CN100532537C - Inserting tube detecting method of soil and fresh water nematode and plant nematode - Google Patents
Inserting tube detecting method of soil and fresh water nematode and plant nematode Download PDFInfo
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- CN100532537C CN100532537C CNB2005100724080A CN200510072408A CN100532537C CN 100532537 C CN100532537 C CN 100532537C CN B2005100724080 A CNB2005100724080 A CN B2005100724080A CN 200510072408 A CN200510072408 A CN 200510072408A CN 100532537 C CN100532537 C CN 100532537C
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Abstract
An intubation detection method of the soil, water and plant nematode, it relates to the detection method of biological group, and it supplies a microorganism which is yeast Yr0503, the classification name of the strain is yeast and the store number is CGMCC No.1336. The detection processes of soil, water and plant nematode induced in the detector tube by microorganism is as fellows: taking the detector tube, loading the slide, adding the cellulous, sterilizing by high pressure and taking out after cooling. Preparing the culture medium, splitting, and the microorganism are inoculated after the culture medium is sterilized and cooled. The culture medium is filled into the detector tube. There are holes in the soil which are filled with sterile water, and the detector tuber is inserted into the hole for some time and it is pulled out to detect. This is conveniently for the inspector to operate, and the plant can avoid being pulled out and be protected. The sensitivity of the detection is high. It can by applied to the census of the soil nematode and the investigation of the plant nematode and it also can be applied to detecting the soil nematode to evaluate the environment.
Description
Technical field
The present invention relates to a kind of detection method of biological group, especially relate to a kind of intubate detection method that is present in the nematode in soil, fresh water and the plant.
Background technology
Nematode is the mcroorganism monoid that occurring in nature extensively distributes, and what have parasitizes in animal, the plant materials, and what have is present in soil and the water body.The former causes disease, causes financial loss; The latter constitutes a ring of human foods chain, also is an integral part of soil and self purification of water body function, still is a link of nature energy cycle simultaneously.The nematode that parasitizes farm crop need detect to obtain cause of disease accurately to be diagnosed; Also can detect to estimate the quality of environment for the nematode in soil and the water body.
Existing detection Plant nematode all is to continue to use the graceful funnel method of Bel.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.The soil that the plant next door is opened in pick downcuts a part of root system, is cut into the segment of 1～2cm after cleaning, and wraps in the quadrate mull, is immersed in the water.Nematode is swum out of from root tissue, 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 to belong to any kind again.(Bi Zhishu, lijin, plant nematology, agriculture press, 211-212).Though this method can be isolated nematode, has significant disadvantages: injure plant root when 1) taking a sample, the peasant is difficult to accept sometimes, particularly near the season of gathering in the crops; 2) time-consuming; 3) need to use a lot of funnels, and take a lot of lab space.
Detect soil nematodes and generally select following two kinds of methods for use:
Soil suspension tilt-pour process:
In bucket, put 3-4L water, add about 500cm then
3Soil and firmly stir to break up soil block, so nematode dissociates out.After approximately leaving standstill 1min, grogs sinks to the bottom, and nematode is just stayed in the suspension.Pour into suspension, by each tool 850,250 and 45 μ m sieve apertures (being equivalent to 20,250 and 325 order/square inches) one the cover be sieved in second bucket, again liquid is refunded first the bucket, repeat this whole process, stir as the front, allow soil avale, pour into and sieve.The water of the second last bucket sieves again, collects nematode then in sieve, promptly with the behind of thin current from sieve they is flushed in the beaker.If soil has a lot of organic substances, can allow nematode dissociate out by a device so, promptly use the cloth fastening beaker mouth of a cleaning, then beaker is placed upside down in the funnel that water arranged.
Hyperbaric solution difference centrifugal separation:
The organic substance of separate out suspended from soil at first, and soil keeps nematode, and then separate nematode with hyperbaric solution.100cm
3Soil is suspended in the 600mL water and stirs well, injects a container to remove stone, palpulus root etc. by scalping.Soil suspension branch is installed to the centrifugal 5min of each centrifuge tube.Soil contains a lot of clays usually, can the adhesion nematode on the soil face when centrifugal.If soil does not have clay, need before centrifugal, to add a small amount of kaolin (Kaolin).Outwell supernatant liquor, dry centrifuge tube top to remove foreign material.Sugared soln is (490 gram sucrose are dissolved in the 1000mL water) in pipe.Make soil resuspending and the same centrifugal in pipe, supernatant liquor is poured into the thinnest sieve and collected nematode with tap water, allow them in water, utilize gravity or recentrifuge and sink.Hyperbaric solution also can be prepared with other materials such as molasses.(1. and 2. draw work from (U.S.) V.H.Dropkin, Pan Cangsang, Lin Jingyi, the plant nematology introduction, the Xiamen: press of Xiamen University, 45-47).
Summary of the invention
Purpose of the present invention aims to provide a kind of microorganism.
Another object of the present invention aims to provide a kind ofly utilizes microorganism that soil and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube.
The said microorganism of the present invention is yeast Yr 0503, yeast Yr 0503 separates to obtain from the mud in infiltration pond of refuse landfill is inspired confidence in east, Xiamen City, Fujian Province, the classification called after yeast of bacterial strain, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, that registers on the books is numbered CGMCC No.1336, strain number: Yr 0503, preservation date: on March 24th, 2005.
The screening of yeast Yr 0503, separation, cultural method are as follows:
Preparation Corn Meal Agar substratum (CMA) is stand-by.Take mud from the bottom, infiltration pond of refuse landfill, take back the laboratory in the container of sterilizing of packing into, a little mud of picking moves in the culture dish (diameter is chosen as 9cm) of dry sterilization in sterilisable chamber.Fusing CMA substratum, treat its be chilled to solidify before (temperature is chosen as 42～45 ℃), pour in the culture dish with aseptic technique, the about 20ml of every ware covers the ware lid, immediately the rotating and culturing ware, leave standstill, treat after the substratum cooled and solidified culture dish to be put upside down, change in 25 ℃ of incubators and cultivate.When treating that agar plate surface grows bacterium colony, under anatomical lens, seek bacterium colony like oil droplet shape (glossy), its single bacterium colony of picking, on CMA or PDA agar plate with the streak inoculation of oblique line method, itself and miscellaneous microbial strains are scatter in agar plate surface, single thalline can be fixed on a bit, behind growth and breeding, form single bacterium colony, separate the purpose of purifying thereby reach.If impure, then also need to proceed separation and purification.In 25～28 ℃ incubator, cultivate behind the purifying.
The present invention is said to utilize microorganism that soil and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, the steps include:
Step 1: line taking worm detector tube, long 3～15cm (preferred 9cm), wide 1.5～5cm (preferred 2.9cm), high 1～3cm (preferred 1.5cm), the sealing of one end, the other end is open;
Step 2: the detector tube slide glass of packing into, in detector tube, add Mierocrystalline cellulose again, the mouth of pipe clogs with filtrate, and through autoclaving, take out the cooling back again;
Step 3: the preparation substratum, substratum is liquid nutrient medium or solid medium;
Step 4: the liquid nutrient medium packing with preparing, clog or encase the mouth of pipe with filtrate, through autoclaving, cooling back microbe inoculation bacterium;
Maybe, encase the mouth of pipe, behind autoclaving, topple over into the plane, microbe inoculation bacterium after the cooled and solidified with masking foil with the solid medium packing for preparing;
Said microbial bacteria is a kind of in yeast (Yr 0503), interlinkage spore (Alternaria spp.), sickle-like bacteria (Fusarium spp.), Cephalosporium species indeterminata (Cephalosporium sp.), shield kind (Phoma lingam) of restraining mould genus species indeterminata (Coniothyrium sp.) or Phoma etc.;
Step 5: cultured substratum is packed in the detector tube that step 2 finishes by part;
Step 6: in all soil of the root of soil to be measured or plant to be measured, aperture is set, in aperture, injects aqua sterilisa, with the detector tube mouth of pipe down, insert in the aperture of soil;
Step 7: the detector tube that inserts in the soil was kept somewhere in soil 3～5 days, extracted inspection then, if transparent detector tube then faces toward the tube wall inspection with anatomical lens;
If opaque detector tube is then extracted the slide glass in the pipe out after uncorking, examine under a microscope.
In step 1, detector tube holds 2 slide glasss and packs into.
In step 2, autoclaved temperature is 121 ℃, and the time is 20min, the long 7.6cm of slide glass, wide 2.6cm.
In step 3, said substratum is selected from molasses culture medium, washing water of rice substratum, potato culture (PDA), malt extract medium, rice bent juice substratum, bean sprouts medium, grass soak juice cultivate pour, at least a in maize powder medium (CMA) and the Cha Peike substratum.
In step 4, with the liquid nutrient medium packing for preparing, loading amount is 1/5～1/3 of a detector tube capacity, and temperature is 121 ℃, 1Kg/cm
2Autoclaving 20min., cooling back microbe inoculation bacterium after 3～7 days, is hidden standby 5～10 ℃ of refrigerator and cooled 25～28 ℃ of following shaking culture.
Maybe the solid medium branch for preparing is packed in vitro, loading amount is about 1/3～1/2 of pipe capacity, seals with masking foil, behind autoclaving, topple over into the plane, microbe inoculation bacterium after the cooled and solidified after cultivating 3～7 days under 25～28 ℃, is hidden standby 5～10 ℃ of refrigerator and cooled.
In step 5, cultured microbial bacteria substratum is packed into by part in the detector tube that step 2 finishes, and whenever loading amount 2～5ml by all means inhales full being advisable with the Mierocrystalline cellulose in managing.
In step 6, in aperture, inject 5～10ml aqua sterilisa.
Except the above-mentioned pipe that can insert slide glass, can also utilize the test tube that varies in size to make detector tube.When making detector tube with test tube, though save step 1 and step 2, check, convenient when identifying not as aforesaid method, must wash the tube wall of nematode come because identify the worm kind from detector tube, move on on the slide glass with suction pipe again and examine.
Any detector tube no matter can be placed in the incubator 25～28 ℃ to it and cultivate and take out microscopy again after 3～4 days, and nematode is bred like this, can improve recall rate greatly.
Detect soil and fresh water nematode and Plant nematode in this way, greatly facilitate the quarantine and examination personnel operation, help extensive detection; Make plant avoid the trouble that root is cut in digging simultaneously, reach the purpose of protective plant.Because the nematode population in soil and the plant is seldom sometimes, the present invention can lure nematode in the pipe, worm amount amplification rapidly on its cultured microorganism, thus improve the sensitivity that detects.The present invention is used for the generaI investigation of soil nematodes and the investigation of certain plants nematodiasiss, and also can utilize present method is that index is carried out environmental evaluation with the nematode that detects soil.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1
Get molasses and clear water, by 1: 8 to 1: 6 dilution proportion, or molasses and waste molasses transferred to 5.5 to 7 with pH after the mixing, with 6～6.5 the bests by 1: 20 to 1: 10 dilution proportion.The liquid nutrient medium branch for preparing is packed in the Erlenmeyer flask, loading amount is about 1/5～1/3 of flask capacity, clog or encase bottleneck with cotton plug with 8 layers of gauze, through autoclaving, cooling back inoculation yeast bacterium, 25～28 ℃ of shaking culture are after 3～7 days, fermented liquid is injected the nematode detector tube, whenever loading amount 2～5ml by all means encases the mouth of pipe with 8 layers of gauze then, perhaps clogs the mouth of pipe with sterilization cotton (or other porose media).
In soil to be measured, dig out or get out an aperture, in the hole, inject 5～10ml aqua sterilisa; Then the detector tube tipping is come to insert in the aperture of soil.
Insert with this 10 of detector tubes having inoculated the molasses fermented liquid of yeast in all soil of root of 10 banyans in the campus, extract after 3 days and take back laboratory examination, in 9 detector tubes a large amount of free-living nematodes are arranged as a result, the positive rate of free-living nematode is 90%.
Embodiment 2
Similar to Example 1, what they were different is to adopt solid medium.With the molasses solution branch that the prepares test tube of packing into, every test tube loading amount is 1/2, fastens the mouth of pipe with tin platinum paper, and encase the tube wall upper edge, behind autoclaving, fall into the plane, inoculation yeast bacterium after the cooled and solidified, 25 ℃ of cultivations are taken out after 3 days and are placed 5～10 ℃ of refrigerator and cooled Tibetan standby.
Insert the root week of 10 mandarin trees in the orchard in the soil with this 10 of the saccharomycetic molasses nutrient agar detector tubes inoculated, extract after 4 days and take back laboratory examination, in every detector tube of result a large amount of nematodes are arranged all, the nematode that little bar class is wherein arranged also has the nematode that minute hand nematode etc. can transmitted virus.The nematode positive rate is 100%.
Embodiment 3
Similar to Example 1, what they were different is substratum washing water of rice substratum, washing water of rice is left standstill for some time hypsokinesis remove supernatant liquor, and its throw out is determined as about 1.4Be with specific gravity hydrometer, and regulating pH is 6.0～6.5.
Root week of 10 strain lantanas of inserting street central authorities green belt with 10 of the detector tubes of this adding yeast washing water of rice fermented liquid extracts after 5 days and takes back laboratory examination in the soil, in 9 detector tubes a large amount of nematodes is arranged as a result, and the positive rate of nematode is 90%.
Embodiment 4
Similar to Example 1, what they were different is substratum potato juice substratum: the 200g potato is cleaned peeling, be cut into small pieces or push into filament, add 1000ml water (W: V) in 1: 5 ratio, (attention prevents burnt to boil 30min., available water-bath), use filtered through gauze, filtered juice is added water to boiling preceding volume.Add 20g sucrose again, transfer about pH to 6.
Root week of lily bulb of inserting 10 parts of imports that Entry-Exit Inspection and Quarantine Bureau provides with 10 of the detector tubes of this adding yeast potato juice fermented liquid is in the soil, extract after 3 days and take back laboratory examination, every detector tube of result all has a large amount of free-living nematodes, and the positive rate of free-living nematode is 100%.
Embodiment 5
Similar to Example 1, what they were different is the substratum malt extract medium: mixed kilned malt powder and water in 1: 4 ratio, at 58～65 ℃ of following saccharification 3～4h, obtain the wort of pol about 10 ° (Bahrain), boil the back filtered through gauze, regulate about pH to 6.
20 of detector tubes with this adding yeast attenuate liquid insert in all soil of root of 10 banana trees in the orchard, extract after 3 days and take back laboratory examination, and every detector tube of result is all had a large amount of nematodes, and the positive rate of nematode is 100%.
Embodiment 6
Similar to Example 1, they are different is substratum with the bent juice substratum of rice: aspergillus is inoculated on the boiled rice, cultivated 4 days in 28 ℃, make the rice song, take by weighing 1kg after air-dry, add water 3L, be incubated 60 ℃ of a few hours, when not having starch reaction till.Boil, use filtered through gauze, add water move to 12BX.
Insert with this 10 of detector tubes that add the bent juice saccharomycetes to make fermentation liquid of rice in all soil of root of 10 longans in the campus, extract after 4 days and take back laboratory examination, in 7 detector tubes a large amount of nematodes are arranged all as a result, the positive rate of nematode is 70%.
Embodiment 7
Similar to Example 1, what they were different is the substratum bean sprouts medium: add the 100g soybean sprout in 1000ml water, boil half an hour, use filtered through gauze, filtrate water is supplemented to 1000ml, sugaring 50g, natural pH.
10 of detector tubes with this adding yeast bean sprouts juice fermented liquid insert in all soil of root of 10 strain sponge gourds in the vegetable garden, extract after 3 days and take back laboratory examination, and every detector tube of result all has a large amount of nematodes, and the positive rate of nematode is 100%.
Embodiment 8
Similar to Example 1, what they were different is that substratum soaks the juice substratum with grass, and its prescription is: hay (pulverizing) 50g or bright careless 200g, KH
2PO
42g, 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.
10 of detector tubes that soak the juice fermented liquid with this adding yeast grass insert in the soil on 10 lawns in the campus, extract after 3 days and take back laboratory examination, in 4 detector tubes free-living nematode are arranged as a result, and the positive rate of free-living nematode is 40%.
Embodiment 9
Similar to Example 1, what they were different is that substratum is the CMA substratum, and its prescription is: corn (corn flakes) 20g, sucrose 20g, agar 17g, distilled water 1000ml.Corn flakes boil 1h under 60 ℃ 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.
10 of detector tubes with this adding yeast CMA fermented liquid insert in all soil of root of 10 calamondins in the flower nursery, extract after 5 days and take back laboratory examination, in 8 detector tubes a large amount of nematodes are arranged all as a result, and the positive rate of nematode is 80%.
Embodiment 10
Similar to Example 1, what they were different is that substratum adopts Cha Peike (Czapek) substratum, and its prescription is: NaNO
33g, K
2HPO
41g, MgSO
47H
2O 0.5g, KCl 0.5g, FeSO
47H
2O 0.01g, sucrose 30g, agar 15g, distilled water 1000ml (sucrose will be put into before facing sterilization).
10 of detector tubes with this adding yeast Cha Peike fermented liquid insert in all soil of root of 10 strain chrysanthemums in the flower nursery, extract after 3 days and take back laboratory examination, in 9 detector tubes a large amount of nematodes are arranged as a result, and the positive rate of nematode is 90%.
Embodiment 11
Similar to Example 1, what they were different is that its microorganism that inoculates is interlinkage spore (Alternaria sp.).
This attaching kind 20 of the potato juice nutrient agar detector tubes of interlinkage spore insert in all soil of root of 10 mandarin trees in the orchard, extract after 3 days and take back laboratory examination, in 16 detector tubes a large amount of nematodes are arranged as a result, the positive rate of nematode is 80%.Aphelenchus avenae (Aphelenchus avenae), wall pellitory aphelenchoides (Aph.parietinus) etc. are wherein arranged, also have more free-living nematode and other nematodes in addition.
Embodiment 12
Similar to Example 1, what they were different is that its microorganism that inoculates is sickle-like bacteria (Fusarium sp.).This attaching kind 20 of the potato juice nutrient agar detector tubes of sickle-like bacteria insert in all soil of root of 10 mandarin trees in the orchard, extract after 5 days and take back laboratory examination, in 18 detector tubes a large amount of nematodes are arranged as a result, the positive rate of nematode is 90%.Aphelenchus avenae (Aphelenchus avenae) etc. is wherein arranged, also have more free-living nematode and other nematodes in addition.
Embodiment 13
Similar to Example 1, what they were different is that its microorganism that inoculates is Cephalosporium species indeterminata (Cephalosporium sp.).This attaching kind 10 of the mould potato juice nutrient agar detector tubes of cephalo root week in the soil of inserting 10 banyans in urban district, extract after 4 days and take back laboratory examination, in 9 detector tubes a large amount of nematodes are arranged as a result, the positive rate of nematode is 90%.More free-living nematode and other nematodes are wherein arranged.
Embodiment 14
Similar to Example 1, what they were different is that its microorganism that inoculates is that shield restrains mould genus species indeterminata (Coniothyrium sp.).This attaching kind 10 mould of potato juice nutrient agar detector tubes of shield gram insert in all soil of root of 10 strain purples Soviet Union in the flower nursery, extract after 4 days and take back laboratory examination, in 9 detector tubes a large amount of nematodes are arranged as a result, the positive rate of nematode is 90%.Root knot nematode (Meloidogyne spp.) larva etc. is wherein arranged, also have more free-living nematode in addition.
Embodiment 15
Similar to Example 1, what they were different is that its microorganism that inoculates is a kind (Phoma lingam) of Phoma.This attaching kind 10 of the mould potato juice nutrient agar detector tubes of stem point insert in all soil of root of 10 strain Flos Celosiae Cristataes in the flower nursery, extract after 3 days and take back laboratory examination, in 8 detector tubes a large amount of nematodes are arranged as a result, the positive rate of nematode is 80%.Root knot nematode (Meloidogyne spp.) larva etc. is wherein arranged, also have more free-living nematode in addition.
Claims (10)
1, a kind of microorganism, it is characterized in that microorganism is yeast Yr0503, the classification called after yeast of bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, that registers on the books is numbered CGMCCNo.1336, strain number: Yr0503, preservation date: on March 24th, 2005.
2, utilize a kind of microorganism as claimed in claim 1 that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that the steps include:
Step 1: line taking worm detector tube, long 3～15cm, wide 1.5～5cm, high 1～3cm, the sealing of one end, the other end is open;
Step 2: the detector tube slide glass of packing into, in detector tube, add Mierocrystalline cellulose again, the mouth of pipe clogs with filtrate, and through autoclaving, take out detector tube cooling back;
Step 3: the preparation substratum, substratum is liquid nutrient medium or solid medium;
Step 4: the liquid nutrient medium packing with preparing, clog or encase the mouth of pipe with filtrate, through autoclaving, cooling back microbe inoculation bacterium;
Maybe, encase the mouth of pipe, behind autoclaving, topple over into the plane, microbe inoculation bacterium after the cooled and solidified with masking foil with the solid medium packing for preparing;
Said microbial bacteria is yeast Yr0503;
Step 5: cultured substratum is packed in the detector tube that step 2 finishes by part, encase the mouth of pipe with 8 layers of gauze then, perhaps clog the mouth of pipe with sterilize cotton or other porose media;
Step 6: in all soil of the root of soil to be measured or plant to be measured, aperture is set, in aperture, injects aqua sterilisa, with the detector tube mouth of pipe down, insert in the aperture of soil;
Step 7: the detector tube that inserts in the soil was kept somewhere in soil 3～5 days, extracted inspection then, to transparent detector tube, faced toward the tube wall inspection with anatomical lens;
To opaque detector tube, extract the slide glass in the pipe after uncorking out, examine under a microscope.
3, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 1 line taking worm detector tube, long 9cm, wide 2.9cm, high 1.5cm, the sealing of one end, the other end is open.
4, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 1 detector tube holds 2 slide glasss and packs into.
5, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 2 autoclaved temperature is 121 ℃, the time is 20min, the long 7.6cm of slide glass, wide 2.6cm.
6, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 3, said substratum is selected from molasses culture medium, the washing water of rice substratum, potato culture, malt extract medium, rice bent juice substratum, bean sprouts medium, grass soak at least a in juice substratum, maize powder medium and the Cha Peike substratum.
7, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 4 with the liquid nutrient medium packing for preparing, loading amount is 1/5～1/3 of a detector tube capacity, 121 ℃, 1Kg/cm
2Autoclaving 20min., cooling back microbe inoculation bacterium after 3～7 days, is hidden standby 5～10 ℃ of refrigerator and cooled 25～28 ℃ of following shaking culture.
8, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 4, the solid medium branch for preparing is packed in vitro, loading amount is 1/3～1/2 of a pipe capacity, seal with masking foil, behind autoclaving, topple over into the plane, microbe inoculation bacterium after the cooled and solidified, after cultivating 3～7 days under 25～28 ℃, hide standby 5～10 ℃ of refrigerator and cooled.
9, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 5, cultured microbial bacteria substratum is packed into by part in the detector tube that step 2 finishes, whenever loading amount 2～5ml by all means inhales full being advisable with the Mierocrystalline cellulose in managing.
10, as claimed in claim 2ly utilize microorganism that soil nematodes and fresh water nematode and Plant nematode are lured the detection method of identifying into detector tube, it is characterized in that in step 6, in aperture, inject 5～10ml aqua sterilisa.
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| CN1275549A (en) * | 2000-07-03 | 2000-12-06 | 吉林大学 | Conversion of domestic refuse into high-effect biological organic fertilizer by using biological engineering technology |
| CN1420180A (en) * | 2002-08-23 | 2003-05-28 | 厦门大学 | Method for early diagnosis and detection of pine nematode disease |
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| CN1275549A (en) * | 2000-07-03 | 2000-12-06 | 吉林大学 | Conversion of domestic refuse into high-effect biological organic fertilizer by using biological engineering technology |
| CN1420180A (en) * | 2002-08-23 | 2003-05-28 | 厦门大学 | Method for early diagnosis and detection of pine nematode disease |
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