CN102318585A - Method for purification culture of sweet potato rot stem nematodes - Google Patents
Method for purification culture of sweet potato rot stem nematodes Download PDFInfo
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- CN102318585A CN102318585A CN201110128860A CN201110128860A CN102318585A CN 102318585 A CN102318585 A CN 102318585A CN 201110128860 A CN201110128860 A CN 201110128860A CN 201110128860 A CN201110128860 A CN 201110128860A CN 102318585 A CN102318585 A CN 102318585A
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 244000017020 Ipomoea batatas Species 0.000 title claims abstract description 28
- 235000002678 Ipomoea batatas Nutrition 0.000 title claims abstract description 28
- 241000399949 Ditylenchus dipsaci Species 0.000 title abstract description 8
- 238000000746 purification Methods 0.000 title abstract description 4
- 241000244206 Nematoda Species 0.000 claims abstract description 45
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 45
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000008223 sterile water Substances 0.000 claims abstract description 9
- 239000012188 paraffin wax Substances 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 238000007789 sealing Methods 0.000 claims abstract description 7
- 239000013536 elastomeric material Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 5
- 230000007614 genetic variation Effects 0.000 abstract description 2
- 239000013013 elastic material Substances 0.000 abstract 1
- 230000003950 pathogenic mechanism Effects 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 230000009897 systematic effect Effects 0.000 abstract 1
- 238000009395 breeding Methods 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 241000123650 Botrytis cinerea Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 238000004080 punching Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 101100008044 Caenorhabditis elegans cut-1 gene Proteins 0.000 description 2
- 241000399948 Ditylenchus destructor Species 0.000 description 2
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- 235000002767 Daucus carota Nutrition 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000427940 Fusarium solani Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
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- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
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- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
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- 238000012364 cultivation method Methods 0.000 description 1
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Abstract
The invention discloses a method for purification culture of sweet potato rot stem nematodes, which comprises the following steps of: (1) nematode preparation: picking female nematode in the rot stem nematode population by a needle to be placed onto a slide under a 40* microscope, further confirming the female nematode to be gestational female nematode under a 100* microscope, and then, placing the gestational female nematode into a container for use; and (2) purification culture: transversely or vertically cutting the whole potato, perforating holes on one cross section, injecting little sterile water into each hole in a standard that the water does not overflow from the holes, then, inoculating the rot stem nematode obtained in the first step into the small holes one by one, then, covering the cut potato blocks onto the potato blocks inoculated with the rot stem nematode as a natural moisture preservation cover, sealing the edge gaps by paraffin wax, placing the potato blocks into a culture box after being bound by elastic materials, carrying out culture for 30 days at 25 DEG C, taking down the small holes inoculated with the rot stem nematode by a perforator with the diameter being 13mm and respectively carrying out nematode separation. The method provided by the invention is simple, the operation is easy, the workload is low, more importantly, the purity of the obtained group is high, the vitality is good, and the assurance is provided for the systematic study work on the biological characteristics, the pathogenic mechanisms, the population groups, the individual genetic variation and the like of the nematode.
Description
Technical field
The invention belongs to the plant pathogeny line insect field of biology, relate to the stem eelworm population high-quality purifying cultured method of rotting.
Technical background
The rotten stem eelworm disease of sweet potato is the important disease on China's agricultural, mainly is distributed in provinces and regions such as Henan, Hebei, Shandong, Shanxi, Anhui, Jiangsu at home.Its pathogen sweet potato rot stem eelworm (
Ditylenchus Destructor) on potato, find, cause potato rotten (Thorne G proposed in 1945).Sweep stem nematode is one of the important disease in China sweet potato producing region, and the rotten stem eelworm of its cause of disease is the important quarantine sex object of China.Improper owing to introducing a fine variety in the sweet potato production in recent years, cause the large tracts of land of sweep stem nematode to spread.Endanger more and more serious, the production that has jeopardized sweet potato.This disease causes by eelworm harm, and cause of disease is through Agricultural University Of Nanjing's fourth good fortune again, evaluations such as Lin Maosong be potato rot stem eelworm (
Ditylenchus destructoR), this is the only pathogen that causes plant dry rot symptom in the plant nematode, and sweet potato receives infecting of the rotten stem eelworm of potato, the lighter's underproduction 10%-30%, and weight person reaches 50%-80%, even total crop failure.This nematode also endangers carrot, peanut, peppermint etc. except harm potato, sweet potato.
Acquisition colony purity is high, the good nematode of vitality is the basis and the prerequisite of work such as this nematode biological property of system research, pathogenesis, population colony and individual inheritance variation.
At present system carries out the method that purifying is cultivated this nematode, generally obtain through following several kinds of channels, as in P D A flat board, insert the rotten sickle-like bacteria of eggplant (
Fusariumsolani), Botrytis cinerea (
Botrytis cinereaPers) and cupreum (
Chaetomiumsp), secretly in 25 ℃ of incubators cultivate the stem eelworm of rotting, in addition people such as Liu Bin report the Botrytis cinerea bacterium of adopting corn culture medium to cultivate (
Botrytis cinerea) the different live body of list that the stem eelworm of rotting is carried out cultivates, indoor can be fast and a large amount of breedings stem eelworm (Liu Bin, Zheng Jingwu, 2006, the agriculture journal in Zhejiang) of rotting; People such as Ruan Long report method (Ruan Long, Wu Fei, the Jiang Jiang etc. that adopt the rotten stem eelworm of sweet potato root of hair list host cultivating system breeding potato; The biotechnology circular; 2006 the 6th phases), above-mentioned purification cultivation method all is to succeed through inoculating a large amount of nematodes, and the successful basis of purifying is the removal of before a large amount of nematode inoculations, carrying out other polypides such as saprophitic nematode; Wait the people to be reported in 400 to 500 rotten stem eelworms of first sucking-off before the inoculation nematode as lacquer is red forever; Then microscopically remove saprophitic nematode (coat with lacquer red forever, Li Xiuhua, horse is beautiful. North China agronomy is reported. 2008).If said method nematode population purity in practical application is not high; And carry the words of other soil nematodess; Removing the required workload of these assorted worms must strengthen, and carries assorted worm unavoidably, has reduced the purity of cultivating; This makes troubles for quick inoculation, high-quality purifying nematode; Brought facility for a large amount of fast breeding nematodes though simultaneously above-mentioned purifying is cultivated propagation method,, possibly change aspect the biological propertyes such as pathogenicity if the stem eelworm of rotting is cultivated (under the situation that the host changes) in breeding over a long time on the fungi.People such as Ruan Long report and adopt the method for the rotten stem eelworm of sweet potato root of hair list host cultivating system breeding potato not only to exist above-mentioned defective, and method is very numerous and diverse.Therefore, high-quality purifying cultivation nematode seems extremely important.
Summary of the invention
The technical problem that the present invention will solve provides a kind of purifying and cultivates the rot method of stem eelworm of sweet potato, not only simple, the operation easily of this method, and also the nematode population purity that obtains is high, vitality is good.
The present invention realizes through following technical scheme:
A kind of purifying is cultivated the method for the rotten stem eelworm of sweet potato, comprises the following steps:
(1) preparation of nematode
Big instar larvae, male worm with the rotten stem eelworm of pin picking under 40 * Stereo microscope are put in the vessel subsequent use;
(2) purifying is cultivated
To put in order potato and laterally or vertically cut, punch on the section therein, in each hole, inject a little sterile water; Be as the criterion with overfolw hole not; The rotten stem eelworm that afterwards step (1) is obtained, a big instar larvae and two male worms are inserted in the aperture, the potato piece that downcuts are covered on the potato piece after inserting the stem eelworm of rotting as natural moisture preserving again; With paraffin that the edge gap sealing is good; And lie in the incubator with after the elastomeric material binding, cultivated 30 days in 25 ℃, using diameter is that aperture that 13 millimeters card punch will be inoculated nematode takes off and carries out nematode respectively and separate.
The further improvement project of the present invention is that a kind of purifying is cultivated the method for the rotten stem eelworm of sweet potato, comprises the following steps:
(1) preparation of nematode
Under 40 * Stereo microscope, be placed on the slide, further confirm as the female worm of becoming pregnant, be put in the vessel subsequent use then at 100 * microscopically with the rotten female worm of stem eelworm population of pin picking;
(2) purifying is cultivated
To put in order potato and laterally or vertically cut, punch on the section therein, in each hole, inject a little sterile water; Be as the criterion with overfolw hole not; The rotten stem eelworm that afterwards step (1) is obtained, one is inserted in the aperture, the potato piece that downcuts is covered on the potato piece after inserting the stem eelworm of rotting as natural moisture preserving again; With paraffin that the edge gap sealing is good; And be placed in the incubator with after the elastomeric material binding, cultivated 30 days in 25 ℃, using diameter is that aperture that 13 millimeters card punch will be inoculated nematode takes off and carries out nematode respectively and separate.
Above-mentioned two kinds of methods, the diameter in hole is 2-6 millimeter (the too big drift easily of diameter, and waste potato piece) on the sweet potato section; The degree of depth is the 10-30 millimeter; The spacing of Kong Yukong is 2.5-4 centimetre (separate as long as the nematode after being convenient to cultivate is taken out, pitch of holes should be smaller as far as possible, practices thrift the potato piece).
In incubator, spray small amount of aseptic water in advance, to keep the moistening growing environment of potato piece.
In each hole, inject a little sterile water, when its purpose was to inoculate nematode, it was free in sterile water by choosing pin to be convenient to nematode, and it is deadly with the extruding of potato piece to be unlikely to be chosen pin.Above-mentioned two kinds of methods repeat respectively 20 times.
The rotten stem eelworm that at every turn obtains is all put into the counting ware separate observation, initial data is carried out standardization, use DPS statistical software deal with data, the calculating horizontal mean also carries out otherness relatively.
The result shows, the purifying culture experiment that the stem eelworm colony of rotting adopts become pregnant the female worm of wall scroll and two kinds of methods of nematode selfing to carry out population is feasible.Single female purifying is cultivated and is compared with this nematode parental autocopulation, and the probability of purifying reproductive success significantly increases, and reaches 65%, cultivates that to separate observation offspring par after 30 days be 84.15, on average raises up seed several 137.4 at P than parental autocopulation
0.05Significantly reduce on the level, think maybe the part pieces of an egg have excreted before the female worm inoculation of wall scroll due to.Two kinds of relative homogeneous of nematode population regularity that method was separated to are also found in research, but it is less relatively to adopt single female purifying to breed required workload, is about 2 hours; And adopt the workload of nematode parental autocopulation relatively large, be about 4 hours; Other discovers that there is not the parthenogenesis phenomenon in the stem eelworm of rotting, and it raises up seed and can only carry out through sexual reproduction.
Table 1 is that single female purifying is cultivated and parental autocopulation purifying cultivation results statistics
Annotate: average back algebraically a, b represent at P respectively in the table
0.05There is significance on the level.Be that 137.4 of the average back of parental autocopulation algebraically and 84.15 of the average back of single female breeding algebraically have 95% confidence level in the performance of quantity variance significance.
The present invention compared with prior art has following obvious advantage:
The present invention cultivates the stem eelworm of rotting with two kinds of method purifying; All obtain effect preferably respectively; Method is simple, operation easily, workload are little; Colony's purity that what is more important obtains is high, vitality is good, is the assurance that provides of the biological property of this nematode of system research, pathogenesis, population colony and individual work such as genetic variation.
Description of drawings
Fig. 1 is potato piece section punching sketch map.
Embodiment
Embodiment 1
Supply the rotten stem eelworm colony of examination to provide in the present embodiment by Agricultural University Of Nanjing Plant Protection cinemas worm laboratory.Used potato piece kind is Su Yu 303 (to the stem eelworm non-resistant that rots), is provided by the academy of agricultural sciences, Jiangsu Province.
A kind of purifying is cultivated the method for the rotten stem eelworm of sweet potato, comprises the following steps:
(1) preparation of nematode
Under 40 * Stereo microscope, be placed on the slide, further confirm as the female worm of becoming pregnant, be put in the concave surface ware subsequent use then at 100 * microscopically with the rotten female worm of stem eelworm population of pin picking;
(2) purifying is cultivated
To put in order potato and vertically cut 1/3rd (obtaining the potato piece 2 of 1/3rd potato piece 2/1sts, three), punching is (as shown in Figure 1 on the section of 2/3rds potato pieces; The diameter in hole 3 is 3 millimeters on the sweet potato section, and the degree of depth is 20 millimeters, and the spacing of Kong Yukong is 3 centimetres); In each hole, inject a little sterile water, be as the criterion the rotten stem eelworm that afterwards step (1) is obtained with overfolw hole not; Article one, insert in the aperture, again 1/3rd potato piece 1 is covered on 2/3rds the potato piece 2 after inserting rotten stem eelworm as natural moisture preserving, with paraffin that the edge gap sealing is good; And with being placed on after the elastomeric material binding in the incubator (before the placement; In incubator, spray small amount of aseptic water in advance, to keep the moistening growing environment of potato piece), obtained some pure rotten stem eelworms in 30 days in 25 ℃ of cultivations.
Selecting the aperture when in the present embodiment, separating nematode for use is that the card punch of 13mm takes out hole 3 and separates.Figure
Shown in, have 20 holes 3.Separating resulting is seen single female breeding in the table 1.
Embodiment 2
A kind of purifying is cultivated the method for the rotten stem eelworm of sweet potato, comprises the following steps:
(1) preparation of nematode
The female worm of above the average age for marriage children, male worm with the rotten stem eelworm of pin picking under 40 * Stereo microscope are put in the vessel subsequent use;
(2) purifying is cultivated
To put in order potato and vertically cut 1/3rd (obtaining the potato piece 2 of 1/3rd potato piece 2/1sts, three), punching is (as shown in Figure 1 on the section of 2/3rds potato pieces; The diameter in hole 3 is 3 millimeters on the sweet potato section, and the degree of depth is 20 millimeters, and the spacing of Kong Yukong is 3 centimetres); In each hole, inject a little sterile water; Be as the criterion with overfolw hole not, the rotten stem eelworm that afterwards step (1) is obtained, a female worm of above the average age for marriage children and two male worms are inserted in the aperture; Again 1/3rd potato piece 1 is covered on 2/3rds the potato piece 2 after inserting the stem eelworm of rotting as natural moisture preserving; With paraffin that the edge gap sealing is good, and (before the placement, in incubator, spray small amount of aseptic water in advance with lying in the incubator after the elastomeric material binding; To keep the moistening growing environment of potato piece), obtained some pure rotten stem eelworms in 30 days in 25 ℃ of cultivations.
Selecting the aperture when in the present embodiment, separating nematode for use is that the card punch of 13mm takes out hole 3 and separates.Figure
Shown in, have 20 holes 3.Separating resulting is seen parental autocopulation in the table 1.
Claims (6)
1. the method that purifying is cultivated the rotten stem eelworm of sweet potato is characterized in that comprising the following steps:
(1) preparation of nematode
Under 40 * Stereo microscope, be placed on the slide, further confirm as the female worm of becoming pregnant, be put in the vessel subsequent use then at 100 * microscopically with the rotten female worm of stem eelworm population of pin picking;
(2) purifying is cultivated
To put in order potato and laterally or vertically cut, punch on the section therein, in each hole, inject a little sterile water; Be as the criterion with overfolw hole not, the rotten stem eelworm that afterwards step (1) is obtained, one is inserted in the aperture; Again the potato piece that downcuts is covered on the potato piece after inserting the stem eelworm of rotting as natural moisture preserving; With paraffin that the edge gap sealing is good, and, obtained some pure rotten stem eelworms in 30 days in 25 ℃ of cultivations with being placed in the incubator after the elastomeric material binding.
2. a kind of purifying as claimed in claim 1 is cultivated the method for the rotten stem eelworm of sweet potato, it is characterized in that: the diameter in inoculation nematode hole is the 2-6 millimeter on the sweet potato section, and the degree of depth is 10 ~ 30 millimeters, and the spacing of Kong Yukong is 2.5-4 centimetre.
3. a kind of purifying as claimed in claim 1 is cultivated the method for the rotten stem eelworm of sweet potato, it is characterized in that: before in incubator, inserting the stem eelworm of rotting, spray small amount of aseptic water in advance, to keep the moistening growing environment of potato piece.
4. the method that purifying is cultivated the rotten stem eelworm of sweet potato is characterized in that comprising the following steps:
(1) preparation of nematode
Big instar larvae, male worm with the rotten stem eelworm of pin picking under 40 * Stereo microscope are put in the vessel subsequent use;
(2) purifying is cultivated
To put in order potato laterally or vertically cuts; Punch on the section therein, in each hole, inject a little sterile water, be as the criterion with overfolw hole not; The rotten stem eelworm that afterwards step (1) is obtained; Article one, big instar larvae and two male worms are inserted in the aperture, the potato piece that downcuts are covered on the potato piece after inserting the stem eelworm of rotting as natural moisture preserving again, and are with paraffin that the edge gap sealing is good; And, obtained some pure rotten stem eelworms in 30 days in 25 ℃ of cultivations with being placed in the incubator after the elastomeric material binding.
5. a kind of purifying as claimed in claim 4 is cultivated the method for the rotten stem eelworm of sweet potato, it is characterized in that: the diameter in inoculation nematode hole is the 2-6 millimeter on the sweet potato section, and the degree of depth is 10 ~ 30 millimeters, and the spacing of Kong Yukong is 2.5-4 centimetre.
6. a kind of purifying as claimed in claim 4 is cultivated the method for the rotten stem eelworm of sweet potato, it is characterized in that: before in incubator, inserting the stem eelworm of rotting, spray small amount of aseptic water in advance, to keep the moistening growing environment of potato piece.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102972355A (en) * | 2012-12-05 | 2013-03-20 | 华南农业大学 | Method for breeding, culturing and storing plant nematodes by using sweet potato calluses |
| CN103798044A (en) * | 2014-02-21 | 2014-05-21 | 江苏徐州甘薯研究中心 | Stem nematode disease resistance identification method for sweet potato related wild species |
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| CN101260422A (en) * | 2008-04-10 | 2008-09-10 | 江苏徐州甘薯研究中心 | Sweet potato stem-nematode expansibility resistance identification method |
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| CN101260422A (en) * | 2008-04-10 | 2008-09-10 | 江苏徐州甘薯研究中心 | Sweet potato stem-nematode expansibility resistance identification method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102972355A (en) * | 2012-12-05 | 2013-03-20 | 华南农业大学 | Method for breeding, culturing and storing plant nematodes by using sweet potato calluses |
| CN103798044A (en) * | 2014-02-21 | 2014-05-21 | 江苏徐州甘薯研究中心 | Stem nematode disease resistance identification method for sweet potato related wild species |
| CN103798044B (en) * | 2014-02-21 | 2015-06-17 | 江苏徐州甘薯研究中心 | Stem nematode disease resistance identification method for sweet potato related wild species |
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