CN104623707A - Surface sterilization method for maintaining hatching activity of root-knot nematode eggs - Google Patents
Surface sterilization method for maintaining hatching activity of root-knot nematode eggs Download PDFInfo
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- CN104623707A CN104623707A CN201510018392.9A CN201510018392A CN104623707A CN 104623707 A CN104623707 A CN 104623707A CN 201510018392 A CN201510018392 A CN 201510018392A CN 104623707 A CN104623707 A CN 104623707A
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Abstract
The invention belongs to the field of phytopathology, and particularly relates to a surface sterilization method for maintaining hatching activity of root-knot nematode eggs. The surface sterilization method comprises the following steps: preparing root-knot nematode eggs suspension, performing surface sterilization, inoculating and detecting. The method has the advantages of being simple to operate, and high in the hatching rate of the sterilized root-knot nematode eggs. The method has extremely important utilization values on the aspects of research on phytopathology and phytonematology, no exogenous culture of root-knot nematodes, and the like.
Description
Technical field
The invention belongs to Plant Pathology field, be specifically related to a kind of processes for disinfecting surfaces keeping root-knot nematode egg hatching activity.
Background technology
Root-knot nematode (Meloidogyne spp.) is a kind of plant pathogeny line insect of height specialized form.The root-knot nematode of known vegetable of causing harm mainly contains Meloidogyne incognita, M hapla, peanut root-knot nematode and javanese root knot nematode etc.Its host range is extensive, and can cause harm 2000 various plants such as vegetable, fruit tree, ornamental plant, cereal crops and Chinese herbal medicine, is especially injured seriously with the industrial crops such as Fructus Lycopersici esculenti, Fructus Cucumidis sativi, causes harm and also causes the Combined Infection of some fungus and bacterium sexually transmitted disease (STD)s evil simultaneously.Root-knot nematode, based on the root of various plant of causing harm, shows as side root and fibrous root increases than normally, and on young root or fibrous root, form the goiter that spherical or irregular shape differs in size, have in beads shape.Short and small, slow, the leaf color anomaly of killed plant above ground portion growth, result is few, yields poorly, and even causes plant dead ahead of time.
Root-knot nematode is one of important pathogen of plant.In the research process of root-knot nematode, if further investigate the Molecular interaction etc. of the mechanism of causing a disease of this cause of disease or itself and host plant, usually relate to cultivating without external source of root-knot nematode, the surface sterilization of root-knot nematode egg and incubation rate are the keys of test success or failure.Therefore, the further investigation of simple method to this disease obtaining maintenance root-knot nematode egg hatching activity is significant.
Summary of the invention
The object of this invention is to provide a kind of processes for disinfecting surfaces keeping root-knot nematode egg hatching activity, to overcome the deficiency that existing inoculation root-knot nematode technology is usually polluted or caused egg hatching rate low.Technical scheme provided by the invention is:
Keep a processes for disinfecting surfaces for root-knot nematode egg hatching activity, it is characterized in that, comprise the following steps:
(1) root-knot nematode egg suspension is prepared: obtain disease plant root system and prepare root-knot nematode egg suspension;
(2) surface sterilization: pour ovum suspension into self-control sieve, then immerses 70% alcoholic solution by the sieve filling line eggs, surface sterilizing, is shifted out by sieve, repeatedly repeats to rinse line eggs remove remaining alcoholic solution with sterilized water;
(3) pollution detection: by the line eggs even spread of a part of surface sterilization in PDA flat board, every processed group repeats for 3 times, is placed in 25 DEG C of calorstats, and observe pollution condition, whether sterilizing is thorough to detect it;
(4) incubation rate detects: the line eggs of another part surface sterilization is placed in 96 porocyte culture plates, 50 ± 10, every hole ovum, repeats, records the initial number of ovum under microscope for 4 times, and within every 3 days, record egg hatching goes out the quantity of second instar larvae, calculates incubation rate.
In the technical program, the concentration obtaining root-knot nematode egg suspension in step (1) is 20-50 grain ovum/μ L.
In the technical program, in step (2), sterilization time is 30-60S.
In the technical program, in step (2) self-control sieve be dismantled and assembled can the sterilization sieve of sterilizing, it is of a size of internal diameter 2cm, screen cloth mesh diameter 30 μm.
Compared with prior art, the beneficial effect that has of the present invention:
1, the present invention adopts alcoholic solution as disinfectant solution, nontoxic, economical, easily obtains and environmentally safe.
2, the present invention is simple to operate, and ovum grain is not lost in operating process.
3, the present invention saves time, and can save time 3-5h compared with nematodes ovum disinfectant solution (as streptomycin sulfate).
Accompanying drawing explanation
Fig. 1 is that root-knot nematode egg utilizes PDA plating medium to detect sterilization effect after 70% alcoholic solution sterilizing;
Main Reference Numerals illustrates:
A, B, C disinfect through 70% alcoholic solution; D, E, F are controlled trial (only through aseptic water washing).
Detailed description of the invention
Below in conjunction with concrete example, the present invention is further detailed explanation.
embodiment 1: the acquisition of root-knot nematode egg suspension and sterilization, operate as follows:
(1) ovum in root-knot nematode egg capsule on the sodium-hypochlorite process collection inoculation susceptible tomato plant root system of latter 25-35 days is utilized;
(2) be mixed with the ovum suspension of respective concentration as required, the ovum grain concentration of this test is 20-50 grain ovum/microlitre;
(3) apparatus such as distilled water, 100mL beaker, sterilization sieve that autoclaving is crossed is prepared before test;
(4) prepare the alcoholic solution 40mL of 70%, be placed in beaker, ovum suspension is slowly poured into sterilization sieve, then sterilization sieve is put into the alcoholic solution beaker filling 70%, the half of submergence sieve height;
(5) disinfecting time is 30-60S, then takes out sterilization sieve, rapidly with aseptic water washing ovum to remove the alcoholic solution of remnants;
(6) with sterilized water, the ovum handled well is moved in another sterilizing small beaker, namely obtain surface sterilization and keep the root-knot nematode egg of egg hatching vigor, may be used for root-knot nematode without external source cultivation etc.
embodiment 2:the dull and stereotyped pollution detection of PDA and incubation rate detection experiment
Testing program: the acquisition of line eggs and sterilization method thereof, with embodiment 1, in order to prove the impact of above-mentioned sterilization method on the incubation rate of ovum, have carried out following test:
(1) pollution detection: by the line eggs even spread of a part of surface sterilization in PDA flat board, every processed group repeats for 3 times, is placed in 25 DEG C of calorstats, and observe pollution condition, whether sterilizing is thorough to detect it;
(2) incubation rate detects: the line eggs of another part surface sterilization is placed in 96-porocyte culture plate, 50 ± 10, every hole ovum, repeats, records the initial number of ovum under microscope for 4 times, and within every 3 days, record hatches the quantity of second instar larvae, calculates incubation rate.
Result of the test:
(1) the dull and stereotyped pollution detection of PDA shows, through the ovum of 70% alcoholic solution surface sterilization without any pollution, and the contrast ovum of non-sterile presents antibacterial and fungal contamination (referring to Fig. 1) on inoculation PDA flat board.
The ovum of (2) 70% alcoholic solution surface sterilizations is compared with the ovum of non-sterile (referring to table 1), the two accumulation incubation rate each time is during incubation all without significant difference (P > 0.05), the 15th day time, surface sterilization processed group accumulation incubation rate is 93.3%, and matched group is 93.9%.Result shows, utilizes 70% hatching of alcoholic solution surface sterilizing on root-knot nematode egg not affect.
Incubation rate after table 1 root-knot nematode egg surface sterilizing
The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.
Claims (4)
1. keep a processes for disinfecting surfaces for root-knot nematode egg hatching activity, it is characterized in that, comprise the following steps:
(1) root-knot nematode egg suspension is prepared: obtain disease plant root system and prepare root-knot nematode egg suspension;
(2) surface sterilization: pour ovum suspension into self-control sieve, then immerses 70% alcoholic solution by the sieve filling line eggs, surface sterilizing, is shifted out by sieve, repeatedly repeats to rinse line eggs remove remaining alcoholic solution with sterilized water;
(3) pollution detection: by the line eggs even spread of a part of surface sterilization in PDA flat board, every processed group repeats for 3 times, is placed in 25 DEG C of calorstats, and observe pollution condition, whether sterilizing is thorough to detect it;
(4) incubation rate detects: the line eggs of another part surface sterilization is placed in 96 porocyte culture plates, 50 ± 10, every hole ovum, repeats, records the initial number of ovum under microscope for 4 times, and within every 3 days, record hatches the quantity of second instar larvae, calculates incubation rate.
2. the processes for disinfecting surfaces keeping root-knot nematode egg hatching activity as claimed in claim 1, is characterized in that, the concentration obtaining root-knot nematode egg suspension in step (1) is 20-50 grain ovum/μ L.
3. the processes for disinfecting surfaces keeping root-knot nematode egg hatching activity as claimed in claim 1, is characterized in that, in step (2), sterilization time is 30-60S.
4. the as claimed in claim 1 processes for disinfecting surfaces keeping root-knot nematode egg hatching activity, is characterized in that, in step (2) self-control sieve be dismantled and assembled can the sterilization sieve of sterilizing, it is of a size of internal diameter 2cm, screen cloth mesh diameter 30 μm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110547259A (en) * | 2019-09-25 | 2019-12-10 | 北京农学院 | Disinfection method suitable for sterile culture of meloidogyne incognita |
| CN115554445A (en) * | 2022-09-09 | 2023-01-03 | 天津市疾病预防控制中心 | Microbial carrier sheet distribution method for on-site monitoring of disinfection effect of imported goods |
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| CN101731103A (en) * | 2009-11-27 | 2010-06-16 | 华南农业大学 | Artificial inoculation method of root-knot nematodes at upper part of tomato plant field |
| CN101983580A (en) * | 2010-10-25 | 2011-03-09 | 南京林业大学 | Forage for artificial breeding of Monochamus alternatus Hope and method for preparing the same |
| CN102487901A (en) * | 2011-12-06 | 2012-06-13 | 华南农业大学 | Method for breeding meloidogynes by utilizing single allosome of separated root system of non-exogenous cultured water spinach |
| CN103548780A (en) * | 2013-11-15 | 2014-02-05 | 重庆市中药研究院 | Method for preventing and controlling paecilomyces disease in artificially breeding ophiocordyceps sinensis larvae host process |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101473808A (en) * | 2009-01-22 | 2009-07-08 | 中国热带农业科学院热带生物技术研究所 | Artificial culture, breeding and preservation method of plant root-knot nematode |
| CN101731103A (en) * | 2009-11-27 | 2010-06-16 | 华南农业大学 | Artificial inoculation method of root-knot nematodes at upper part of tomato plant field |
| CN101983580A (en) * | 2010-10-25 | 2011-03-09 | 南京林业大学 | Forage for artificial breeding of Monochamus alternatus Hope and method for preparing the same |
| CN102487901A (en) * | 2011-12-06 | 2012-06-13 | 华南农业大学 | Method for breeding meloidogynes by utilizing single allosome of separated root system of non-exogenous cultured water spinach |
| CN103548780A (en) * | 2013-11-15 | 2014-02-05 | 重庆市中药研究院 | Method for preventing and controlling paecilomyces disease in artificially breeding ophiocordyceps sinensis larvae host process |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110547259A (en) * | 2019-09-25 | 2019-12-10 | 北京农学院 | Disinfection method suitable for sterile culture of meloidogyne incognita |
| CN115554445A (en) * | 2022-09-09 | 2023-01-03 | 天津市疾病预防控制中心 | Microbial carrier sheet distribution method for on-site monitoring of disinfection effect of imported goods |
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| CN104623707B (en) | 2017-11-28 |
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