CN103202268A - Biological assay method of microbial volatile compound for influencing approach behavior of nematodes - Google Patents
Biological assay method of microbial volatile compound for influencing approach behavior of nematodes Download PDFInfo
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Abstract
The invention relates to a biological assay method of a microbial volatile compound for influencing approach behavior of nematodes. The biological assay method comprises the following steps of: taking a thin tube with an inner cavity volume of 10 to 300 cu.mm.; opening a small opening in the middle of the thin tube for dropping distilled water; placing a small block of microbial culture medium for generating active volatile organic compounds (VOCs) or a medium of sample solution (such as filter paper, culture medium and glass wool) containing the VOCs on one side of the thin tube, and marking as a processing end; placing an unvaccinated microbe or a small block of culture medium or medium without containing active volatile organic compounds (VOCs) on the other side, marking as a contrast end; then sealing both sides of the thin tube, dropping nematode liquid from the small opening in the middle of the thin tube by using a micro sample injector, and placing in a incubator for culturing; separating the nematodes after 10 hours, 24 hours and 36 hours respectively, carrying out microscopic examination after 24 hours of standing at the temperature of 28 DEG C, counting the amount of separated nematodes and carrying out data analysis. According to the biological assay method, micro or even nanogram level of compounds with luring activity can be conveniently found out.
Description
Technical field
The present invention relates to a kind of living survey method that influences the microorganism volatile compound (VOCs) of nematode approach behavior, belong to the biometric techniques field.
Background technology
The chemiecology of research pine wood nematode has great significance to pathogenesis and the process of understanding loose wilt disease.Wherein, it is essential to give birth to the survey means.To aspect the reaction of smell, (Electroantennogram EAG) is very important bioassay method (Zhao Xincheng etc., 2004) to EAG at fast detecting insect feeler.Gas-chromatography and tentaculum electric potential (GC-EAD) technology used in conjunction can be identified compound by the chemical sensor of insect feeler, detects active component wherein.Be subject to present means, what the research means of the pheromones of nematode can't be as insect, can not directly detect nematode to the neuroelectric pulse signal of active component with instrument.Therefore have only VOCs to be analyzed is analyzed and identify the structure of all components through GC-MS, buy all possible compounds or synthesize, again nematode is given birth to survey (Zhao etc., 2007; Kaplan etc., 2008; Niu etc., 2010).
The living survey means of pine wood nematode commonly used generally adopt the agar plate bioassay, are matrix with water agar plate or juggle etc., study dividing a word with a hyphen at the end of a line of pine wood nematode.Concrete method is as follows: in culture dish, pour 2% agar water liquid into, treat that agar solidifies after, in culture dish back side line, be initial point with dull and stereotyped center with marking pen, flat board is divided into four quadrants uniformly.First or third quadrant put into processing, be generally fungi fritter or sample solution etc.; Second or fourth quadrant put into contrast, be generally medium fritter or solvent etc.Splash into nematode liquid at dull and stereotyped center then, detect the situation of dividing a word with a hyphen at the end of a line of nematode after a period of time.Mamiya (Mamiya, 2006) the water agar plate material of measuring and monitoring the growth of standing timber of making a living has been studied the viable bacteria silk of timber saprophytic fungus to the effect of pine wood nematode.Lin Zhao Li (Zhao etc., 2007) is matrix with 0.8% agar plate, and the volatile matter of masson pine and Monochamus alternatus Hope is studied the sucking action of nematode.Long Ruimin (Long Ruimin, 2007) is matrix with fresh water sand and the water agar plate of sterilization respectively, has studied dividing a word with a hyphen at the end of a line of pine wood nematode.Tan Jiajin (Tan Jiajin etc., 2009) has studied the sucking action of pine tree volatile matter to pine wood nematode at water agar plate and juggle.
Above-mentioned agar plate bioassay has two big deficiencies, and at first, it is very big to the amount requirement of giving birth to the test sample product, to form the higher concentration gradient, is felt by pine wood nematode; Secondly, adopted nematode to carry out in the method for agar surface motion and since different nematodes in natural world habitat difference, the medium of survival is also different, the motion of agar surface and not exclusively suitable nematode, particularly most nematodes are to move in medium, rather than move at dielectric surface.Therefore the motion of these method affect nematodes, sensitivity and the accuracy of surveying given birth in influence then.
Ishikawa (Ishikawa M, Shuto Y, Watanabe H. β-Myrcene, a Potent Attractant Component of Pine Wood for the Pine Wood Nematode, Bursaphelenchus xylophilus (Pesticide Chemistry). Agricultural and biological chemistry, 1986,50 (7): 1863-1866.) set up a kind of living survey method, for detection of the volatile matter of test compound (5 mg) sucking action to pine wood nematode.The living survey device that he uses is T type glass tube, includes the agar of 9 ml, 1.5 %.The cavity body of this device is about π * r
2* h=3.14 * (5 mm)
2* 200 mm=15700 mm
3And individual siphon pipe is arranged at T type pipe top, is used for outwards bleeding.Use this method, it is very high to the concentration requirement of the sample in managing, the amount of the compound of surveying that namely needs very big (the milligram mg order of magnitude).Use this life survey method can't detect GC separate obtain pine wood nematode is had bioactive micro-volatile matter (compound of the nanogram ng order of magnitude).
Zhang Tianyuan ((Zhang Tianyuan, Niu Qiuhong, Ying Shusong, Liu Yan, Wang Min, Wang Zhenzhen, it is fine to cross rain. and bacterium Bacillus sp. NS-3 and Botrytis cinerea are drawn luring of pine wood nematode. Nanyang Normal College's journal, 2011 (06): 47-50.) adopt " water agar plate divide a word with a hyphen at the end of a line method " and " glass-tube method ", studied the ability that pine wood nematode antagonistic bacterium Bacillus sp. NS-3 and Botrytis cinerea are lured nematode.Its method is the glass-tube method of improvement, with reference to the method from Ishikawa.Adopt glass-tube and installed siphon pipe thereon additional, externally bleed.Cavity volume in the method in the glass-tube is π * r
2* h=3.14 * (45 mm)
2* 7.5 mm=47688.75 mm
3, this method is very big to the amount requirement of giving birth to the test sample product equally, and very high requirement is arranged.
In addition, present living survey method is often carried out in conjunction with gaschromatographic mass spectrometry (GC-MS) combined instrument, the VOCs that collects is carried out after gaschromatographic mass spectrometry (GC-MS) combined instrument measures, the all substances of identify are synthesized or buy compound known, then pine wood nematode is given birth to survey, final structure and content (Zhao etc., 2007 of determining active component; Kaplan etc., 2008; Niu etc., 2010).This method is not only complicated, and buys probably less than all compounds, the feasible compound that can not identify all biologically actives.
In sum, existing living survey method has tangible restriction to the concentration of active volatility metabolite, and it requires active volatility metabolite (VOCs) that certain amount is arranged, and to form the higher concentration gradient, is felt by pine wood nematode.The living survey method of siphonal glass-tube is installed in existing employing additional, and is very high to the concentration requirement of the sample in managing, the amount of the compound of surveying that namely needs very big (the milligram mg order of magnitude).For the sample of active volatility metabolite (VOCs) trace, maybe can't measure, though or can detect certain biologically active, the statistical disposition result difference is (p〉0.05) not significantly.Use the existing survey method of giving birth to detect directly obtain pine wood nematode is had bioactive micro-volatile matter (compound of the nanogram ng order of magnitude) by the GC separation.Need be improved.
Summary of the invention
The present invention aims to provide a kind of method that can determine the microorganism volatile compound (VOCs) that influences the nematode approach behavior of trace.
Technical solution of the present invention is as follows:
A kind of living survey method that influences the microorganism volatile compound (VOCs) of nematode approach behavior may further comprise the steps:
Get cavity volume at 10 to 500 mm
3Tubule, open an osculum that is used for dripping distilled water and nematode liquid in the tubule middle, be sidelong the medium fritter that generation active volatility metabolite (VOCs) microorganism is arranged at one of tubule, perhaps contain the medium of the sample solution of VOCs, be labeled as end for process; Opposite side is put into a not microbe inoculation or do not contain the medium fritter of active volatility metabolite (VOCs), is labeled as the contrast end, the tubule both sides is sealed again, and splashes into nematode liquid with microsyringe from the osculum in the middle of the tubule, places incubator to cultivate.Respectively at 10 h, 24 h separate nematode behind 36 h, leave standstill microscopy behind 24 h under 28 ℃, the go forward side by side statistical analysis of line data of the nematode number that statistics is separated to.
The tubule that above-mentioned tubule can adopt the plastic or other material of glass, metal, non-volatile thing to make preferably adopts the transparent silicon sebific duct.
The invention has the advantages that: " silicone tube method " of the present invention is sensitiveer, and very little cavity volume and the environment of sealing guarantee that " silicone tube method " has very high detection sensitivity for the volatile matter of trace.
Further technical solution is: the placed in the middle placement is suitable for the nematode medium of movement in pipe, is positioned between two parties in the silicone tube centered by the osculum place in the middle of silicone tube, and distilled water is slowly injected at the osculum place in the middle of silicone tube, makes medium just all moistening.
The overwhelming majority has adopted nematode to carry out in the method for agar surface motion in the existing living survey method in the past, since different nematodes in natural world habitat difference, the medium of survival is also different, the motion of agar surface and not exclusively suitable nematode, particularly most nematodes are to move in medium, rather than move at dielectric surface.Therefore the motion of these method affect nematodes, sensitivity and the accuracy of surveying given birth in influence then.The present invention has taked specific nematode use is suitable for the nematode medium of movement, makes nematode move in the medium that is fit to, and has improved sensitivity and the accuracy of living survey.
Further technical solution is again: described medium also can be cotton thread, filter paper, glass fiber for nematode real life environments soil, wood chip through deactivation, and preferably adopting diameter is the filter paper rod of transparent silica gel bore.
Above-mentioned medium is to be suitable for the nematode medium of movement, and the filter paper rod is easily obtained and made.
Further technical solution is again: the internal diameter of described transparent silicon sebific duct is 1-3 mm, and length is 3-5 cm, and cavity volume is 23.55 mm
3-353.25 mm
3
The present technique solution makes can give birth to survey to collected mouthful sample of the trace of collecting by GC, and method is simple, can find out the compound with attractive activity of nanogram magnitude.Experiment showed, that detection sensitivity exceeds several orders of magnitude than living survey method commonly used, can detect the compound less than the pine wood nematode biologically active of 1 ng.
Further technical solution is again: the medium of the sample solution of the above-mentioned VOCs of containing is filter paper, medium, glass fiber.
The living survey method (hereinafter to be referred as " silicone tube method ") that the present invention sets up, can be in conjunction with the GC coupling, leading to the detector pillar at GC installs the branch pillar additional and collects mouth, and on the basis of the split ratio that changes the GC chromatographic column, collect mouth from GC and collect minor compound, and can give birth to survey with material that this trace separates, and determine its biologically active, identify the structure of active component at last by GC-MS.Can be used for detecting the biologically active of the micro-pine wood nematode of VOCs; Identifying micro-organisms is lured the active volatility metabolite of nematode, provides certain theoretical foundation in the hope of biological control and the earlier detection diagnosis of the disease that causes for nematode.
Description of drawings
Fig. 1 silicone tube method is measured
E. vermicolaThe VOCs of fungi fritter * attracts the effect of pine wood nematode.
Embodiment
Lure the real experiment of give birth to surveying of active volatility metabolite (VOCs) of nematode the present invention will be further described below with reference to fungi Esteya vermicola CNU 120806.
The sensitivity that the effect of the VOCs attraction pine wood nematode of experiment 1 test fungi E. vermicola and comparison " silicone tube method " and " water agar plate method " detect micro substance.
1, test material:
For examination fungi: Esteya vermicola CNU 120806 (being called for short Esteya vermicola), Taean, Korea university is so kind as to give
For trying nematode: pine wood nematode (Bursaphelenchus xylophilus)
Material and reagent:
Transparent silicon sebific duct (Nanjing is apart from one-tenthization glass Co., Ltd for internal diameter 3 mm, external diameter 6 mm);
Slide measure D15TN (Mitutoyo Corporation);
Fast qualitative filter paper (Hangzhou Paper Co., Ltd of Xinhua);
Knife blade (operating theater instruments factory of Shanghai medicine equipment Co., Ltd);
Miillpore filter Ф 0.22 μ m (new Asia, Shanghai City purifies device factory);
Seal film Parafilm (Hangzhou microorganism reagent Co., Ltd);
Culture dish (the Nanjing auspicious trade Co., Ltd of gold celebrating);
Roundlet filter paper (r=0.6 cm, self-control);
Fresh corn powder (country fair purchase);
Sucrose (Chemical Reagent Co., Ltd., Sinopharm Group);
Agar powder (Chemical Reagent Co., Ltd., Sinopharm Group);
Streptomycin sulphate (Nanjing Sheng Xing Bioisystech Co., Ltd);
Benzylpenicillin sodium salt (Nanjing Sheng Xing Bioisystech Co., Ltd);
30% hydrogen peroxide (Nanjing Chemistry Reagent Co., Ltd.);
Benzinum (boiling range 60-90 ℃, analyze pure, Nanjing Chemistry Reagent Co., Ltd.);
Absolute ethyl alcohol (analyze pure, Nanjing Chemistry Reagent Co., Ltd.);
Absolute ethyl alcohol (spectroscopic pure, Aladdin reagent Shanghai Co., Ltd);
Carrene (analyze pure, Nanjing Chemistry Reagent Co., Ltd.);
Australene (92%, chemical engineering institute of Nanjing Forestry University);
Nopinene (92%, chemical engineering institute of Nanjing Forestry University)
Medium:
Potato sucrose agar medium (PSA);
Water agar (2% WA; 0.8% WA);
Weak nutrition maize flour medium (LCMA);
The two anti-medium (two anti-LCMA) of weak nutrition maize flour agar;
Instrument and equipment:
Thermo Finnpipette adjustable pipette (thermoelectric Shanghai Instr Ltd.);
Analytical balance FA1104N (Shanghai precision instrument science Co., Ltd);
Light microscope YS2 (Japanese Nikon company);
Vertical pressure steam sterilizer YXQ-LS-50S11 (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.);
Constant incubator MLR-351 (electrical machinery of Japanese sanyo company);
Vertical laminar flow clean bench CA-920-2 (going up marine clean cleaning equipment Co., Ltd);
Thermostat water bath HH-2 digital display (Changzhou Guohua Electric Appliance Co., Ltd.);
The automatic dual pure water distiller of SZ-93 (Shanghai Yarong Biochemical Instrument Plant);
Refrigerator BCD-215KC F (Qingdao HaiEr Co., Ltd);
800 type centrifugation devices (Shanghai Surgical Operation Equipment Factory).
2, test method.
2.1 cultivate and separate pine wood nematode.
2.2 preparation potato sucrose agar medium (PSA), water agar (2% WA), water agar (0.8% WA), weak nutrition maize flour medium (LCMA), the two anti-medium (two anti-LCMA) of weak nutrition maize flour agar.
2.3
E. vermicolaPurifying:
E. vermicolaPurification process as follows: under aseptic condition, scrape with transfer needle and to get cultural hypha on Ф 7.5 cm LCMA flat boards, cultivate 5 d down for 26 ℃.At the other 10 μ l nematode liquid (containing 100 nematodes approximately) that splash into of the bacterium colony that newly grows up to.Behind 2 d, under light microscope, the nematode of being infected by fungal spore with the fine needle picking is dripped last 33 % H in advance on the slide to sterilized slide
2O
2With 2 sterile waters.With nematode with 3 % H
2O
2Wash 3 times, each 10 s, use aseptic washing 2 times again after, be transferred on the two anti-LCMA flat boards of new Ф 7.5 cm, with the pollution (Li Tianfei etc., 2000) of removing bacterium, each is dull and stereotyped to insert 1 nematode, flat board places 26 ℃ to cultivate 2 d down.
Behind the 2d, can observe under light microscopic, mycelia is grown from infecting in the nematode body, and nutrient hypha crawls on medium, and aerial hyphae leaves upwards growth of medium, and the viscosity spore on the doleiform conidiophore that is born in aerial hyphae.The viscosity spore is irregular ellipse, and semilune caves inward, and conidium includes the structure (Li Tianfei etc., 2000) of a similar endoparasitism spore.Under light microscopic, use fine needle to scrape gently and get aerial hyphae, the viscosity spore just adheres to needle point, and with syringe needle gently line on new Ф 9 cm PSA flat boards, spore can be scattered on the flat board at random.Under high power lens (400 times), observe, if find semi-moon shaped single spore, and near no mycelia and other spores, just use marking pen in dull and stereotyped rear indicia.Place 26 ℃ to cultivate 1-2 d down flat board.Observe down at high power lens (400 times), if single spore is normally sprouted, will contain the monosporous medium with sterile razor blade and downcut, be forwarded in the test tube that contains PSA, cultivate 8 d down in 26 ℃.
Purifying
E. vermicolaOriginally colony edge is white in color, closely, and peripheral some flocculence, but color becomes grayish green gradually, finally deepens green.Bacterium colony produces dark-brown pigment.After mycelia grows well, place 4 ℃ to preserve down in test tube.
2.4 making silicone tube:
Get the long transparent silicon sebific duct (internal diameter 3 mm, external diameter 6 mm) of 5 cm, open an osculum with knife blade in the silicone tube middle, osculum is used for dripping distilled water and nematode liquid.The fast qualitative filter paper is made the filter paper rod of 3 mm, fill in gently in the silicone tube, the placed in the middle placement.Slowly inject 140 μ l distilled water with the osculum place of liquid-transfering gun in the middle of silicone tube, make the filter paper rod all moistening, and inhale and remove circulating water unnecessary in the filter paper rod.
2.5 give birth to the preparation of surveying with the bacterium piece
Inoculate at Ф 9 cm PSA flat boards
E. vermicolaFungi is cultivated 8 d down in 26 ℃.
Cut several fritters with sterile razor blade at colony edge, about 0.1 cm of the area of mycelia on each fritter
2, the mycelia amount is about 0.05 mg, is called for short the fungi fritter; On the PSA flat board of inoculated fungi not, cut the fritter of equal volume equally, be called for short the PSA fritter, in contrast.
Survey experiment 2.6 give birth to
The silicone tube that contains moistening filter paper rod, one is sidelong into a fungi fritter, is labeled as end for process with marking pen; Opposite side is put into a PSA fritter, is labeled as the contrast end.Seal with sealing film Parafilm the silicone tube both sides.Experiment repeats 3 times.Splash into 10 μ l nematode liquid (about 300 nematodes) with microsyringe from the osculum in the middle of the silicone tube, leave standstill a moment, place 28 ℃ incubator secretly to cultivate.
2.7 separate nematode
Respectively at 10 h, 24 h separate nematode behind 36 h.The method of separating nematode from silicone tube is as follows:
Rapidly end for process and the contrast end (equal 2 cm are long) of silicone tube are downcut with knife blade.Filter paper rod that respectively will be wherein takes out, and together with corresponding silicone tube and fritter, places the graceful funnel of shellfish to separate, and leaves standstill microscopy behind 24 h under 28 ℃, the nematode number that statistics is separated to.
2.8 interpretation of result
In the same silicone tube, the nematode number (bar) that end for process and contrast end are separated to is designated as a and b respectively.The computing formula of end for process ratio and contrast end ratio is as follows:
The nematode number that end for process ratio (%)=end for process is separated to/(nematode that end for process and contrast end are separated to is counted summation) * 100=a/ (a+b) * 100
The nematode number that end for process ratio (%)=the contrast end is separated to/(nematode that end for process and contrast end are separated to is counted summation) * 100=a/ (a+b) * 100
Use the check of SPSS 13.0 independent sample t, come the end for process ratio of 3 repetitions of comparison and the difference between the contrast end ratio average, significant difference is judged with the two-tailed test value (p value) of independent sample t check.
If p〉0.05, there was no significant difference between end for process ratio and the contrast end ratio is described, namely
E. vermicolaThe pine wood nematode of VOCs do not have the attraction effect;
P＜0.05, and end for process ratio〉50%, illustrating between end for process ratio and the contrast end ratio has significant difference, namely
E. vermicolaThe attraction effect of the nematode of VOCs of mycelia is compared with contrast PSA medium, and significant difference is arranged;
P＜0.01, and end for process ratio〉50%, illustrating between end for process ratio and the contrast end ratio has utmost point significant difference, namely
E. vermicolaThe nematode of the VOCs of mycelia has and attracts effect extremely significantly.
3, result and analysis.
1.2.1 the VOCs of fungi E. vermicola attracts the effect of pine wood nematode
End at silicone tube is put into
E. vermicolaThe fungi fritter is labeled as end for process; The other end is put into the PSA fritter, is labeled as the contrast end.Osculum place in the middle of silicone tube adds nematode liquid, and respectively at 10 h, 24 h separate nematode behind 36 h.Interpretation of result is represented with the two-tailed test value (p value) of end for process ratio and independent sample t check.The results are shown in Table 1-1 and Fig. 1.
From showing 1-1 and Fig. 1 as can be seen
E. vermicolaThe pine wood nematode of VOCs very strong attraction effect is arranged, this result and Wang Chunyan giving birth on the water agar plate surveyed result consistent (Wang etc., 2010).After the fungi fritter placed silicone tube 10 h, end for process was separated to 241.33 ± 76.59 nematodes, and the end for process ratio is 88.45% ± 0.04〉50%.The independent sample t check analysis, p=0.000＜0.01 illustrates compared with the control,
E. vermicolaThe VOCs of fungi has and attracts effect extremely significantly.As time goes on, the result that 24 h separate with 36 h shows that the fungi fritter is still keeping very high attraction effect to pine wood nematode, and the end for process ratio is respectively 86.55% ± 0.10 and 85.16% ± 0.09 (p＜0.001).
In the transparent silicon sebific duct, about 0.1 cm of the mycelia area of fungi fritter
2, mycelia is measured about 0.05 mg.Fungi fritter and PSA fritter directly do not touch filter paper rod and nematode wherein, and therefore, what " silicone tube method " detected is the effect of the nematode of VOCs.
E. vermicolaThe VOCs of mycelia forms certain concentration difference gradually in the side accumulation of silicone tube moistening filter paper rod, attract the pine wood nematode of dropping in the middle of the filter paper rod by filter paper, moves to the fungi fritter.The result of preliminary experiment shows before, will
E. vermicolaMycelia spread to little group, put into silicone tube, nematode is had equally attract effect extremely significantly.Detect behind 24 h, end for process is separated to 174.00 ± 31.32 nematodes, and the end for process ratio is 85.02% ± 0.03.p=0.000<0.01。Above result shows
E. vermicolaThe live body mycelia can produce the volatile materials of luring pine wood nematode.
Table 1-1 silicone tube method is measured
E. vermicolaThe VOCs of fungi fritter * attracts the effect of pine wood nematode
| Standing time | A (bar) | B (bar) | The end for process ratio | Contrast end ratio | P value * * |
| 10h | 241.33±76.59 | 33.00±20.88 | 88.45%±0.04 | 11.55%±0.04 | 0.000 |
| 24h | 186.67±47.37 | 27.33±18.88 | 86.55%±0.10 | 13.45%±0.10 | 0.001 |
| 36h | 90.67±60.34 | 12.00±2.65 | 85.16%±0.09 | 14.84%±0.09 | 0.001 |
Annotate: the data in the table are mean+SD, and a and b represent the nematode number that end for process and contrast end are separated to respectively
* the about 0.1cm of mycelia area of fungi fritter
2, the about 0.05mg of mycelia amount
* P value is the two-tailed test value of independent sample t check.
According to experimental result, the nematode disengaging time of this life survey method is set at 10 h, because along with the prolongation of standing time of fungi fritter, attract effect not occur rising appreciably, the nematode number that end for process is separated to is on a declining curve.10h to 36h, the nematode that end for process is separated to drops to 90.67 ± 60.34 from 241.33 ± 76.59.The reason that this phenomenon occurs may be, nematode is attracted by the VOCs of fungi fritter, moves to fungi gradually, and along with the prolongation in processing time, a lot of nematodes can be infected by the viscosity spore on the mycelia, and its motility is affected, and is difficult to separate from the graceful funnel of shellfish.
From separating resulting, also have the fraction nematode can be mobile to contrast end, and the VOCs of fungi the concentration of contrast end a little less than, so whether the nematode of the VOCs of PSA medium have sucking action still to can not determine, can determine in subsequent experimental.Because what each root silicone tube dripped can not guarantee unanimity for examination nematode number, when therefore separating nematode, statistics reaches the nematode number of filter paper rod both sides (equal 1 cm is long) respectively, and carries out statistical test with percentage.
The silicone tube method is measured
E. vermicolaThe VOCs of fungi fritter * attracts effect (about 0.1 cm of the mycelia area of * fungi fritter of pine wood nematode
2, mycelia is measured about 0.05 mg) and see Fig. 1.
Test the comparison of 2 two kinds of living survey method sensitivity
Sample solution by observing gradient concentration is to the attraction effect of pine wood nematode, comes the relatively difference of " silicone tube method " and " water agar plate method " these two kinds living survey method sensitivity.
2. the preparation of the sample solution of 1 gradient concentration
With australene and nopinene mass ratio (Zhao etc., 2007 according to 9:1; Zhao etc., 2009), be made into gradient solution.Get 90 μ l australenes and 10 μ l nopinenes and mix, be dissolved in the absolute ethyl alcohol (spectroscopic pure), be mixed with the solution of 78 mg/ml.Gradient dilution to 10 mg/ml successively, 1 mg/ml, 10
-1Mg/ml, 10
-2Mg/ml, 10
-3Mg/ml, 10
-4Mg/ml, 10
-5The sample solution of seven concentration such as mg/ml.Sample solution under each concentration is all got 5 μ l and is dripped on little filter paper (r=0.6 cm), and contrast is the anhydrous ethanol solvent of 5 μ l.Give birth to two kinds of living survey methods respectively and survey, detect sample under each concentration to the attraction effect of pine wood nematode, experiment repeats 3 times.
2.2 giving birth to of " silicone tube method " surveyed
Give birth to the same 1.1.2.5 of survey method, the same 1.1.2.4.5 of interpretation of result.
2.3 giving birth to of " water agar plate method " surveyed
Existing method is adopted in the making of 0.8% agar plate.Ruling at the culture dish back side with ruler and marking pen, is initial point with dull and stereotyped center, and flat board is divided into four quadrants uniformly.5 μ l samples under each concentration are dropped on the little filter paper (r=0.6 cm) with microsyringe.Leave standstill a moment, treat that solvent evaporates is intact, filter paper is placed in first on the agar plate or second quadrant gently, filter paper is from culture dish edge 1 cm; Contrast is the anhydrous ethanol solvent of 5 μ l, drops in equally on the little filter paper, treats that solvent evaporates is intact, and is staggered relatively with processing, is placed in dull and stereotyped the 3rd or the four-quadrant.With the culture dish cover lid, seal with Parafilm at once, 28 ℃ of following lucifuges leave standstill 12 h.Card punch with Ф 1cm behind 12 h takes out filter paper and corresponding agar block, binds up with gauze, and places the graceful funnel of shellfish to separate, and leaves standstill microscopy behind 24 h under 28 ℃.The filter paper of dropping sample is designated as end for process together with the agar block of corresponding Ф 1cm.The filter paper that drips anhydrous ethanol solvent is done the contrast end together with the agar block note of corresponding Ф 1cm.The interpretation of result method is the same.
2.4 result and analysis
2.4.1 the VOCs of fungi E. vermicola attracts the effect of pine wood nematode
End at silicone tube is put into
E. vermicolaThe fungi fritter is labeled as end for process; The other end is put into the PSA fritter, is labeled as the contrast end.Osculum place in the middle of silicone tube adds nematode liquid, and respectively at 10 h, 24 h separate nematode behind 36 h.Interpretation of result is represented with the two-tailed test value (p value) of end for process ratio and independent sample t check.The results are shown in Table 1-1 and Fig. 1.
From showing 1-1 and Fig. 1 as can be seen
E. vermicolaThe pine wood nematode of VOCs very strong attraction effect is arranged, this result and Wang Chunyan giving birth on the water agar plate surveyed result consistent (Wang etc., 2010).After the fungi fritter placed silicone tube 10 h, end for process was separated to 241.33 ± 76.59 nematodes, and the end for process ratio is 88.45% ± 0.04〉50%.The independent sample t check analysis, p=0.000＜0.01 illustrates compared with the control,
E. vermicolaThe VOCs of fungi has and attracts effect extremely significantly.As time goes on, the result that 24 h separate with 36 h shows that the fungi fritter is still keeping very high attraction effect to pine wood nematode, and the end for process ratio is respectively 86.55% ± 0.10 and 85.16% ± 0.09 (p＜0.001).
In the transparent silicon sebific duct, about 0.1 cm of the mycelia area of fungi fritter
2, mycelia is measured about 0.05 mg.Fungi fritter and PSA fritter directly do not touch filter paper rod and nematode wherein, and therefore, what " silicone tube method " detected is the effect of the nematode of VOCs.
E. vermicolaThe VOCs of mycelia forms certain concentration difference gradually in the side accumulation of silicone tube moistening filter paper rod, attract the pine wood nematode of dropping in the middle of the filter paper rod by filter paper, moves to the fungi fritter.The result of preliminary experiment shows before, will
E. vermicolaMycelia spread to little group, put into silicone tube, nematode is had equally attract effect extremely significantly.Detect behind 24 h, end for process is separated to 174.00 ± 31.32 nematodes, and the end for process ratio is 85.02% ± 0.03.p=0.000<0.01。Above result shows
E. vermicolaThe live body mycelia can produce the volatile materials of luring pine wood nematode.
Table 1-1 silicone tube method is measured
E. vermicolaThe VOCs of fungi fritter * attracts the effect of pine wood nematode
| Standing time | A (bar) | B (bar) | The end for process ratio | Contrast end ratio | P value * * |
| 10h | 241.33±76.59 | 33.00±20.88 | 88.45%±0.04 | 11.55%±0.04 | 0.000 |
| 24h | 186.67±47.37 | 27.33±18.88 | 86.55%±0.10 | 13.45%±0.10 | 0.001 |
| 36h | 90.67±60.34 | 12.00±2.65 | 85.16%±0.09 | 14.84%±0.09 | 0.001 |
Annotate: the data in the table are mean+SD, and a and b represent the nematode number that end for process and contrast end are separated to respectively
* the about 0.1cm of mycelia area of fungi fritter
2, the about 0.05mg of mycelia amount
* P value is the two-tailed test value of independent sample t check.
According to experimental result, the nematode disengaging time of this life survey method is set at 10 h, because along with the prolongation of standing time of fungi fritter, attract effect not occur rising appreciably, the nematode number that end for process is separated to is on a declining curve.10h to 36h, the nematode that end for process is separated to drops to 90.67 ± 60.34 from 241.33 ± 76.59.The reason that this phenomenon occurs may be, nematode is attracted by the VOCs of fungi fritter, moves to fungi gradually, and along with the prolongation in processing time, a lot of nematodes can be infected by the viscosity spore on the mycelia, and its motility is affected, and is difficult to separate from the graceful funnel of shellfish.
From separating resulting, also have the fraction nematode can be mobile to contrast end, and the VOCs of fungi the concentration of contrast end a little less than, so whether the nematode of the VOCs of PSA medium have sucking action still to can not determine, can determine in subsequent experimental.Because what each root silicone tube dripped can not guarantee unanimity for examination nematode number, when therefore separating nematode, statistics reaches the nematode number of filter paper rod both sides (equal 1 cm is long) respectively, and carries out statistical test with percentage.
2.5 the comparison of two kinds of living survey method sensitivity
Existing bibliographical information, volatile matter australene and the nopinene of masson pine, the weight proportion mixing according to 27-90: 73-10 has very strong sucking action (Zhao etc., 2007 to reproduction type pine wood nematode; Zhao etc., 2009).Therefore, australene and nopinene being made into the sample solution of gradient concentration according to the mass ratio of 9:1, is respectively 10 mg/ml, 1 mg/ml, 10
-1Mg/ml, 10
-2Mg/ml, 10
-3Mg/ml, 10
-4Mg/ml, 10
-5Seven concentration of mg/ml, each concentration is all got 5 μ l, drips on little filter paper, with two kinds of living survey methods (silicone tube method and 0.8% agar plate method) pine wood nematode is cooked to give birth to respectively and surveys, by the biologically active of the sample under the observation gradient concentration to nematode, come the sensitivity of two kinds of methods of comparison.
Giving birth to the survey method is " 0.8% agar plate method ", when being 10 mg/ml for the sample concentration that tries, between handling and contrasting utmost point significant difference (p=0.000＜0.01) is arranged all, and the end for process ratio is 61.51% ± 0.03, and other each concentration (1 mg/ml to 10
-4Mg/ml) sample under, difference that there are no significant between handling and contrasting.
Presentation of results " silicone tube method " has very high sensitivity to the detection of micro substance, apparently higher than " water agar plate method ", is applicable to the VOCs that detects micro-example.Be standard with the biologically active (p＜0.05) that detects the firpene sample, " silicone tube method " detected least concentration is 10
-4Mg/ml, sample size is 0.5 ng.Be 10 mg/ml and " water agar plate method " detects minimum concentration, sample size is 50 μ g.Two kinds of methods can detected sample size differ 5 orders of magnitude." silicone tube method " is simple to operate, and giving birth to the survey time is 10 h.Less and the sealing in space in the silicone tube, so the VOCs of sample obtains keeping therein, is easy to form the higher concentration gradient, felt by pine wood nematode.For the sample of the trace of this experiment, utilize " water agar plate method " though can detect certain biologically active, the statistical disposition result difference is (p〉0.05) not significantly.Having only the sample concentration of working as is 10 mg/ml, when sample size is 50 μ g, just shows tangible biologically active.
The sample solution * of table 1-3 variable concentrations gives birth to the result who surveys with the silicone tube method
| Give birth to the concentration of surveying | A (bar) | B (bar) | The end for process ratio | Contrast end ratio | P value * * |
| 1 mg/ml | 33.00±16.82 | 19.00±8.54 | 61.29%±0.21 | 38.71%±0.21 | 0.257 |
| 10 -1 mg/ml | 36.00±6.24 | 14.33±1.53 | 71.39%±0.01 | 28.61%±0.01 | 0.000 |
| 10 -2 mg/ml | 25.33±3.79 | 14.00±1.00 | 64.21%±0.04 | 35.79%±0.04 | 0.001 |
| 10 -3 mg/ml | 21.67±2.89 | 15.33±1.15 | 58.45%±0.03 | 41.55%±0.03 | 0.002 |
| 10 -4 mg/ml | 33.67±10.41 | 25.33±6.03 | 56.64%±0.03 | 43.36%±0.03 | 0.004 |
| 10 -5 mg/ml | 9.50±8.64 | 7.50±6.22 | 57.48%±0.23 | 45.52%±0.23 | 0.292 |
Annotate: the data in the table are mean+SD, and a and b represent the nematode number that end for process and contrast end are separated to respectively
* sample solution refers to australene and nopinene according to the mixed gradient solution of 9:1 mass ratio, gives birth to the volume of surveying and is 5 μ l
* P value is the two-tailed test value of independent sample t check.
The sample solution * of table 1-4 variable concentrations gives birth to the result who surveys with 0.8% agar plate method
| Give birth to the concentration of surveying | A (bar) | B (bar) | The end for process ratio | Contrast end ratio | P value * * |
| 10 mg/ml | 18.67±6.11 | 11.67±4.04 | 61.51%±0.03 | 38.49%±0.03 | 0.000 |
| 1 mg/ml | 14.00±3.00 | 10.67±4.04 | 57.31%±0.13 | 42.69%±0.13 | 0.244 |
| 10 -1 mg/ml | 10.67±4.16 | 8.00±3.00 | 56.79%±0.06 | 43.21%±0.06 | 0.052 |
| 10 -2 mg/ml | 14.00±3.61 | 11.33±3.06 | 55.17%±0.12 | 44.83%±0.12 | 0.356 |
| 10 -3 mg/ml | 16.00±8.19 | 11.33±2.31 | 56.27%±0.13 | 43.73%±0.13 | 0.310 |
| 10 -4 mg/ml | 16.33±9.29 | 16.67±7.51 | 49.50%±0.15 | 50.50%±0.15 | 0.939 |
Annotate: the data in the table are mean+SD, and a and b represent the nematode number that end for process and contrast end are separated to respectively
* sample solution refers to australene and nopinene according to the mixed gradient solution of 9:1 mass ratio, gives birth to the volume of surveying and is 5 μ l
* P value is the two-tailed test value of independent sample t check.
Experimental result shows that the detection sensitivity of " silicone tube method " exceeds several orders of magnitude than living survey method commonly used, can detect the compound less than the pine wood nematode biologically active of 1 ng.This method is applicable to the biologically active that detects micro-example.
Below be a plurality of embodiment of the present invention.
Embodiment 1: a kind of living survey method that influences the microorganism volatile compound (VOCs) of nematode approach behavior may further comprise the steps: getting internal diameter is 1 mm, and length is 3cm, and cavity volume is 23.55 mm
3The transparent silicon sebific duct, open an osculum that is used for dripping distilled water and nematode liquid in transparent silicon sebific duct middle, placement diameter placed in the middle is the filter paper rod of transparent silica gel bore in pipe, distilled water is slowly injected at osculum place in the middle of silicone tube, make the filter paper rod just all moistening, be sidelong the medium fritter that produces active volatility metabolite (VOCs) microorganism into having one by one at one of tubule, perhaps contain the medium (as filter paper, medium, glass fiber) of the sample solution of VOCs, be labeled as end for process; Opposite side is put into a not microbe inoculation or do not contain the medium fritter of active volatility metabolite (VOCs), is labeled as the contrast end, the tubule both sides is sealed again, and splashes into nematode liquid with microsyringe from the osculum in the middle of the tubule, places incubator to cultivate.Respectively at 10 h, 24 h separate nematode behind 36 h, leave standstill microscopy behind 24 h under 28 ℃, the go forward side by side statistical analysis of line data of the nematode number that statistics is separated to.
Embodiment 2: a kind of living survey method that influences the microorganism volatile compound (VOCs) of nematode approach behavior may further comprise the steps: getting internal diameter is 3 mm, and length is 5 cm, and cavity volume is 353.25 mm
3Glass tube, open an osculum that is used for dripping distilled water and nematode liquid in the glass tube middle, the nematode real life environments soil of placing through deactivation placed in the middle in pipe, distilled water is slowly injected at osculum place in the middle of silicone tube, make soil just all moistening, be sidelong the medium fritter that produces active volatility metabolite (VOCs) microorganism into having one by one at one of tubule, perhaps contain the medium (as filter paper, medium, glass fiber) of the sample solution of VOCs,, be labeled as end for process; Opposite side is put into a not microbe inoculation or do not contain the medium fritter of active volatility metabolite (VOCs), is labeled as the contrast end, the tubule both sides is sealed again, and splashes into nematode liquid with microsyringe from the osculum in the middle of the tubule, places incubator to cultivate.Respectively at 10 h, 24 h separate nematode behind 36 h, leave standstill microscopy behind 24 h under 28 ℃, the go forward side by side statistical analysis of line data of the nematode number that statistics is separated to.
Claims (7)
1. a living survey method that influences the microorganism volatile compound of nematode approach behavior may further comprise the steps: get cavity volume at 10 to 500 mm
3Tubule, open an osculum that is used for dripping distilled water and nematode liquid in the tubule middle, be sidelong the medium fritter that the active volatility metabolite microorganism of generation is arranged at one of tubule, perhaps contain the medium of the sample solution of active volatility metabolite, be labeled as end for process; Opposite side is put into a not microbe inoculation or do not contain the medium fritter of active volatility metabolite, be labeled as the contrast end, again the tubule both sides are sealed, splash into nematode liquid with microsyringe from the osculum in the middle of the tubule, place incubator to cultivate, respectively at 10 h, 24 h, separate nematode behind 36 h, leave standstill microscopy behind 24 h under 28 ℃, the go forward side by side statistical analysis of line data of the nematode number that statistics is separated to.
2. the living survey method that influences the microorganism volatile compound of nematode approach behavior according to claim 1 is characterized in that: the tubule that described tubule can adopt the plastic or other material of glass, metal, non-volatile thing to make.
3. the living survey method that influences the microorganism volatile compound of nematode approach behavior according to claim 2, it is characterized in that: described tubule is the transparent silicon sebific duct.
4. the living survey method that influences the microorganism volatile compound of nematode approach behavior according to claim 1, it is characterized in that: the described placement between two parties in pipe is suitable for the nematode medium of movement, be positioned in the silicone tube between two parties centered by the osculum place in the middle of silicone tube, distilled water is slowly injected at osculum place in the middle of silicone tube, makes medium just all moistening.
5. the living survey method that influences the microorganism volatile compound of nematode approach behavior according to claim 4 is characterized in that: described medium is for through the nematode real life environments of deactivation soil, wood chip, cotton thread, filter paper rod.
6. the living survey method that influences the microorganism volatile compound of nematode approach behavior according to claim 1, it is characterized in that: the internal diameter of described transparent silicon sebific duct is 1-3 mm, and length is 3-5 cm, and cavity volume is 23.55 mm
3-353.25 mm
3
7. the living survey method that influences the microorganism volatile compound of nematode approach behavior according to claim 1, it is characterized in that: the described medium that contains the sample solution of active volatility metabolite is filter paper, medium, glass fiber.
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