CN1167806C - Method for early diagnosis and detection of pine nematode disease - Google Patents
Method for early diagnosis and detection of pine nematode disease Download PDFInfo
- Publication number
- CN1167806C CN1167806C CNB021299447A CN02129944A CN1167806C CN 1167806 C CN1167806 C CN 1167806C CN B021299447 A CNB021299447 A CN B021299447A CN 02129944 A CN02129944 A CN 02129944A CN 1167806 C CN1167806 C CN 1167806C
- Authority
- CN
- China
- Prior art keywords
- pine
- nematode
- test tube
- substratum
- fungi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Catching Or Destruction (AREA)
Abstract
The present invention relates to a method for detecting pine nematode diseases. Pestalotia (the strain number of which is Ps 0202) or other microbe fungi are inoculated on solid or liquid mold media; circular holes are chiseled and drilled on trunks of pines to be detected; then, test tubes provided with bacterium bodies are inserted in the circular holes arranged on the trunks; and after 3 to 5 days, the test tubes are taken out and are observed and identified by a microscope. When the method is used for detecting pine nematode diseases, the method can realize early diagnosis and can enable the pines to be free of skin peeling, and thereby, the goal for protecting pinewood landscapes is achieved. Because the amount of the pine nematode which is the origin of the disease is less at the early stage of the onset, the method can tempt the nematode to enter the test tubes in order to enable the amount of the nematode to increase greatly on the cultivated fungi, and thereby, the sensitivity of the detection is improved. Simultaneously, the method is convenient for operation, and is favorable for large-scale detection and general survey of the pine nematode diseases at the early stage.
Description
(1) technical field
The present invention relates to a kind of detection method of pine nematode.
(2) background technology
Pine wood nematode (Bursaphelenchus xylophilus) disease is a kind of destructive disease of pine tree, and it can make large stretch of pine tree dead fast, causes the tremendous economic loss; Yet, because pine nematode their early stage symptom is not obvious, often misses and prevent and treat good opportunity, cause financial loss and disease popular.So its early diagnosis is just seemed particularly important.
Originally, detecting pine wood nematode all is to continue to use Bel's Man funnel method.Its basic device is the funnel of a suitable diameter, and one section rubber tubing of funnelled pipe termination is with the switching of spring water stopper control tube.Funnel is placed on the annulus of brandreth, fills with clear water in it.Split the next section bark from the pine tree trunk, be cut into small pieces, wrap in the quadrate mull, be immersed in the water.Nematode is swum out of from Cortex Pini, sinks at the bottom of funnelled pipe through gauze.Behind diel, open spring pinchcock, flow out less water, be collected in the glass dish, water contains nematode.The nematode that contains with microscopic examination water is a pine wood nematode again.(Bi Zhishu, lijin compile " plant nematology ", agriculture press, 211-212).Though this method can be isolated nematode, has significant disadvantages: 1) split many barks, pine tree becomes and is difficult to see, destroys woodland scenery; 2) time-consuming; 3) need to use a lot of funnels, and take a lot of lab space.
(3) summary of the invention
The present invention aims to provide a kind of detection method of utilizing microbial fungi pine wood nematode to be elicited evaluation.Used microbial fungi dish crinosity (Pestalotia sp.) fungi of the present invention is to separate from the Ramulus et folium taxi cuspidatae of Longyan, Fujian Province to obtain, the classification name of bacterial strain: the dish crinosity belongs to, this thalline has been stored in China Committee for Culture Collection of Microorganisms common micro-organisms center, that registers on the books is numbered CGMCC NO.0756, strain number: Ps 0202, preservation date: on June 27th, 2002.
Technical scheme of the present invention is as follows: used microbial bacteria is dish crinosity (Pestalotia sp.), Botrytis cinerea (Botrytiscinerea Pers.), Canada dish lead fungi (Aleurodiscus canadensis skolko), loose shell capsule spore (Cytosporapini Desm.) or Krafft Jeff cheese bacteria (Tyromyces kravtzevianus Bondarzew ﹠amp; Parmasto inParmasto), its step is as follows:
Step 1: the preparation mould medium, the substratum that is suitable for mould-growth has potato juice substratum (PDA), and grass soaks juice substratum, maize powder medium (CMA), the foster base of Cha Peike, washing water of rice substratum and molasses culture medium, and substratum can be solid or liquid;
Step 2: pack into the solid mould medium branch for preparing in vitro small-bore, loading amount is 1/3～1/2 of a pipe capacity, seal with tin platinum paper, behind autoclaving, topple over into the plane, microbe inoculation fungi after the cooled and solidified, cultivate after 3～5 days (mycelia covers 1/3 agar plate approximately) for 20～28 ℃, 5～10 ℃ of refrigerations are standby;
Maybe the liquid nutrient medium for preparing is packed in the container, beyond the Great Wall cotton plug or encase the container finish autoclaving, cooling back microbe inoculation fungi with gauze, cultivate the small-bore test tube of packing after 3～5 days for 25～28 ℃, every loading amount 2～3ml seals with tin platinum paper, places vertically standby.
In addition, in vitro also can add the branch of pine tree, put in vitro behind the autoclaving (or disinfecting in alcohol).
Step 3: it is identical with test tube to dig out or get out diameter on pine tree trunk to be measured, and the degree of depth is the circular hole of test tube length 1/3.Throw off the tin platinum paper of test tube mouth, fill in the cotton that a little group sterilized, and drip 2～5ml aqua sterilisa, then test tube is inserted the circular hole on the trunk;
When temperature was lower than 24 ℃, test tube inserted the back and wraps up with black bag or cloth, and tightens, to improve invisible spectro temperature.
Step 4: detector tube inserts the back to be kept 3～5 days on the tree body, extracted then, faced toward the tube wall inspection with anatomical lens earlier, general nematode crawling on tube wall, find that than being easier to particularly the nematode great majority accumulate in the position that aggegation water is arranged on the tube wall, and the place of moisture film is perhaps arranged.
Can be placed in the incubator 25 ℃ to detector tube and cultivate and take out microscopy again after 3～4 days, nematode is bred like this, can improve recall rate greatly.
Detect pine nematode in this way, both can realize early diagnosis, can make pine tree avoid the trouble of peeling again, reach the purpose of protection pine forest view.Because pine tree wilt disease their early stage, its cause of disease one pine wood nematode quantity are seldom, present method can lure nematode in the pipe, worm amount amplification rapidly on the fungi of its cultivation, thereby improve the sensitivity that detects, be convenient to operation simultaneously, help extensive the detection and the sick early stage generaI investigation of pine wilt nematode.
(4) embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
The 200g potato is cleaned peeling, be cut into small pieces or push into filament, (w: v), boil 30min. (attention prevents burnt, available water-bath), use filtered through gauze, filtered juice is added water to boiling preceding volume to add 1000ml water by 1: 5 ratio.Add 200g sucrose again, 17g agar is transferred about pH6.
With pack into the test tube of 18 * 180mm of prepared culture medium branch, every test tube loading amount 5ml fastens the test tube mouth with tin platinum paper, and encases the test tube wall upper edge.Through 1.0kg/cm
2Sterilization 30min. topples over into the plane then, and inoculation dish crinosity (Pestalotia sp.) after the cooled and solidified was cultivated 3 days for 25 ℃, and when treating that mycelia covers 1/3 agar plate approximately, it is standby that taking-up places 5～10 ℃ of refrigerator and cooled to hide.
The pine tree that need to detect is dug out or gets out the circular hole of a diameter 18mm, the about 60mm of the degree of depth on trunk with chisel or awl.Then the detector tube that grows up to mycelia is inserted in the circular hole, face when inserting and throw off tin platinum paper, make the mouth of pipe aim at circular hole.The pipe of all detections is all filled in cotton that a little group sterilized in nearly mouth of pipe place, and then is dripped the 2.5ml aqua sterilisa before insertion.To guarantee its water supply.
Cause temperature at that time is lower than 24 ℃, all wraps up with the black small plastic bag after every test tube inserts, and tightens with bungee, to improve invisible spectro temperature.
With the detector tube of this potato juice nutrient agar of having inoculated the dish crinosity 20, carry out first test on the trunk of 20 pine trees in the insertion woods, insert and took back the laboratory in the tree in 3 days, be placed in 25 ℃ of incubators and check with anatomical lens after 4 days, the result lures from 6 pine trees and has drawn nematode, and positive rate is 30%.
With 15 of similar detection pipes, carry out second batch of test on the trunk of 15 pine trees in the insertion woods, took back the laboratory through 5 days in the tree, that night, microscopy found in 3 arms nematode is arranged, positive rate is 20%.
And same method is inserted the tree body to the clear water control group, and positive rate is 0.
Embodiment 2
Adopt liquid nutrient medium as different from Example 1, add the 200g potato in promptly every 1000ml solution, 20g sucrose is formulated.The liquid nutrient medium branch that is made into is packed in the Erlenmeyer flask of capacity 500ml, every bottled amount 100ml.Cotton plug or encase bottleneck beyond the Great Wall then with 8 layers of gauze.Through autoclaving, the cooling back is by aseptic method inoculation dish crinosity, and shaking culture is 3 days on shaking table, divides the test tube of the 18 * 180mm that packs into, and every test tube loading amount 2ml seals with tin platinum paper, places vertically standby.
With 20 of the detector tubes of this potato juice liquid substratum of having inoculated the dish crinosity, carry out first test on the trunk of 20 pine trees in the insertion woods, in the tree through taking back the laboratory in 3 days, be placed in 25 ℃ of incubators and check with anatomical lens after 4 days, the result lures from 2 pine trees and has drawn nematode, and positive rate is 10%.
With 15 of similar detection pipes, carry out second batch of test on the trunk of 15 pine trees in the insertion woods, took back the laboratory through 5 days in the tree, that night, microscopy found in 3 arms nematode is arranged, positive rate is 20%.
Embodiment 3
Added the branch of pine tree as different from Example 1 in substratum, method is: get the branch on the healthy pine tree of no nematode, and the little stub of processing growth 50mm, thick 3～5mm, autoclaving is put into pipe then.
With this inoculation dish crinosity and added 3 of the detector tubes of the potato juice nutrient agar of pine branch bar, insert on the trunk of 3 pine trees in the woods, take back the laboratory after 3 days, checked immediately the same day, just from wherein having found nematode 1 arm.Be placed in 25 ℃ of incubators and check after 4 days, from wherein finding nematode 2 arms.The result shows to lure from 2 pine trees and has drawn nematode that positive rate is 66.7%.
Embodiment 4
Adopt grass to soak the juice substratum as different from Example 1, its prescription is: hay (pulverizing) 50g or bright careless 200g, KH
2PO
12g, agar 15g, distilled water 1000ml, pH5.5～7.Hay (usually using straw) or bright grass (available turf prune get off grass) decoct the 30min after-filtration down at 120 ℃ earlier in high-pressure sterilizing pot.
Soak 20 of the detector tubes of juice nutrient agar with this grass of having inoculated the dish crinosity, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and drawn nematode, and positive rate is 30%.
Embodiment 5
Join substratum with molasses as different from Example 1, promptly molasses are diluted to 1/8～1/6, or waste molasses mixes 1/10～1/20 molasses, mix back pH and transfer to 5.5～7, with 6～6.5 the bests.
With this attaching kind 20 of the detector tubes of molasses nutrient agar of dish crinosity, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and has drawn nematode, positive rate is 30%.
Embodiment 6
Substratum is the CMA substratum as different from Example 1, and its prescription is: corn (corn flakes) 20g, sucrose 20g, agar 17g, distilled water 1000ml.Corn flakes boiled under 60 ℃ 1 hour in 500ml water.Filter with cloth, in the agar solution that is dissolved in filtrate adding in the 500ml water, add water again and supply 1000ml.
This 20 of detector tubes having inoculated the CMA substratum of dish crinosity are inserted on the trunk of 20 pine trees in the woods, and the result lures from 6 pine trees and has drawn nematode, and positive rate is 30%.
Embodiment 7
With Cha Peike (Czapek) substratum, its prescription is: NaNO
33g, K
2HPO
41g, MgSO
47H
2O0.5g, KCl0.5g, FeSO
47H
2O0.01g, sucrose 30g, agar 15g, distilled water 1000ml (sucrose will be put into before facing sterilization).All the other are with embodiment 1.
20 of the detector tubes of this Cha Peike substratum of having inoculated the dish crinosity, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and drawn nematode, and positive rate is 30%.
Embodiment 8
Its fungi that inoculates is Botrytis cinerea (Botrytis cinerea Pers.) as different from Example 1
20 of this potato juice nutrient agar detector tubes of having inoculated Botrytis cinerea, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and has drawn nematode, and positive rate is 30%.
Embodiment 9
The fungi of inoculation is Canada's dish lead fungi (Aleurodiscus canadensis skolko), and all the other are with embodiment 1.
20 of this potato juice nutrient agar detector tubes of having inoculated Canadian dish lead fungi, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and has drawn nematode, and positive rate is 30%.
Embodiment 10
Its fungi that inoculates is loose shell capsule spore (Cytospora pini Desm.) as different from Example 1
20 of this potato juice nutrient agar detector tubes of having inoculated loose shell capsule spore, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and has drawn nematode, and positive rate is 30%.
Embodiment 11
With embodiment 1 not to be that its fungi that inoculates is Krafft Jeff cheese bacteria (Tyromyceskravtzevianus Bondarzew ﹠amp; Parmasto in Parmasto)
20 of this potato juice nutrient agar detector tubes of having inoculated Krafft Jeff cheese bacteria, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and has drawn nematode, and positive rate is 30%.
Embodiment 12
Substratum is the washing water of rice substratum as different from Example 1, its working method is: washing water of rice leaves standstill for some time hypsokinesis and removes supernatant liquor, precipitation is determined as about 1.4Be with specific gravity hydrometer, and regulating pH is 6.5～7.0, then by 1.5～2.0% ratio adding agar and heating for dissolving it.
20 of the detector tubes of this washing water of rice substratum of having inoculated the dish crinosity, insert on the trunk of 20 pine trees in the woods, the result lures from 6 pine trees and drawn nematode, and positive rate is 30%.
Claims (5)
1, the method for early diagnosis of pine nematode is characterized in that used microbial bacteria is dish crinosity (Pestalotia sp.), Botrytis cinerea (Botrytis cinerea Pers.), Canada dish lead fungi (Aleurodiscus canadensis skolko), loose shell capsule spore (Cytospora pini Desm.) or Krafft Jeff cheese bacteria (Tyromyces kravtzevianusBondarzew ﹠amp; Parmasto in Parmasto), said microbial fungi dish crinosity: the strain of (Pestalotia sp.) number: Ps 0202, CGMCC NO.0756, and its step is as follows:
Step 1: the preparation mould medium, what be suitable for has a potato juice substratum (PDA), and grass soaks juice substratum, maize powder medium (CMA), Cha Peike substratum, washing water of rice substratum or molasses culture medium, and substratum is solid or liquid;
Step 2: the fixedly mould medium branch that will prepare is packed in vitro small-bore, and loading amount is 1/3～1/2 of a pipe capacity, seals with tin platinum paper, behind autoclaving, topple over into the plane, microbe inoculation fungi after the cooled and solidified was cultivated 3～5 days for 20～28 ℃, and 5～10 ℃ of refrigerations are standby:
Maybe the liquid nutrient medium for preparing is packed in the container, autoclaving cools off back microbe inoculation fungi, cultivates the small-bore test tube of packing after 3～5 days for 20～28 ℃, and every loading amount 2～3ml seals with tin platinum paper, places vertically standby;
Step 3: on tested pine tree trunk, dig out or get out that diameter is identical with test tube, the degree of depth is the circular hole of test tube length 1/3, throw off the tin platinum paper of test tube mouth, fill in cotton, and drip 2～5ml aqua sterilisa, then test tube is inserted in the circular hole;
Step 4: take out test tube after 3～5 days, anatomical lens is observed down, is identified.
2, the method for early diagnosis of pine nematode as claimed in claim 1 is characterized in that in the step 3, test tube external application black bag or cloth parcel after the intubate.
3, the method for early diagnosis of pine nematode as claimed in claim 1 is characterized in that in the step 4, and the test tube of taking-up is cultivated after 4～6 days under 25 ℃ in incubator and taken out microscopy again.
4, the method for early diagnosis of pine nematode as claimed in claim 1 is characterized in that in the step 2, in vitro puts into the branch of pine tree.
5, the method for early diagnosis of pine nematode as claimed in claim 4 is characterized in that the branch of said pine tree is the pine branch on the healthy pine tree of no nematode, the processing growth 50mm that gets, and the little stub of thick 3～5mm is through autoclaving or alcohol disinfecting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021299447A CN1167806C (en) | 2002-08-23 | 2002-08-23 | Method for early diagnosis and detection of pine nematode disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021299447A CN1167806C (en) | 2002-08-23 | 2002-08-23 | Method for early diagnosis and detection of pine nematode disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1420180A CN1420180A (en) | 2003-05-28 |
| CN1167806C true CN1167806C (en) | 2004-09-22 |
Family
ID=4746357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021299447A Expired - Fee Related CN1167806C (en) | 2002-08-23 | 2002-08-23 | Method for early diagnosis and detection of pine nematode disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1167806C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1645135B (en) * | 2005-01-21 | 2010-09-08 | 江西出入境检验检疫局检验检疫综合技术中心 | Quarantine method for nematode larva of dormant pine wood |
| CN100532537C (en) * | 2005-05-11 | 2009-08-26 | 厦门大学 | Inserting tube detecting method of soil and fresh water nematode and plant nematode |
| CN100487454C (en) * | 2006-04-27 | 2009-05-13 | 浙江省农业科学院 | Quick diagnosis method for brown stem rot of broccoli |
| CN104818217B (en) * | 2015-04-14 | 2018-05-08 | 福建农林大学 | One plant can promote the endogenetic fungus that aleurite montana biomass increases |
| CN105432566B (en) * | 2015-10-30 | 2018-10-26 | 泸县农林局 | A kind of method of quick detection Bursaphelenchus xylophilus |
-
2002
- 2002-08-23 CN CNB021299447A patent/CN1167806C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1420180A (en) | 2003-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105543152B (en) | A kind of pseudomonas putida and its application in promoting katsura tree growth | |
| CN101899408A (en) | Bacillus pumilus and application thereof for killing bursaphelenchus xylophilus with poison | |
| CN105368720B (en) | Cotton endogenetic fungus CEF-082 and its application in cotton verticillium wilt prevention and treatment | |
| CN101948782B (en) | Rhodopseudomonas spheroides strain and liquid inoculant thereof as well as preparation method and application thereof | |
| CN108117998A (en) | A kind of Xylariaceae fungi and its application | |
| HU214457B (en) | Method for screening antifungal activity, compositions containing antifungal microorganisms screened with this method, and method for inhibiting plant fungal diseases | |
| CN101779611A (en) | Method for breeding entomopathogenic nematodes | |
| CN1167806C (en) | Method for early diagnosis and detection of pine nematode disease | |
| CN107235752A (en) | It is a kind of using cow dung and edible fungi residues as the Light medium of raw material | |
| CN105439657B (en) | A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry | |
| CN1354252A (en) | Culture of ramaria mycorrhizal fungi by using glass bead as culture medium | |
| CN102598977B (en) | Method for improving chromium resistance and cadmium resistance of ryegrass with compost fermentation liquid | |
| CN104962491B (en) | The degradation bacteria strains of D of herbicide 2,4 a kind of and its microbial inoculum and the application of production | |
| Dick et al. | Species of Fusarium on Pinus radiata in New Zealand | |
| CN102577806B (en) | Method for using Bacillus thruingiensis fermentation broth to improve salt resistance of lawn plants | |
| CN102599196B (en) | Tall fescue growth adjusting method of employing bacillus thuringiensis fermentation broth | |
| Chouhan et al. | Production and Assessment of Stick-Shaped Spawns of Oyster Mushroom from Banana Leaf-Midribs | |
| CN108575600A (en) | A method of quick and precisely comparing tomato variety lower temperature resistance | |
| CN103060207A (en) | Preparation method for bacteria liquid beneficial for growth-development and mycorrhiza formation of rhododendron plants | |
| CN114644331A (en) | Biomass charcoal-based bacterial fertilizer and preparation method and application thereof | |
| CN100532537C (en) | Inserting tube detecting method of soil and fresh water nematode and plant nematode | |
| Anan et al. | Effect of K1, K2 anti-bacterial agents on tobacco Ralstonia solanacearum | |
| CN101948903B (en) | Method for detecting whether to carry bacterial brown stripe germs in rice seed | |
| CN110301314A (en) | A kind of field acquisition Sweet Potato Germplasm processing method | |
| Klein‐Gebbinck et al. | Fireweed (Epilobium angustifolium) as a possible inoculum reservoir for root‐rotting Armillaria species |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20040922 Termination date: 20110823 |