that suspends, the 10g nematode adds the 30mL aseptic deionized water at last;
E. place on the mixture of ice and water nematode suspension ultrasonic: 50% intensity, 6s ON, 3s OFF, 10 minutes; Place after 30 minutes, ultrasonic once more, 6s ON, 3s OFF, 5 minutes;
F.12,000g, centrifugal 30 minutes collection supernatant liquors;
G. use the membrane filtration supernatant liquor of 0.22 μ m, the supernatant liquor after the filtration is the nematode extract, be stored in after the packing-80 ℃ stand-by;
(3). the cultivation of few spore nodal plexus spore
A.CMA and PDA substratum
The CMA substratum: take by weighing the 20g Semen Maydis powder, add the 1000mL deionized water, 100 ℃ were boiled 20 minutes, collected liquid with 6 layers of filtered through gauze, added 20g agar, sterilized 20 minutes for 121 ℃;
The PDA substratum: peeling potato 200g, stripping and slicing, to boil 30 minutes, 6 layers of filtered through gauze are collected supernatant liquors, add glucose 20g, add water and replenish volume to 1000mL, sterilize 20 minutes for 121 ℃;
The activation of b. few spore nodal plexus spore and spore are cultivated
The few spore nodal plexus spore of inoculation was cultivated 10 days in 26 ℃ of constant incubators on the CMA solid medium, to obtain a large amount of spores, with the few spore nodal plexus spore mycelia on the aseptic water washing CMA flat board; With pipettor the liquid on the flat board is transferred to the funnel inner filtration that aseptic lens wiping paper is housed, collects spore; Concentration with blood counting chamber counting and calculating spore;
The liquid culture of c. few spore nodal plexus spore and the collection of fresh mycelia
Preparation PDA liquid nutrient medium is sub-packed in the triangular flask of 500mL 200mL substratum/bottle, inoculating spores in the liquid medium within, 100 spore/mL liquid nutrient mediums, shaking table is cultivated: 26 ℃, 160rpm, 5-6 days, collect mycelia with 6 layers of aseptic filtered through gauze, and wash mycelia 3-4 time, remove the substratum composition with aseptic deionized water, with 40mL aseptic deionized water suspension mycelia, the liquid that is used for three-dimensional bacterium net is induced;
(4). inducing of few spore nodal plexus spore trapping organs
After the fresh mycelia of few spore nodal plexus spore that collection is obtained suspends with aseptic deionized water, induce at three-dimensional bacterium net to add the nematode extract in the experimental group, add-on is 1: 10, v/v; In the vegetative hyphae control group, add isopyknic aseptic deionized water; Plate is statically placed in 26 ℃ of constant incubators hatches 24-48h, the three-dimensional bacterium net of a large amount of trapping organs produces; After inducing end, filter the mycelia of collecting control group and experimental group respectively, and wash mycelia 3-4 time with aseptic deionized water with 4 layers of aseptic gauze; With the moisture in the aseptic filter paper removal mycelia; Mycelia is with after the liquid nitrogen freeze-drying, be stored in-80 ℃ stand-by.
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| CN105112295A (en) * | 2015-04-07 | 2015-12-02 | 云南大学 | Fresh water fungus Arthrobotrys nonseptata and application thereof |
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