CN100542397C - Utilize the termite-proof method of the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte - Google Patents
Utilize the termite-proof method of the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte Download PDFInfo
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- CN100542397C CN100542397C CNB2004100440830A CN200410044083A CN100542397C CN 100542397 C CN100542397 C CN 100542397C CN B2004100440830 A CNB2004100440830 A CN B2004100440830A CN 200410044083 A CN200410044083 A CN 200410044083A CN 100542397 C CN100542397 C CN 100542397C
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Abstract
Utilize the termite-proof method of the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte, it relates to a kind of termite-proof method of utilizing the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte.The present invention is performed such: separate in the termite body and have the fungal component of lignin and/or cellulose-decomposing ability as indicator bacteria; From the termite-proof trees, separate the trees endophyte, it is fermented, obtain trees endophyte zymotic fluid; Utilize trees endophyte zymotic fluid to adopt the Oxford agar diffusion method that the symbiosis of termite bacterium is made bacteriostatic test; There is inhibiting trees endophyte zymotic fluid to soak filter paper with fungal component, termite is carried out feeding experiment termite.Through statistical results show: the experimental group termite is got the amount of food filter paper and obviously lacks than control group, and the lethality of experimental group termite is obviously more than control group, and difference is extremely remarkable; A little less than the termite activity of experimental group in view of the active situation of termite is than control group, thus contain in the explanation zymotic fluid can termite-proof compound.
Description
Technical field:
The present invention relates to a kind of separating method of endophyte, be specifically related to a kind of termite-proof method of utilizing the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte.
Background technology:
The existing medicament of killing termite is chemical agent, and there is very big toxic and side effect in the former capital, causes serious environmental to pollute.
Summary of the invention:
The purpose of this invention is to provide the termite-proof method that a kind of zymotic fluid that utilizes the trees endophyte suppresses the symbiosis of termite bacterium, this method by micro-organisms nontoxic or low toxicity to people and animals can termite-proof compound.Thereby the present invention utilizes the zymotic fluid of trees endophyte to suppress the symbiosis of termite bacterium to realize the termite-proof method like this: (one) separates in the termite body and has the fungal component of lignin and/or cellulose-decomposing ability as indicator bacteria; (2) from the termite-proof trees, separate the trees endophyte, it is fermented, obtain trees endophyte zymotic fluid; (3) utilize trees endophyte zymotic fluid to adopt the Oxford agar diffusion method that the symbiosis of termite bacterium is made bacteriostatic test; (4) use fungal component to have inhibiting trees endophyte zymotic fluid to soak filter paper, termite is carried out feeding experiment termite; Wherein said termite-proof trees are Western Red Cedar and port-orford-cedar; The method of separating the trees endophyte in the step 2 is achieved in that with cutter the branch of termite-proof trees is cut into 1.0～1.5 centimetres segment, the wood particle of handling is inserted water agar soak in the juice medium, grows bacterium on 3～5d isolation medium; Respectively bacterium is inserted corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, dextrose peptone medium with three zoning collimation methods, isolate single bacterium colony, respectively the bacterium that is separated to is inserted in corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, the glucose peptone inclined-plane then, obtain trees endophyte bacterial classification, Preliminary Identification is divided into bacterium, fungi, actinomycetes and saccharomycete again; The endophyte of trees described in step 2 zymotic fluid is preparation like this: the trees endophyte bacterial classification of separation and purification gained is inoculated into respectively in corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, the glucose peptone liquid nutrient medium, bacterial culture 2～3d under 28 ℃, the condition of 100r/min, fungi, actinomycetes and saccharomycete are cultivated 5～6d; Culture fluid with 3500r/min centrifugation 10～15min, is got supernatant, the aseptic conical flask of impouring, under aseptic condition, under 40 ℃ of waters bath with thermostatic control, 150r/min condition, be concentrated into 1/10th of its volume with Rotary Evaporators, put into 4 ℃ of refrigerators and preserve, as standby zymotic fluid.The present invention is from separating its endophyte among the trees of termite-proof, obtain its metabolite by fermenting and producing, utilize zymotic fluid to suppress to have in the termite body fungal component of strong lignin, cellulose-decomposing ability, and then do the feeding experiment of termite, the zymotic fluid that result's proof can suppress the trees endophyte of symbiosis of termite bacterium also has good inhibitory effect to termite.The present invention adopts the fungal component that strong lignin, cellulose-decomposing ability are arranged that separates in the termite body as indicator bacteria, make bacteriostatic test by the Oxford agar diffusion method earlier and check the tunning of from Western Red Cedar and Port-orford-cedar, isolating endophyte whether the symbiosis of termite bacterium is had inhibitory action, and then use fungal component to have inhibiting tunning immersion filter paper that termite is carried out feeding experiment termite.Through the test of the feeding with medicine of termite, and through statistical results show: the experimental group termite is got the amount of food filter paper and obviously lacks than control group, and the lethality of experimental group termite is obviously more than control group, and difference is extremely remarkable; A little less than the termite activity of experimental group in view of the active situation of termite is than control group, thus contain in the explanation zymotic fluid can termite-proof compound.Fungal component to termite has better inhibiting tunning that termite is also had good killing effect, and the two has linear trend, and this just provides a kind of succinct and feasible new approach for the screening and the fast detecting of killing the termite biological agent.
Embodiment:
Embodiment one: thus present embodiment is to utilize the metabolite of trees endophyte to suppress the symbiosis of termite bacterium like this to realize the termite-proof method: and (one) separates in the termite body and has the fungal component of lignin and/or cellulose-decomposing ability as indicator bacteria; (2) from the termite-proof trees, separate the trees endophyte, it is fermented, obtain trees endophyte metabolite; (3) utilize trees endophyte metabolite to adopt the Oxford agar diffusion method that the symbiosis of termite bacterium is made bacteriostatic test; (4) use fungal component to have inhibiting trees endophyte metabolite to soak filter paper, termite is carried out feeding experiment termite.Described termite-proof trees are Western Red Cedar and port-orford-cedar.
Embodiment two: thus present embodiment is to utilize the metabolite of trees endophyte to suppress symbiosis of termite bacterium realization termite-proof method like this:
One, choosing of material:
1, Western Red Cedar (on September 10th, 2003 took a sample in U.S. Louis An Na state)
2, port-orford-cedar (take a sample in Ore. in September, 2003)
3, Odontotermes formosanus (Odontotermes formosanus) (sampling) in the Chinese Luo Jiashan of Wuhan City
4, U.S. termite (sampling) in U.S. Louis An Na state university
Two, culture medium preparation:
(1) indicator bacteria medium:
1, cellulose medium:
1.1 bacteria cellulose medium:
CMC-Na (sodium carboxymethylcellulose is called for short CMC) 10g, NH
4H
2PO
40.5g, K
2HPO
42.0g, MgSO
42.0g, MnSO
4H
2O 2.5mg, FeSO
47H
2O 7.5mg, NaCl 0.3g, peptone 10g, agar 20g, water 1000ml.
1.2 fungin medium:
CMC-Na 10g, NaNO
32g, K
2HPO
41g, KCl 0.5g, MgSO
40.5g, FeSO
40.01g, peptone 4g, urea 1g, agar 20g, water 1000ml.
1.3 actinomycetes cellulose medium:
CMC-Na 10g, KNO
31g, NaCl 0.4g, K
2HPO
40.5g, MgSO
40.5g, FeSO
47H
2O 0.01g, agar 20g, water 1000ml.
1.4 membranin medium:
CMC-Na 5g, yeast extract 1g, KCl 1.8g, sodium acetate 8.2g, agar 20g, water 1000ml.
2, lignin medium:
2.1 the preparation of lignin:
Take by weighing the 2g sawdust, wrap, put into Soxhlet extractor, add benzene alcohol mixed liquor (benzene: ethanol=2:1), put extracting 6 hours (control extract cycle-index per hour 4 times) in the boiling water bath with qualitative filter paper; After extracting is intact, sample is taken out air-dry, change in the 250ml tool plug conical flask of the concentrated sulfuric acid that 12～15 ℃ of precooling contain 15ml72%, the jam-pack bottle stopper sways 1min, makes sample all be immersed in the acid solution, constant temperature is incubated 2.5 hours down at 18～20 ℃ then, and often shakes conical flask; Change over to after the insulation in the 1000ml conical flask, adding 345ml distilled water diluting to acid concentration is 3%, keeps that its concentration is constant boiled 4 hours, leaves standstill, and filters, and filter residue is dried constant weight, is lignin.
2.2 bacterium lignin medium:
Lignin 10g, NH
4H
2PO
40.5g, K
2HPO
42.0g, MgSO
42.0g, MnSO
4H
2O2.5mg, FeSO
47H
2O 7.5mg, NaCl 0.3g, peptone 10g, agar 20g, water 1000ml.
2.3 fungi lignin medium:
Lignin 10g, NaNO
32g, K
2HPO
41g, KCl 0.5g, MgSO
40.5g, FeSO
40.01g, peptone 4g, urea 1g, agar 20g, water 1000ml.
2.4 actinomycetes lignin medium:
Lignin 5g, KNO
31g, NaCl 0.4g, K
2HPO
40.5g, MgSO
40.5g, FeSO
47H
2O 0.01g, agar 20g, water 1000ml.
2.5 saccharomycete lignin medium:
Lignin 5g, yeast extract 1g, KCl 1.8g, sodium acetate 8.2g, agar 20g, water 1000ml.
(2) isolation medium of trees endophyte:
1, water agar soaks the juice medium:
Scalpel with the bacterium of going out is cut into small pieces 5g red cypress material, adds 100ml water boil 30min, gets the 20ml trees and soaks juice, adds 15～20g agar, adds water to 1000ml, and behind 121 ℃ of high pressure steam sterilization 30min, branch installs in the culture dish standby.
2, Cha Shi medium:
NaNO
32g, K
2HPO
41g, KCl 0.5g, MgSO
40.5g, FeSO
40.01g, agar 20g, water 1000ml.
3, PDA medium:
Potato 200g, sucrose 20g, water 1000ml, agar 20g.
4, S-7 medium:
Glucose 1g, fructose 3g, sucrose 6g, sodium acetate 1g, peptone 1g, Cobastab
11mg, biotin 1mg, Cobastab
61mg, calcium pantothenate 1mg, MgSO
43.6mg, Ca (NO
3)
26.5mg, Cu (NO
3)
21mg, ZnSO
42.5mg, MnCl
25mg, FeCl
3The KH of 2mg, phenyl alanine 5mg, Sodium Benzoate 100mg, 1mol/l
2PO
4Buffer solution 1ml, water 1000ml, pH nature.
(4) the used medium of bacteriostatic test:
Double-deck medium: the plain agar of impouring 10ml in plate, spend the night in 37 ℃ of incubators and examine bacterium, aseptic Oxford cup is stood on the plain agar plate, impouring 15ml is mixed with the semisolid culturemedium of bacteria suspension, after treating that semisolid culturemedium solidifies, extract the Oxford cup with aseptic nipper.
Three, method:
1, disjunctive symbiosis bacterium from termite:
A, the termite that to collect from ant nest cuts head and chest, belly places sterilized culture dish, with 0.1% mercuric chloride sterilization, 1～2min or with ethanol disinfection 3～4min of 75%, behind aseptic water washing 3 times, sterile working changes 20 termite bellies in the sterile mortar over to, grind to form homogenate, a series of diluted for use is made in homogenate, meanwhile, whether in counting the wiping of rolling on the culture medium flat plate, this flat board is cultivated with the counting flat board with sterile termite belly, thorough with the surface sterilization of check termite, if it is not thorough to sterilize, need operation again;
B, get the homogenate of 0.2ml termite with liquid-transfering gun, add respectively in bacterium, fungi, saccharomycete, actinomycetic cellulose medium and the lignin culture medium flat plate, with being coated with the rod coating evenly, bacterium is cultivated down at 37 ℃, and other bacterium is cultivated down at 28 ℃;
Above-mentioned cellulose medium and lignin medium grow bacterium behind c, the 3～7d, isolate single bacterium colony with three zoning collimation methods, change the inclined-plane over to after the power of evaluation enzymatic productivity and preserve, and select the higher relatively bacterial strain of enzymatic productivity as standby indicator bacteria;
2, from WesternRed Cedar and port-orford-cedar, separate the trees endophyte:
A, with cutter branch is cut into 1.0～1.5 centimetres segment;
B, the mercuric chloride with 0.1% are handled trees segment 1～4min, outwell mercuric chloride, clean with distilled water and remove residual mercuric chloride 3 times, perhaps use Ethanol Treatment trees segment 3～8min of 75%, clean 3 times with distilled water again;
C, the wood particle handled is inserted water agar soak in the juice medium, grow bacterium on 3～5d isolation medium;
D, bacterium is inserted respectively in corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, the glucose peptone solid culture medium with three zoning collimation methods, isolate single bacterium colony, respectively the bacterium that is separated to is inserted in corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, the glucose peptone inclined-plane then, obtain trees endophyte bacterial classification, Preliminary Identification is divided into bacterium, fungi, actinomycetes, saccharomycete four big classes.
3, the Oxford agar diffusion method is made bacteriostatic test:
A, preparation of fermentation liquid:
The trees endophyte bacterial classification of separation and purification gained is inoculated into respectively in corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, the glucose peptone liquid nutrient medium, bacterial culture 2～3d under 28 ℃, the condition of 100r/min, other bacterium are cultivated 5～6d; Culture fluid with 3500r/min centrifugation 10～15min, is got supernatant, the aseptic conical flask of impouring, under aseptic condition, under 40 ℃ of waters bath with thermostatic control, 150r/min condition, be concentrated into 1/10th of its volume with Rotary Evaporators, put into 4 ℃ of refrigerators and preserve, as standby zymotic fluid.
B, make bacteriostatic experiment with the Oxford agar diffusion method:
, get the described zymotic fluid of a step and do bacteriostatic experiment as indicator bacteria with the bacterium that the lignocellulose capacity of decomposition is arranged that in termite body, is separated to as bateriostatics:
Prepare plain agar plate: 1000ml water adds the agar of 20g, and sterilization is dull and stereotyped, and each dull and stereotyped plain agar of 10～15ml stands in the Oxford cup on the plain agar plate.
The preparation of bacteria suspension: get the 5ml sterile water, be added on the inclined-plane of indicator bacteria (fungal component with termite is an indicator bacteria), with oese scraping gently, inhale with aseptic pipette and beat mixing on the inclined-plane, it is standby to do serial dilution.
The preparation of semisolid culturemedium: get Cha Shi, beef extract-peptone, Ma Dingshi, Maxwell, PDA, S-7, glucose peptone liquid nutrient medium respectively, add 0.75% agar.
Get the 20ml semisolid culturemedium and add in the fine taper bottle, to be cooledly add 2ml during to 50 ℃ and dilute good bacteria suspension, mixing is poured on the plain agar plate that is inserted with the Oxford cup, obtains double-deck medium.Treat to extract the Oxford cup after the culture medium solidifying, in aperture, add the zymotic fluid that 100 μ l concentrate with liquid-transfering gun, in 4 ℃ of refrigerators, place and spend the night, make zymotic fluid fully be diffused in the medium, bacterium is cultivated 72h at 37 ℃, other bacterium down at 28 ℃ then, observes and measures antibacterial circle diameter with slide measure.
4. the termite experiment of feeding:
From ant nest, get 30 termites (25 worker ants, 5 soldier ants) and place under about 25 ℃ and the container of preserving moisture, filter paper is weighed the back with the zymotic fluid immersion, dry in the shade, weigh, give the termite feeding.Termite is divided into control group and experimental group, the filter paper that the control group feeding is soaked in water, the death toll of checking experiment every day group and control group termite is observed active situation.Meter is death toll down, and removes dead termite.Filter paper taken out the weighing that dries in the shade in the 8th day.
Through the test of the feeding with medicine of termite, and to the result through statistical results show: the experimental group termite is got the amount of food filter paper and obviously lacks than control group, and the lethality of experimental group termite is obviously more than control group, and difference is extremely remarkable; A little less than the termite activity of experimental group in view of the active situation of termite is than control group, thus contain in the explanation zymotic fluid can termite-proof compound.
Claims (5)
1, utilize the zymotic fluid of trees endophyte to suppress the termite-proof method of symbiosis of termite bacterium, it is characterized in that it carries out according to following step: (one) separates in the termite body and has the fungal component of lignin and/or cellulose-decomposing ability as indicator bacteria; (2) from the termite-proof trees, separate the trees endophyte, it is fermented, obtain trees endophyte zymotic fluid; (3) utilize trees endophyte zymotic fluid to adopt the Oxford agar diffusion method that the symbiosis of termite bacterium is made bacteriostatic test; (4) use fungal component to have inhibiting trees endophyte zymotic fluid to soak filter paper, termite is carried out feeding experiment termite; Wherein said termite-proof trees are Western Red Cedar and port-orford-cedar; The method of separating the trees endophyte in the step (two) is achieved in that with cutter the branch of termite-proof trees is cut into 1.0～1.5 centimetres segment, the wood particle of handling is inserted water agar soak in the juice medium, grows bacterium on 3～5d isolation medium; Respectively bacterium is inserted corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, dextrose peptone medium with three zoning collimation methods, isolate single bacterium colony, respectively the bacterium that is separated to is inserted in corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, the glucose peptone inclined-plane then, obtain trees endophyte bacterial classification, Preliminary Identification is divided into bacterium, fungi and actinomycetes again; Trees endophyte zymotic fluid described in the step (two) is preparation like this: the trees endophyte bacterial classification of separation and purification gained is inoculated into respectively in corresponding beef extract-peptone, Cha Shi, Ma Dingshi, Gao Shi, PDA, S-7, the glucose peptone liquid nutrient medium, bacterial culture 2～3d under 28 ℃, the condition of 100r/min, fungi and actinomycetes are cultivated 5～6d; Culture fluid with 3500r/min centrifugation 10～15min, is got supernatant, the aseptic conical flask of impouring, under aseptic condition, under 40 ℃ of waters bath with thermostatic control, 150r/min condition, be concentrated into 1/10th of its volume with Rotary Evaporators, put into 4 ℃ of refrigerators and preserve, as standby zymotic fluid.
2, the termite-proof method of utilizing the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte according to claim 1 is characterized in that the fungi described in the step (two) is a saccharomycete.
3, the termite-proof method of utilizing the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte according to claim 1, it is characterized in that adopting described in the step (three) the Oxford agar diffusion method to be performed such: to get the 20ml semisolid culturemedium and add in the fine taper bottle as bacteriostatic test, the 2ml indicator bacteria bacteria suspension that adds during to 50 ℃ to be cooled, mixing is poured on the plain agar plate that is inserted with the Oxford cup; Treat to extract the Oxford cup after the culture medium solidifying, add 100 μ l trees endophyte zymotic fluids with liquid-transfering gun in aperture, place in 4 ℃ of refrigerators and spend the night, bacterium is at 37 ℃ then, and fungi and actinomycetes are cultivated 72h down at 28 ℃; Semisolid culturemedium is preparation like this: get Cha Shi, beef extract-peptone, Ma Dingshi, Maxwell, PDA, S-7, glucose peptone liquid nutrient medium respectively, add 0.75% agar; Described plain agar plate is to prepare like this: 1000ml water adds the agar of 20g, and sterilization is dull and stereotyped, and each dull and stereotyped plain agar of 10～15ml stands in the Oxford cup on the plain agar plate.
4, the termite-proof method of utilizing the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte according to claim 3, it is characterized in that described indicator bacteria bacteria suspension is preparation like this: get the 5ml sterile water, be added on the inclined-plane of indicator bacteria, on the inclined-plane,, inhale with aseptic pipette again and beat mixing with oese scraping gently.
5, the termite-proof method of utilizing the zymotic fluid inhibition symbiosis of termite bacterium of trees endophyte according to claim 3 is characterized in that the fungi described in the step (three) is a saccharomycete.
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| CN105002220A (en) * | 2015-07-27 | 2015-10-28 | 黑龙江大学 | Preparation method of endophyte mixed biological fermentation product for preventing and controlling termites |
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| CN102321555B (en) * | 2011-09-05 | 2013-05-01 | 黑龙江大学 | Endophyte producing termite-killing/resistant compound |
| CN102392071B (en) * | 2011-10-19 | 2014-02-12 | 黑龙江大学 | Method for inhibiting azotobacter in intestinal canal of termite by utilizing metabolic products of tree endophyte |
| CN102363758A (en) * | 2011-10-19 | 2012-02-29 | 黑龙江大学 | Azotobacteria separated from termite intestines |
| CN111357770B (en) * | 2020-05-12 | 2020-08-28 | 广东省生物资源应用研究所 | Application of serratia marcescens in preparation of termite antifeedant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105002220A (en) * | 2015-07-27 | 2015-10-28 | 黑龙江大学 | Preparation method of endophyte mixed biological fermentation product for preventing and controlling termites |
| CN105002220B (en) * | 2015-07-27 | 2018-02-13 | 黑龙江大学 | For the preparation method for the endophyte mixed biologic tunning for preventing and treating termite |
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