CN101042334A - Chemical dyeing method for differentiating diffuse type pine wood nematode - Google Patents
Chemical dyeing method for differentiating diffuse type pine wood nematode Download PDFInfo
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- CN101042334A CN101042334A CNA2006100584427A CN200610058442A CN101042334A CN 101042334 A CN101042334 A CN 101042334A CN A2006100584427 A CNA2006100584427 A CN A2006100584427A CN 200610058442 A CN200610058442 A CN 200610058442A CN 101042334 A CN101042334 A CN 101042334A
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- wood nematode
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Abstract
This invention relates to one chemical dyeing method to identify the diffusion of breeding pine wire worm, which comprises the following steps: a, solving the stimulant into dimethyl sulfoxide liquid with two weight proportion as 2-50:100 and element A, B and C weight proportion as 54-90:9-43:1-5 with A for alpha- firpene, B for beta- firpene and C for longifolene; b, dripping the stimulant into the water liquid with the worm and adding 10 mu L coomassie brilliant blue with intensity of 0.2% and putting in room temperature for 24 hours and observing under microscopes for 24 hours.
Description
Technical field
The present invention relates to be used to differentiate the chemical staining method of diffuse type pine wood nematode, relate in particular to and be used for chemical staining method that the diffuse type pine wood nematode disease is monitored and discerned.
Background technology
Pine nematode is commonly called as pine tree " cancer ", and in a single day pine tree infects this disease, and is long then half a year, short then two months will be dead.This disease from nineteen eighty-two since being found first near the Zhongshan Tomb, China Nanjing, spread to and comprised provinces and cities surplus Jiangsu, Anhui, Guangdong, Guangxi, Zhejiang, Fujian, Shanghai, Chongqing, Hubei, the Shandong etc. 10,86 counties and cities, injured area reaches 1,260,000 mu, and the 500000000 mu of pine forests in south have been constituted serious threat.And occurrence scope is also enlarging year by year.State Administration of Forestry's statistical data shows: annual pine tree more than 600 ten thousand strains that cause death, cause 2,500,000,000 yuan of direct economic losses, and indirect loss reaches 25,000,000,000 yuan.At present, main dependence is removed ill tree and is administered, and seeking the suitable method of preventing and treating from pathogenesis becomes the task of top priority.
The cause of disease of pine nematode---pine wood nematode is one of most important Alien invasive species of China, and it infects circulation can be divided into reproduction type and diffused two parts.The reproduction type nematode breeds in a large number in healthy pine tree and pine tree is fallen ill.Along with ecological deterioration, diffused third-instar larvae (L
III) begin to form, thereby enter the diffusion circulation.L
IIIAround the larva burrow of Monochamus alternatus and pupa chamber, assemble, peel along with the emergence of longicorn become four age persistent form larva (L
IV).L
IVAlong with longicorn is got food and is carried to new healthy pine tree, begin the circulation of infecting of a new round.L
IVEnter resin canal by the wound of getting food or on bark, caused when laying eggs, decortication becomes adult then, these are female, lay eggs after the male imago mating, become reproduction type larva and a large amount of breeding after the ovum hatching and make the very fast withered flavescence of pine tree become the object that Monochamus alternatus is laid eggs once more, the Monochamus alternatus larva of the hatching of laying eggs enters again in the pine nematode tree and gets the food burrow.Both cooperation provide required condition the history of life for the other side finishes, and the condition of utilizing the other side to create is finished self growth.A large amount of breedings of reproduction type nematode are that pine wood nematode and Monochamus alternatus are united the prerequisite that causes pine tree dead fast, if can detect the existence of diffused nematode as early as possible, still just can find disease and take measures then that prevention is in the expansion in forest zone nonevent latent period in symptom.
At present investigate and cultivate when observing in the port quarantine of pine wood nematode and woodland spot, the identification of reproduction type and diffused nematode all is to use microscope polypide observation of characteristics method: need the investigator to have the ABC of nematode polypide structure, and reproduction type and diffused nematode be not easily distinguishable, more apparent difficulty when particularly adding up the ratio of reproduction type and diffused nematode.In addition, the pine wood nematode preliminary judgement does not still have reliability index at present, and some reliable biochemical identification method complicated operations and equipment and the condition that need be correlated with are difficult for carrying out in basic unit and field.The present invention stems from the terpenoid substance that utilizes the pine tree host can stimulate the diffused nematode to secrete specific protein from amphid specifically, and available Kumasi light blue dyes, thereby can convenient and simple discriminating diffused nematode.The method can be utilized in the evaluation of the pine wood nematode of the fresh pine tree of port quarantine, also can be used on field study and pine wood nematode and cultivates when observing, and the reproduction type nematode is on the diffused nematode investigation statistics of transition.Since simple to operate, be easy to judge that the method also can be used as index of pine wood nematode preliminary judgement.
Summary of the invention
The object of the invention is to provide a kind of chemical dyeing method that is used to differentiate diffuse type pine wood nematode, non-index and complicacy that solution is judged the diffused nematode in the investigation sampling and the differentiation in the pine wood nematode training of the sampling of port quarantine and pine wilt disease spot, and the reliability of preliminary judgement pine wood nematode nematode during the investigation of spot.
Technical scheme of the present invention is as follows:
The chemical dyeing method that is used to differentiate diffuse type pine wood nematode provided by the invention, prepare a kind of stimulant by selected specific chemical constitution and proportioning thereof, this stimulant can stimulate the diffused nematode to secrete specific protein from amphid, and secreted specific protein dyeed with the Kumasi light blue, thereby differentiate the diffused nematode.
The chemical dyeing method that is used to differentiate diffuse type pine wood nematode provided by the invention, its step is as follows:
1, the stimulant that will contain component A, B and component C is dissolved in the organic solvent; The weight portion proportioning of described stimulant and organic solvent is 2-50: 100;
Component A, the B in the described stimulant and the weight portion proportioning of component C are 1-21: 1-56: 23-98;
Described component A is an australene, and component B is a nopinene, and component C is a longifolene;
2, the stimulant that will be dissolved in the organic solvent splashes in the aqueous solution that contains pine wood nematode, and add the Kumasi light blue (brillant-blue P-250) of 10 μ L, at room temperature placed at least 24 hours, and observed at microscopically: what the specific protein of its secretion was colored is diffuse type pine wood nematode.
The proportioning of described component A, component B and component C is 21: 56: 23, best results.
The present invention is used to differentiate that the method for operating of the chemical dyeing method of diffused is:
After will being dissolved in 20 μ L dimethyl sulfoxide (DMSO)s (DMSO) by the 1.5 μ g stimulants that component A, component B and the component C of above-mentioned weight portion proportioning are formed, be added to 100 μ L and contain in the aqueous solution of pine wood nematode, and the Kumasi light blue that adds 10 μ L is at room temperature placed the specific protein that is colored that just can see the secretion of diffused nematode head in 24 hours at microscopically.The weight portion proportioning of component A, component B and component C is 21: 56: 23 o'clock, best results.
The chemical dyeing method that does not still have at present the discriminating diffused, the present invention is used to differentiate that the excellent part of the chemical dyeing method of diffused is:
1) stimulation diffused nematode provided by the invention is derived from the pine tree volatile matter from the stimulant of amphid secretion specific protein, belongs to natural organic substance; Its Stability Analysis of Structures, chemical property is stable under the normal temperature and pressure, and is nontoxic, free from environmental pollution;
2) stimulant provided by the invention contains can stimulate the compound terpenoid of diffused nematode from amphid secretion specific protein, is easy to buy on market, and preparation is simple;
3) the present invention is easy and simple to handle reliable, is applicable to the investigation work of basic unit.
4) the present invention has reached the effect of good discriminating diffused and pine wood nematode preliminary judgement in port and woodland investigation and laboratory.
Embodiment
Embodiment 1-3
The stimulation 1.5 μ g that form by the listed weight portion proportioning of table 1 are dissolved in the dimethyl sulfoxide (DMSO) (DMSO) of 20 μ L, add 100 μ L then and contain in the aqueous solution of pine wood nematode and the 0.2%brillant-blue P-250 (Kumasi light blue) that adds 10 μ L at room temperature places 24 hours (each handle do 10 repeat); And observed Color listed in table 1.
Diffuse type pine wood nematode is handled and observed Color.List in table 1.Each is handled and does 10 repetitions.
The different Colors of forming proportioning (weight portion) to diffuse type pine wood nematode of table 1
| Australene A | Nopinene B | Longifolene C | 0.2％ brillant-blue P-250 | Coloration result | |
| Embodiment 1 | 21 | 56 | 23 | 10μl | Have |
| Embodiment 2 | 1 | 1 | 98 | 10μl | Have |
| Embodiment 3 | 10 | 30 | 60 | 10μl | Have |
Embodiment 4-6
The stimulation 0.5 μ g that forms by the listed weight portion proportioning of table 2 is dissolved in the triethylene glycol diacetate of 20 μ L, adds 100 μ L then and contains in the aqueous solution of pine wood nematode and 0.2% brillant-blueP-250 (Kumasi light blue) that adds 10 μ L at room temperature places 24 hours (each handle do 10 repeat); And observed Color listed in table 2.
Diffuse type pine wood nematode is handled and observed Color.List in table 2.Each is handled and does 10 repetitions.
The different Colors of forming proportioning (weight portion) to diffuse type pine wood nematode of table 2
| Australene A | Nopinene B | Longifolene C | 0.2％ brillant-blue P-250 | Coloration result | |
| Embodiment 4 | 21 | 56 | 23 | 10μl | Have |
| Embodiment 5 | 15 | 20 | 75 | 10μl | Have |
| Embodiment 6 | 10 | 40 | 50 | 10μl | Have |
Embodiment 7-9
The stimulation 10.5 μ g that form by the listed weight portion proportioning of table 3 are dissolved in the methylene chloride of 20 μ L, add 100 μ L then and contain in the aqueous solution of pine wood nematode and the 0.2%brillant-blue P-250 (Kumasi light blue) that adds 10 μ L at room temperature places 24 hours (each handle do 10 repeat); And observed Color listed in table 3.
Diffuse type pine wood nematode is handled and observed Color.List in table 3.Each is handled and does 10 repetitions.
The different Colors of forming proportioning (weight portion) to diffuse type pine wood nematode of table 3
| Australene A | Nopinene B | Longifolene C | 0.2％ brillant-blue P-250 | Coloration result | |
| Embodiment 7 | 21 | 56 | 23 | 10μl | Have |
| Embodiment 8 | 5 | 20 | 75 | 10μl | Have |
| Embodiment 9 | 8 | 32 | 60 | 10μl | Have |
As seen from table, use the chemical dyeing method that is used to differentiate diffuse type pine wood nematode provided by the invention, have good identification result, for quarantine and the investigation of China pine wood nematode provides a kind of new method.
Claims (2)
1, a kind of chemical dyeing method that is used to differentiate diffuse type pine wood nematode, its step is as follows:
1) stimulant that will contain component A, B and component C is dissolved in the organic solvent; The weight portion proportioning of described stimulant and organic solvent is 2-50: 100;
Component A, the B in the described stimulant and the weight portion proportioning of component C are 1-21: 1-56: 23-98;
Described component A is an australene, and component B is a nopinene, and component C is a longifolene;
2) stimulant that will be dissolved in the dimethyl sulfoxide solvent splashes in the aqueous solution that contains diffuse type pine wood nematode, and the concentration expressed in percentage by weight that adds 10 μ L is 0.2% Kumasi light blue, at room temperature placed at least 24 hours, and observed at microscopically: what the specific protein of its secretion was colored is diffuse type pine wood nematode.
2, by the described chemical staining method that is used to differentiate diffuse type pine wood nematode of claim 1, the proportioning of described component A, component B and component C is 21: 56: 23.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100584427A CN100504354C (en) | 2006-03-24 | 2006-03-24 | Chemical dyeing method for differentiating diffuse type pine wood nematode |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100584427A CN100504354C (en) | 2006-03-24 | 2006-03-24 | Chemical dyeing method for differentiating diffuse type pine wood nematode |
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| Publication Number | Publication Date |
|---|---|
| CN101042334A true CN101042334A (en) | 2007-09-26 |
| CN100504354C CN100504354C (en) | 2009-06-24 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102213672A (en) * | 2010-04-03 | 2011-10-12 | 李红玉 | Method and kit for setting survival rate of nematode quickly |
| CN113749054A (en) * | 2021-08-24 | 2021-12-07 | 中国林业科学研究院林业新技术研究所 | Identification method of diffusion type 3-year-old pine wood nematodes |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105259089B (en) * | 2015-11-03 | 2017-09-15 | 三峡大学 | A kind of method for identifying wood quality |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002227917B2 (en) * | 2000-11-09 | 2007-06-21 | Cenix Bioscience Gmbh | Genes required for viability and/or reproduction in C. elegans and their use in the development of anti-nematode agents |
| CN1184479C (en) * | 2003-01-21 | 2005-01-12 | 厦门大学 | Fast separation device of pine material nematode and its detection method |
| CN1226421C (en) * | 2003-10-21 | 2005-11-09 | 南京农业大学 | Pinewood nematoda detection reagent-box and detection method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102213672A (en) * | 2010-04-03 | 2011-10-12 | 李红玉 | Method and kit for setting survival rate of nematode quickly |
| CN102213672B (en) * | 2010-04-03 | 2015-12-02 | 李红玉 | A kind of method of fast quantification nematode survival rate and kit |
| CN113749054A (en) * | 2021-08-24 | 2021-12-07 | 中国林业科学研究院林业新技术研究所 | Identification method of diffusion type 3-year-old pine wood nematodes |
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| CN100504354C (en) | 2009-06-24 |
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