CN1300817A - Liquid culture method for pathogenic nematode of insect - Google Patents
Liquid culture method for pathogenic nematode of insect Download PDFInfo
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- CN1300817A CN1300817A CN 99117297 CN99117297A CN1300817A CN 1300817 A CN1300817 A CN 1300817A CN 99117297 CN99117297 CN 99117297 CN 99117297 A CN99117297 A CN 99117297A CN 1300817 A CN1300817 A CN 1300817A
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Abstract
A liquid culture method for the pathogenic nematode of insecticide, used as biologic insecticide features that the antibacterial substance is added to the liquid medium in the key development phase of said nematode to change somatic density, induce the symbiotic bacteria to generate variation, control the infection-phase nematodes and increase the output of infection-phase nematodes (up to 95% in 8-16 days).
Description
The present invention relates to the entomopathogenic nematode of liquid cultivating method mass production as biotic pesticide.
Insect cause of disease Si Shi Steinernematidae of section and different Rhabditidae Heterorhabditidae nematode are the new bio sterilants.This class nematode has host range widely; To host's tool active search capability, particularly to dwelling property of soil and borer pest; To people and animals, environmental safety.In recent years, be widely used in insects such as control farming, woods, herbage and health, be subjected to the great attention of Chinese scholars and commercial department, and move towards commercialization.
This class nematode is with period of infection worm attitude (infective juveniles, IJ) enter in the insect body with host's food or from natural opening (as anus, pore), the intersegmental membrane of insect, discharge subsequently that the Xenorhabdus that carries in the enteric cavity belongs to (at present with the nematode symbiosis of Si Shi section) or Photorhabdus belongs to the symbiotic bacterium of (at present and the symbiosis of different Rhabditidae nematode).Nematode and symbiotic bacterium excretory toxin (virulence factor) cause insect death.
Commercialization is moved towards in the production of the big area field application requiring nematode of entomopathogenic nematode.At present, the industrialization culture systems of nematode adds single symbiotic bacterium by aseptic technique in various artificial mediums and aseptic nematode is finished, i.e. the outer culture systems of single thalline of nematode.Can be divided into solid culture [Bedding, R.A. (1984) Annals of Applied Biology 104,117-120 according to culture medium; Wouts, W.M. (1981) Journal of Nematology 13,467-469] and liquid culture [Pace et al. (1986) PCT Patent Application No.86/01074; Friedman et al. (1991) PCT PatentApplication No.89/04602; Surrey ﹠amp; Davies (1996) Journal of InvertebratePathology 67,92-99; Han, R.C. (1996) Nematologica 42,546-553; Ehlerset al. (1998) Biocontrol 43:77-86; Tachibana et al. (1997) US patent, Application No.403879].The solid culture method technology content is relatively low, and operation can adapt to multiple nematode easily.But need a large amount of manpowers, space, send the amplification quantity refuse.Liquid culture has overcome the deficiency of solid culture, is the main method that the nematode industrialization is produced.
Comprise that ovum, is to four-age larva and adult stage the life history of entomopathogenic nematode.This type of nematode all has the period of infection worm attitude in one three age.The nematode in this length of time is not got food, can survive in soil one period long period, can invade the host insect that may exist at any time.In the culture systems, the typical development models of nematode is as follows outside single thalline of nematode.Behind nematode and symbiotic bacterium access substratum, semiochemicals (Food signal) the inductive infection phase elegans development by symbiotic bacterium produces becomes three length of times (L3) from period of infection, and four length of times, (L4) was to adult.After the ovum hatching, from first-instar young (L1), second instar larvae (L2), good as nutritional condition, continue to grow to L3 L4 and adult of new generation; Worsen as nutritional condition, L2 can grow and be the period of infection nematode.A new generation period of infection nematode still can continue to grow for L3, carries out new life cycle.The Stahli line worm is adopted dioecious modes of reproduction; And the first-generation adult that heterorhabditis indica is grown from infective stage larva is strict telianthus, and s-generation adult can exist dioecism and hermaphroditic mixing individuality.The infective stage larva of entomopathogenic nematode is the worm age that can infect insect host.On using, have only the period of infection nematode that environmental factor is had certain patience, and influential to insect.Therefore, during the external liquid culture of nematode, ratio and culture cycle that the period of infection nematode accounts for other worm age are most important.
In general, in nutritious substratum, add highdensity nematode will help obtaining maximum number in the shortest time period of infection nematode.But it is add the breeding rate that highdensity nematode will reduce nematode, and uneconomical economically.If nematode can reach the period of infection worm attitude of maximum quantity in the first-generation, will shorten incubation time and reduce the cultivation cost.Pace et al. (1986) (PCT Patent Application No.86/01074) report is reduced to 15 ℃ with sterile distilled water dilution nutrient solution and with culture-liquid temp, can obtain the period of infection nematode of 60-90%.But during the operation of this method, not only inconvenience but also increase expense.Described the substratum of nematode in the patent of Friedman et al. (1989) Tachibana et al. (1997), and controlled the method for stir speed (S.S.) the different length of time according to nematode, but not mentioned control infection phase method of forming of nematode.
The objective of the invention is to propose a kind of liquid cultivating method that can control and improve the entomopathogenic nematode of period of infection nematode production.
The liquid cultivating method of entomopathogenic nematode provided by the present invention, its content comprises: in artificial bacteria culture medium commonly used, insert symbiotic bacterium and the nematode of the phase of coming into being nematode down in aseptic condition, under the condition of stirring, oxygen supply, for example in shaking table concussion cultivation or oxygen supply fermentor tank, carrying out the nematode liquid culture, being characterized in the growth period nematode, mainly is to grow in the key of nematode to add antimicrobial substance (being antibiotic) length of time.The antibiotic that uses comprises that all have inhibiting kind to nematode symbiosis bacterial strain, as duomycin (Chlorotetracycline), Streptomycin sulphate (Streptomycin), paraxin (Chloramphenicol), penbritin (Ampicillin), Nalidixic acid etc., what preferentially select is Streptomycin sulphate.The antibiotic agent weight range is 0.01-1 μ g/ml, according to different symbiosis bacterial strains and antibiotic kind and different.The antibiotic of this dosage range can make part bacterial strain (＜20%CFU; CFU=colony forming unit) the generation type becomes.After the key of described nematode is grown and is typically chosen in age nematode L1 appearance of new generation the length of time.
The nematode of being inserted in artificial bacteria culture medium commonly used described in the inventive method can be the nematode in the various length of times, and is the best to insert the period of infection nematode.
The present invention is in the growth length of time of nematode liquid culture, the anomaly that utilizes antimicrobial substance in nutrient solution, to reduce bacterial density and induce symbiotic bacterium, reduced the nutritional information of bacterium liquid, change the physiological status of part symbiotic bacterium, formation with control and raising period of infection nematode, nematode larval is grown to period of infection nematode direction, in addition, also controlled the further growth of the period of infection nematode that had formed already.The present invention can improve the ratio of period of infection nematode in the nutrient solution greatly, shortens incubation time, improves the liquid culture effect of entomopathogenic nematode.Present method can make nematode (different according to line insect types) in 8-16 days reach a high proportion of period of infection worm attitude (＞95%).
Following experiment and operational instances are further to explanation of the present invention.Be noted that these examples all are illustrative, should not be used as limitation of the present invention.Only describe S.carpocapsae A24 nematode and H.bacteriophora H06 nematode in the example, but the method for this patent is applicable to all nematode kind and strains.
Embodiment one
In the 500ml triangular flask, add 100ml liquid nutrient medium (2% analysis for soybean powder, 1% flour, 0.5% yeast extract paste, 1.2% powdered egg, 3% Semen Maydis oil and 92.3% water), 121 ℃ of following autoclave sterilizations 30 minutes.Insert the nascent type fungal component (5 * 10 of isolated X.nematophilus from S.carpocapsae A24 nematode then
8Thalline), place under 25 ℃, 150rpm shaking table to cultivate 48 hours, insert 5000/ml S.carpocapsae period of infection nematode again, insert behind the nematode second day and rise to take a sample and check the developmental state of nematode in anatomical lens down, cultivate after 4 days adding 0.05 μ g/ml Vetstrep in culturing bottle.After 48 hours, take a sample and check down the developmental state of nematode in anatomical lens, connect behind the worm the 8th, 10,12 and 16 days counting nematode productions and period of infection nematode ratio.
Embodiment two
In the 500ml triangular flask, add 100ml liquid nutrient medium (2% analysis for soybean powder, 1% flour, 0.5% yeast extract paste, 1.2% powdered egg, 3% Semen Maydis oil and 92.3% water), 121 ℃ of following autoclave sterilizations 30 minutes.Insert the nascent type fungal component (5 * 108 thalline) of isolated P.luminescens from the H.bacteriophora nematode then, place under 25 ℃, 150rpm shaking table to cultivate 48 hours, insert 5000/mlH.bacteriophora period of infection nematode again, insert behind the nematode second day and rise to take a sample and check the developmental state of nematode in anatomical lens down, cultivate after 6 days adding 0.05 μ g/ml Vetstrep in culturing bottle.After 48 hours, take a sample and check down the developmental state of nematode in anatomical lens, connect behind the worm the 8th, 10,12 and 16 days counting nematode productions and period of infection nematode ratio.
The isolating method of the nascent type symbiotic bacterium of the foregoing description one and embodiment two described Xenorhabdus and Photorhabdus is as follows: from the greater wax moth end larva Galleria mellonella in age (seven ages) that entomopathogenic nematode infects, place under 25 ℃, behind the insect death (general 2 to 4 days), with the 70% alcohol body surface dead worm of sterilizing, cut off polypide with sterile scissors then, get hemolymph and draw NBTA, NA and Mai Kangkai flat board.(Journal of General Microbiology 121,303-309) method of describing is identified the nematode symbiotic bacterium according to Akhurst (1980).From S.carpocapsae A24 nematode, isolate the X.nematophilus fungal component; From the H.bacteriophora nematode, isolate the P.luminescens fungal component.
Embodiment one and embodiment two described entomopathogenic nematode liquid cultivating methods and ordinary method (promptly not adding antimicrobial substance) comparative structure shows: after adding Vetstrep, the period of infection nematode ratio of S.carpocapsae nematode in the time of 8 days reaches 95%, and the period of infection nematode production is 267 * 1000/ml; And be 6% with the period of infection nematode ratio of the culturing bottle of Vetstrep, the period of infection nematode production is 18 * 1000/ml, though the nematode ultimate production is 304 * 1000/ml.After 12 days, the period of infection nematode ratio that adds the culturing bottle of Vetstrep reaches 97%, and is 32% with the period of infection nematode ratio of the culturing bottle of Vetstrep.As seen, after the adding Vetstrep, nematode is grown to period of infection worm attitude, shortened incubation time.
After adding Vetstrep in the H.bacteriophora culture of nematodes liquid, the period of infection nematode ratio in the time of 10 days reaches 96%, and the period of infection nematode production is 197 * 1000/ml; And be 84% with the period of infection nematode ratio of the culturing bottle of Vetstrep, the period of infection nematode production is 178 * 1000/ml.After 16 days, the period of infection nematode ratio that adds the culturing bottle of Vetstrep reaches 98%, and is 89% with the period of infection nematode ratio of the culturing bottle of Vetstrep.As seen, after the adding Vetstrep, nematode is grown to period of infection worm attitude, shortened incubation time.
Claims (5)
1. the liquid cultivating method of an entomopathogenic nematode, be included in the artificial medium, insert nascent molded lines worm symbiotic bacterium and nematode down in aseptic condition, under the culture environment of ventilation, stirring, carry out the nematode liquid culture, it is characterized in that adding antimicrobial substance in the growth period of nematode.
2. the liquid cultivating method of entomopathogenic nematode according to claim 1, the joining day that it is characterized in that described antimicrobial substance is the crucial length of time of growth after the first-instar young L1 of nematode a new generation occurs.
3. the liquid cultivating method of entomopathogenic nematode according to claim 1, the add-on that it is characterized in that described antimicrobial substance is every milliliter of substratum 0.01～1 μ g.
4. according to the liquid cultivating method of claim 1 or 2 or 3 described entomopathogenic nematodes, it is characterized in that described antimicrobial substance is a Streptomycin sulphate.
5. the liquid cultivating method of entomopathogenic nematode according to claim 1, the period of infection nematode of the nematode of in artificial medium, being inserted in it is characterized in that cultivating.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307299C (en) * | 2001-06-29 | 2007-03-28 | 鱼生化技术有限公司 | Process for storing enriched nematodes |
| CN1320107C (en) * | 2005-06-16 | 2007-06-06 | 西北农林科技大学无公害农药研究服务中心 | Separation method of entomopathogenic nematode symbiotic bacteria |
| CN1325631C (en) * | 2004-11-26 | 2007-07-11 | 中国农业科学院农业环境与可持续发展研究所 | Culture of entomiasis primitive nematode by enzymolyed culture medium |
| CN100415873C (en) * | 2006-10-17 | 2008-09-03 | 广东省昆虫研究所 | Bacteria strain (X-7 strain) for culturing bait eelworm and its eelworm culture method |
| CN100560717C (en) * | 2005-03-10 | 2009-11-18 | 中国科学院等离子体物理研究所 | The method of agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm |
| CN101041810B (en) * | 2006-10-17 | 2010-09-22 | 广东省昆虫研究所 | Bacteria strain (KG strain) for culturing bait nematode and nematode culture method |
| CN101485300B (en) * | 2008-01-18 | 2011-05-11 | 中国科学院沈阳应用生态研究所 | Method for cultivating Acrobeloides nanus |
| CN104222023A (en) * | 2014-08-30 | 2014-12-24 | 北京安和亿泰生物工程技术有限公司 | Steinernema batch breeding and culture method |
| CN109169534A (en) * | 2018-10-15 | 2019-01-11 | 南开大学 | Nematode solid medium and its preparation method and application |
| CN109644945A (en) * | 2018-12-14 | 2019-04-19 | 广东省生物资源应用研究所 | A kind of method of fast quick-recovery Steinernema carpocapsae infection phase nematode |
| US10683121B2 (en) | 2015-08-18 | 2020-06-16 | Biosystems Technology Limited | Method for preparation of research organisms |
| CN113796463A (en) * | 2021-08-19 | 2021-12-17 | 云南省烟草公司玉溪市公司 | Application of dimethyl sulfoxide in preparation of preparation for improving yield of entomopathogenic nematodes |
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1999
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307299C (en) * | 2001-06-29 | 2007-03-28 | 鱼生化技术有限公司 | Process for storing enriched nematodes |
| CN1325631C (en) * | 2004-11-26 | 2007-07-11 | 中国农业科学院农业环境与可持续发展研究所 | Culture of entomiasis primitive nematode by enzymolyed culture medium |
| CN100560717C (en) * | 2005-03-10 | 2009-11-18 | 中国科学院等离子体物理研究所 | The method of agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm |
| CN1320107C (en) * | 2005-06-16 | 2007-06-06 | 西北农林科技大学无公害农药研究服务中心 | Separation method of entomopathogenic nematode symbiotic bacteria |
| CN100415873C (en) * | 2006-10-17 | 2008-09-03 | 广东省昆虫研究所 | Bacteria strain (X-7 strain) for culturing bait eelworm and its eelworm culture method |
| CN101041810B (en) * | 2006-10-17 | 2010-09-22 | 广东省昆虫研究所 | Bacteria strain (KG strain) for culturing bait nematode and nematode culture method |
| CN101485300B (en) * | 2008-01-18 | 2011-05-11 | 中国科学院沈阳应用生态研究所 | Method for cultivating Acrobeloides nanus |
| CN104222023A (en) * | 2014-08-30 | 2014-12-24 | 北京安和亿泰生物工程技术有限公司 | Steinernema batch breeding and culture method |
| US10683121B2 (en) | 2015-08-18 | 2020-06-16 | Biosystems Technology Limited | Method for preparation of research organisms |
| CN109169534A (en) * | 2018-10-15 | 2019-01-11 | 南开大学 | Nematode solid medium and its preparation method and application |
| CN109644945A (en) * | 2018-12-14 | 2019-04-19 | 广东省生物资源应用研究所 | A kind of method of fast quick-recovery Steinernema carpocapsae infection phase nematode |
| CN109644945B (en) * | 2018-12-14 | 2022-07-26 | 广东省生物资源应用研究所 | Method for rapidly recovering nematodes in Steinernema carpocapsae infection stage |
| CN113796463A (en) * | 2021-08-19 | 2021-12-17 | 云南省烟草公司玉溪市公司 | Application of dimethyl sulfoxide in preparation of preparation for improving yield of entomopathogenic nematodes |
| CN113796463B (en) * | 2021-08-19 | 2023-07-18 | 云南省烟草公司玉溪市公司 | Application of dimethyl sulfoxide in preparation of preparation for improving yield of entomopathogenic nematodes |
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