CN103789216A - Artificial infection method for in-vitro inoculation of tobacco bacterial wilt - Google Patents
Artificial infection method for in-vitro inoculation of tobacco bacterial wilt Download PDFInfo
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- CN103789216A CN103789216A CN201410006432.3A CN201410006432A CN103789216A CN 103789216 A CN103789216 A CN 103789216A CN 201410006432 A CN201410006432 A CN 201410006432A CN 103789216 A CN103789216 A CN 103789216A
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- 238000011081 inoculation Methods 0.000 title claims abstract description 53
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 35
- 241000208125 Nicotiana Species 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000000338 in vitro Methods 0.000 title claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 10
- 208000015181 infectious disease Diseases 0.000 title abstract 5
- 201000010099 disease Diseases 0.000 claims abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 12
- 208000024891 symptom Diseases 0.000 claims abstract description 12
- 241000813090 Rhizoctonia solani Species 0.000 claims description 16
- 235000019504 cigarettes Nutrition 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 229920001817 Agar Polymers 0.000 claims description 10
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- -1 18 grams Substances 0.000 claims description 4
- 244000061176 Nicotiana tabacum Species 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
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- 239000011521 glass Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
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- 241000894006 Bacteria Species 0.000 abstract 1
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Abstract
The invention discloses an artificial infection method for in-vitro inoculation of tobacco bacterial wilt. The method comprises the following steps: (1) preparing a main appliance for inoculating; (2) cultivating tobacco seedlings; (3) preparing inoculation mycelia; (4) collecting in-vitro leaves; (5) treating the in-vitro leaves; (6) inoculating bacteria; (7) treating after inoculating. The artificial infection method has the advantages that 1, the used appliance is simple and the inoculating infection process has short time and few occupied space field; disease symptoms are clear and easy to identify, and the repeatability is good; (2) the implementation and operation are simple, easy and convenient; the partial condition of the inoculating infection is easy to control and the standardization of a technical method is easy to realize.
Description
Technical field
The present invention relates to agrotechnique and biotechnology.A specifically artificial onset's method that grows tobacco damping-off in vitro inoculation.
Background technology
In the anti-research of Plant diseases control, the research of the research of many underlying issues or most important theories problem all be unable to do without artificial inoculation this basic means of falling ill.Plant diseases artificial onset can inoculate on plants, also can on Vitro Plant organ material, inoculate.Adopt the result of study of plants inoculation morbidity, although more can reflect these looks that host-germ does mutually, on live body, inoculate, often need more time and place, space, expend more energy and financial resources, and in many cases, the difficulty that local condition controls is larger; And adopt Vitro Plant organ material to inoculate morbidity, and often need less time and space requirement, simple, the local condition of inoculation morbidity is easier to control, and easily improves the scale and efficiency of test; Many plant pathology problems, adopt To body material inoculation morbidity means to study, and also can reach research purpose, and in other words, adopting inoculation on To body material is consistent with the result of study of inoculating on plants.Therefore, in many important research of plant disease such as rice blast, the sheath and culm blight of rice, wheat scab, wheat powdery mildew, soybean rust, the late blight of potato, downy mildew of garpe and melon epidemic disease, downy mildew of cucurbits, various crop anthrax, all there is the in vitro inoculation of employing method to carry out relevant research.
Infecting by pathogenic fungi Rhizoctonia solani the tobacco damping-off causing is one of important disease of tobacco production, mainly fall ill in seedling stage, cause dead seedling, when serious, cause some fields to be short of seedling in a large number, in recent years in the tobacco strain phase also common generation, particularly some high-yield culturing districts, often there is higher sickness rate, owing to mainly causing the basal part of stem necrosis of tobacco plant in this disease of strain phase, thereby cause whole strain to be injured, cause larger loss, this has become to produce the major issue facing, address this problem, still depend on the fundamental research of strengthening tobacco damping-off, and artificial onset is this disease about one of basic means of research.Although this disease main harm cigarette strain basal part of stem, but contriver finds, rhizoctonia solani also can infect tobacco upper blade and cause downright bad scab clearly, this pathogenic property can be used in artificial inoculation morbidity, and do not find so far tobacco rhizoctonia solani can infect the pathogenic advantageous feature of blade, in artificial onset's practice, be applied and set up supporting technology.
Summary of the invention
The object of this invention is to provide an artificial onset's method that grows tobacco damping-off in vitro inoculation.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
The one artificial onset's method that grows tobacco damping-off in vitro inoculation be in the culture dish tilting by tobacco Isolated leaf inoculation tobacco rhizoctonia solani, cause leaf tissue downright bad and show disease symptom clearly, the step of artificial onset's method is as follows:
1. inoculation is prepared with main utensil: do inoculation with common culture dish and use main utensil, and for subsequent use after cleaning-sterilizing.
2. cigarette seedling is cultivated: cultivate according to a conventional method cigarette seedling, conventional water and fertilizer management, until the culture dish diameter of cigarette seedling leaf length and step 1 when close, can use.
3. the mycelial preparation of inoculation: rhizoctonia solani is transplanted to the dull and stereotyped center of the common fungi culture medium of PSA, PSA medium component is: 200 grams of potatos, 20 grams of sucrose, 18 grams, agar, water 1000mL, at 28 ℃, be cultured to and grow compared with macrocolony, cut-off footpath is that 6mm punch tool is beaten and got mycelia agar nahlock of the same size in flat-plate bacterial colony periphery, makes inoculum with this mycelia piece.
4. the collection of excised leaf: use clean scissors, the blade that the blade length on clip step 2 tobacco plant is close with the culture dish diameter of step 1, reinstall with clean container indoor, stand-by rinse blade surface well gently under tap water after.
5. the disposal of excised leaf: the culture dish slant setting that step 1 is got ready, open ware lid, get excised leaf a slice that step 4 prepares and launch to put into culture dish, the top of blade is placed in the eminence of inclination culture dish, the cardinal extremity of blade is placed in the lowest part of culture dish, and with the short glass stick of one section of clean sterilizing, the cardinal extremity of blade is pushed down, then get clean clear water and inject culture dish, clear water submergence blade base.
6. inoculation germ: the mycelia piece that picking step 3 is prepared, be placed with gently in step 5 and operate on complete blade, cover back culture dish lid.
7. after inoculation, dispose: the culture dish that step 6 is loaded to inoculation blade is placed in the general room cultivation that possesses normal scattered light, and culture temperature maintains 26～28 ℃.
Inoculation result causes blade to occur the downright bad symptom of the blade being perfectly clear, and generally inoculates latter 30 hours and starts to occur downright bad symptom, whole blade total necrosis after 4～6 days.
Advantage of the present invention is:
1) utensil used is simple, and the inoculation pathogenic process time is short, and the place that takes up room is few.
2) disease symptom is clear easy to identify, favorable reproducibility.
3) implementation and operation is simple and easy convenient.
4) inoculation morbidity local condition easily controls, and easily realizes the stdn of technological method.
Accompanying drawing explanation
Fig. 1 be the present invention inoculate use culture dish in heeling condition.
Fig. 2 is modes of emplacement and the postvaccinal state of excised leaf of the present invention.
In figure, arrow refers to inoculum agar block.
Fig. 3 is the morbidity performance process sample 1 of tobacco Isolated leaf inoculation rhizoctonia solani of the present invention.
In figure, A～C portion is the performance that same blade morbidity gently weighs to the state of an illness from the state of an illness successively; D portion is the performance of inoculation contrast, and arrow indication is blank medium agar piece.
Fig. 4 is the morbidity performance process sample 2 of tobacco Isolated leaf inoculation rhizoctonia solani of the present invention.
In figure, A～C portion is the performance that same blade morbidity gently weighs to the state of an illness from the state of an illness successively; D portion is the performance of inoculation contrast, and arrow indication is blank medium agar piece.
Fig. 5 is the performance of serious morbidity after tobacco leaf inoculation rhizoctonia solani of the present invention.
In figure, A～C portion is different blade morbidity sample; D portion is the performance of inoculation contrast, and arrow indication is blank medium agar piece.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
Carrying out Isolated leaf inoculation morbidity and often have following technical requirements, 1) blade can stablize normal expansion; 2) maintenance of blade cardinal extremity otch and contact with moisture but avoid again water immersion to steep whole blade; 3) transparent blade daylighting and the PD be convenient to of utensil observed; 4) utensil is simple and to implement inoculation easy to operate, can keep again subenvironment in high wet condition.Contriver utilizes the simple operations adding water in slant setting culture dish, reaches dexterously above-mentioned technical requirements.The present invention, take this technical essential as core, sets up a kind of matching method of inoculating damping-off on the in vitro leaf of tobacco, take tilted-putted culture dish as main utensil, as shown in Figure 1, places excised leaf and inoculates germ, as shown in Figure 2 in culture dish.After tobacco Isolated leaf inoculation rhizoctonia solani, under 25～28 ℃ of conditions, cultivate visible downright bad appearance of excised leaf after general 30 hours, as shown in the A portion of Fig. 3, passing in time, scab expands toward surrounding from vaccination, finally can cause whole blade downright bad rotten, as shown in Fig. 3～Fig. 5.From being inoculated into whole blade downright bad 4～5 days consuming time conventionally.
Inoculation causes speed or the weight of PD, and relevant with rhizoctonia solani inoculation mycelium cell age state, also relevant with postvaccinal culture temperature, state of an illness weight can be weighed by indexs such as Lesion sizes.
Embodiment 1
The one artificial onset's method that grows tobacco damping-off in vitro inoculation is inoculated tobacco rhizoctonia solani bacterial strain Rs-1 on cigarette seedling kind cloud and mist 87 excised leafs, as follows operation:
1. main utensil is prepared: the common culture dish that employing diameter is 15cm is made inoculation utensil, for subsequent use after cleaning-sterilizing.
2. cigarette seedling is cultivated: cultivate according to a conventional method cigarette seedling kind cloud and mist 87, conventional water and fertilizer management, until the culture dish diameter of cigarette seedling leaf length and step 1 is when consistent (blade length is about 14cm), can use.
3. the mycelial preparation of inoculation: bacterial strain Rs-1 is transplanted to PSA culture medium flat plate center, and PSA nutrient media components is: 200 grams of potatos, 20 grams of sucrose, 18 grams, agar, water 1000mL; At 28 ℃ of temperature, cultivate and grow compared with macrocolony for 48 hours, cut-off footpath is that 6mm punch tool is beaten and got mycelia agar nahlock of the same size in flat-plate bacterial colony periphery, makes inoculum with this mycelia piece.
4. the collection of excised leaf: use clean scissors, the blade length on clip step 2 tobacco plant is about the blade of 14cm, reinstall with clean container indoor, stand-by rinse blade surface well gently under tap water after.
5. the disposal of excised leaf: the culture dish slant setting of as shown in Figure 1 step 1 being got ready; Open ware lid, get excised leaf a slice that step 4 prepares and launch to put into culture dish, the top of blade is placed in the eminence of inclination culture dish, the cardinal extremity of blade is placed in the lowest part of culture dish, and the cardinal extremity of blade is pushed down with the short glass stick of one section of clean sterilizing, then get the clean clear water of 30mL and inject culture dish, clear water submergence blade base, as shown in Figure 2.
6. inoculation germ: the mycelia piece that picking step 3 is prepared, be placed with gently in step 5 and operate on complete blade, as shown in Figure 2, then cover back culture dish lid, make to form in culture dish the little space of a stable high humidity.
7. after inoculation, dispose: the culture dish that step 6 is loaded to inoculation blade is placed on the general room culturing rack that possesses normal scattered light and cultivates, and culture temperature maintains 25～27 ℃.
Bearing performance goes out the symptom of blade necrosis clearly as Fig. 3～Fig. 5; Inoculating latter 72 hours downright bad scab diameters is 4.5cm.
Embodiment 2
The one artificial onset's method that grows tobacco damping-off in vitro inoculation, on cigarette seedling kind cloud and mist 87 excised leafs, inoculate tobacco rhizoctonia solani bacterial strain Rs-1, operation steps is pressed step 1 to step 7 operation of embodiment 1 and is implemented, wherein change in the operation with embodiment 1 step 7 of the operation of step 7, after the step 7) inoculation of embodiment 1, culture temperature maintains 25～27 ℃, and after the step 7) inoculation of the present embodiment, culture temperature maintains 19～21 ℃.Result also shows the symptom of blade necrosis clearly as Fig. 3～Fig. 5, but scab expansion speed is slower than the speed of embodiment 1, and inoculating latter 72 hours downright bad scab diameters is 1.7cm.
Embodiment 3
Adopt grow tobacco artificial onset's method of damping-off in vitro inoculation of the present invention one, on cigarette seedling kind cloud and mist 87 excised leafs, inoculate tobacco rhizoctonia solani bacterial strain Rs-1, operation steps is pressed step 1) to the step 7) operation of embodiment 1 and is implemented, wherein change in the operation with embodiment 1 step 3) of the operation of step 3), the cultivation duration of the step 3) inoculum mycelia of embodiment 1 is 48 hours, and the cultivation duration of the step 3) inoculum mycelia of the present embodiment is 96 hours.Result also shows the symptom of blade necrosis clearly as Fig. 3～Fig. 5, but scab expansion speed is slower than the speed of embodiment 1, and inoculating latter 72 hours downright bad scab diameters is 1.9cm.
Embodiment 4
Adopt grow tobacco artificial onset's method of damping-off in vitro inoculation of the present invention one, on cigarette seedling kind cloud and mist 87 excised leafs, inoculate tobacco rhizoctonia solani bacterial strain Rs-1, operation steps is pressed step 1) to the step 3) operation of embodiment 1 and is implemented, but in step 3), the inoculum mycelium culture duration of the present embodiment is 96 hours; Press step 4) to the step 7) operation of embodiment 1 and implement, but in step 7), the postvaccinal culture temperature of the present embodiment maintains 19～21 ℃; Result shows the symptom of blade necrosis clearly as Fig. 3～Fig. 5 equally, but scab expansion speed is much slower than the speed of embodiment 1, and inoculating latter 72 hours downright bad scab diameters is 0.9cm.
Claims (1)
1. an artificial onset's method that grows tobacco damping-off in vitro inoculation, is characterized in that, in the culture dish tilting, by tobacco Isolated leaf inoculation tobacco rhizoctonia solani, causes leaf tissue downright bad and show disease symptom clearly;
Operation steps is as follows:
1) inoculation is prepared with main utensil: do inoculation with common culture dish and use main utensil, and for subsequent use after cleaning-sterilizing;
2) cigarette seedling is cultivated: cultivate according to a conventional method cigarette seedling, conventional water and fertilizer management, until the culture dish diameter of cigarette seedling leaf length and step 1) when close, can use;
3) inoculate mycelial preparation: rhizoctonia solani is transplanted to the dull and stereotyped center of the common fungi culture medium of PSA, PSA medium component is: 200 grams of potatos, 20 grams of sucrose, 18 grams, agar, water 1000mL, at 28 ℃, be cultured to and grow compared with macrocolony, cut-off footpath is that 6mm punch tool is beaten and got mycelia agar nahlock of the same size in flat-plate bacterial colony periphery, makes inoculum with this mycelia piece;
4) collection of excised leaf: use clean scissors, clip step 2) blade that the blade length on tobacco plant is close with the culture dish diameter of step 1), reinstall with clean container indoor, stand-by rinse blade surface well gently under tap water after;
5) disposal of excised leaf: the culture dish slant setting that step 1 is got ready, open ware lid, get excised leaf a slice of step 4) preparation and launch to put into culture dish, the top of blade is placed in the eminence of inclination culture dish, the cardinal extremity of blade is placed in the lowest part of culture dish, and with the short glass stick of one section of clean sterilizing, the cardinal extremity of blade is pushed down, then get clean clear water and inject culture dish, clear water submergence blade base;
6) inoculation germ: the mycelia piece that picking step 3) is prepared, be placed with gently in step 5) and operate on complete blade, cover back culture dish lid;
7) inoculate rear disposal: the culture dish that step 6) is loaded to inoculation blade is placed in the general room cultivation that possesses normal scattered light, and culture temperature maintains 26～28 ℃;
Inoculation result causes blade to occur the downright bad symptom of the blade being perfectly clear, and generally inoculates latter 30 hours and starts to occur downright bad symptom, whole blade total necrosis after 4～6 days.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439551A (en) * | 2018-12-17 | 2019-03-08 | 靖西市秀美边城农业科技有限公司 | A method of using Isolated leaf inoculation grey leaf spot disease of maize and morbidity |
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| WO2001007596A1 (en) * | 1999-07-23 | 2001-02-01 | Wisconsin Alumni Research Foundation | Arabidopsis thaliana cyclic nucleotide-gated ion channel/dnd genes; regulators of plant disease resistance and cell death |
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Patent Citations (5)
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| JPH0769823A (en) * | 1993-09-03 | 1995-03-14 | Sumitomo Chem Co Ltd | Method for controlling bacterial blight in water culture and microorganism for controlling bacterial blight used therefor |
| US6080565A (en) * | 1996-09-04 | 2000-06-27 | Cornell Research Foundation, Inc. | Cutinases as inducers of plant defense reactions and agents for the control of plant diseases |
| WO2001007596A1 (en) * | 1999-07-23 | 2001-02-01 | Wisconsin Alumni Research Foundation | Arabidopsis thaliana cyclic nucleotide-gated ion channel/dnd genes; regulators of plant disease resistance and cell death |
| CN101993822A (en) * | 2010-09-08 | 2011-03-30 | 广西大学 | Device for inducing isolated organ of plant to cause disease by inoculating and application method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109439551A (en) * | 2018-12-17 | 2019-03-08 | 靖西市秀美边城农业科技有限公司 | A method of using Isolated leaf inoculation grey leaf spot disease of maize and morbidity |
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