CN103789216B - The one artificial onset's method growing tobacco damping-off in vitro inoculation - Google Patents
The one artificial onset's method growing tobacco damping-off in vitro inoculation Download PDFInfo
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- CN103789216B CN103789216B CN201410006432.3A CN201410006432A CN103789216B CN 103789216 B CN103789216 B CN 103789216B CN 201410006432 A CN201410006432 A CN 201410006432A CN 103789216 B CN103789216 B CN 103789216B
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- 238000011081 inoculation Methods 0.000 title claims abstract description 53
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000000338 in vitro Methods 0.000 title claims abstract description 13
- 241000208125 Nicotiana Species 0.000 title claims abstract 5
- 241000918585 Pythium aphanidermatum Species 0.000 title claims abstract 3
- 235000019504 cigarettes Nutrition 0.000 claims abstract description 15
- 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
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 244000061176 Nicotiana tabacum Species 0.000 claims description 29
- 241000813090 Rhizoctonia solani Species 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
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- 230000001580 bacterial effect Effects 0.000 claims description 8
- 230000017074 necrotic cell death Effects 0.000 claims description 8
- 239000002054 inoculum Substances 0.000 claims description 7
- 240000001131 Nostoc commune Species 0.000 claims description 3
- 235000013817 Nostoc commune Nutrition 0.000 claims description 3
- 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
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 238000007796 conventional method Methods 0.000 claims description 3
- 239000003337 fertilizer Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
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- 241000233866 Fungi Species 0.000 claims description 2
- 239000012533 medium component Substances 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 5
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- 206010039509 Scab Diseases 0.000 description 6
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Abstract
The invention discloses an artificial onset's method growing tobacco damping-off in vitro inoculation.The step of the method is as follows: 1) inoculation major appliances prepares; 2) cigarette seedling is cultivated; 3) mycelial preparation is inoculated; 4) collection of excised leaf; 5) disposal of excised leaf; 6) germ is inoculated; 7) dispose after inoculation.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; Disease symptom is clear easy to identify, favorable reproducibility.2) implementation and operation is simple and easy convenient; Inoculation morbidity local condition easily controls, the stdn of easy actualizing technology method.
Description
Technical field
The present invention relates to agrotechnique and biotechnology.A specifically artificial onset's method growing tobacco damping-off in vitro inoculation.
Background technology
In the anti-research of Plant diseases control, the research of many underlying issues or the research of most important theories problem all be unable to do without artificial inoculation and to fall ill this basic means.Plant diseases artificial onset can inoculate on plants, also can inoculate on Vitro Plant organ material.Adopt the result of study of plants inoculation morbidity, although these looks that host-germ does mutually more can be reflected, live body is inoculated, often need more Time and place place, 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, often need less Time and place demand, 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, also can reach research purpose, in other words, adopt inoculation on To body material to be consistent with the result of study inoculated 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 have and adopt in vitro inoculation method to carry out relevant research.
Infecting by pathogenic fungi Rhizoctonia solani the tobacco damping-off caused is one of important disease of tobacco leaf production, mainly fall ill in seedling stage, cause dead seedling, some fields are caused to be short of seedling in a large number time serious, in recent years at tobacco Adult plant also common generation, particularly some high-yield culturing districts, often there is higher sickness rate, owing to mainly causing the basal part of stem of tobacco plant downright bad in this disease of Adult plant, thus cause whole strain to be injured, cause larger loss, this has become the major issue of producing and facing, address this problem, still the fundamental research strengthening tobacco damping-off is depended on, and artificial onset is this disease about one of the 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 to be infected the pathogenic advantageous feature of blade, be applied in artificial onset's practice and set up supporting technology.
Summary of the invention
The object of this invention is to provide an artificial onset's method growing 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 growing tobacco damping-off in vitro inoculation is that cause leaf tissue downright bad and show disease symptom clearly, the step of artificial onset's method is as follows by tobacco Isolated leaf inoculation tobacco rhizoctonia solani in the culture dish tilted:
1. inoculation major appliances prepares: make inoculation major appliances with Nostoc commune Vanch ware, for subsequent use after cleaning-sterilizing.
2. cigarette seedling is cultivated: cultivate cigarette seedling according to a conventional method, conventional water and fertilizer management, until during the culture dish similar diameters of cigarette seedling leaf length and step 1, can use.
3. inoculate mycelial preparation: rhizoctonia solani is transplanted to the common fungi culture medium plate center of PSA, PSA medium component is: potato 200 grams, sucrose 20 grams, 18 grams, agar, water 1000mL, be cultured at 28 DEG C and grow comparatively macrocolony, cut-off footpath is that 6mm punch tool is beaten in flat-plate bacterial colony periphery and got mycelia agar nahlock of the same size, makes inoculum with this mycelia block.
4. the collection of excised leaf: use clean scissors, the blade of the blade length on clip step 2 tobacco plant and the culture dish similar diameters of step 1, reinstalls indoor with clean container, 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, the excised leaf a slice of getting step 4 preparation is launched 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. inoculate germ: the mycelia block that picking step 3 prepares, be placed with gently and operate on complete blade in step 5, cover back culture dish lid.
7. dispose after inoculation: culture dish step 6 being loaded inoculation blade is placed in the general room possessing normal scattered light and cultivates, and culture temperature maintains 26 ~ 28 DEG C.
The downright bad symptom of the blade that inoculation result causes blade to occur being perfectly clear, general inoculation starts to occur downright bad symptom for latter 30 hours, 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, the stdn of easy actualizing technology method.
Accompanying drawing explanation
Fig. 1 is that the culture dish that the present invention inoculates is in heeling condition.
Fig. 2 is the modes of emplacement of excised leaf of the present invention and postvaccinal state.
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 be the morbidity of same blade successively from the state of an illness gently to the performance that the state of an illness is heavy; D portion is the performance of inoculation contrast, and arrow indication is blank cultures agar block.
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 be the morbidity of same blade successively from the state of an illness gently to the performance that the state of an illness is heavy; D portion is the performance of inoculation contrast, and arrow indication is blank cultures agar block.
Fig. 5 is the performance of serious morbidity after tobacco leaf of the present invention inoculation rhizoctonia solani.
In figure, A ~ C portion is different blade morbidity sample; D portion is the performance of inoculation contrast, and arrow indication is blank cultures agar block.
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 normally expansion; 2) blade cardinal extremity otch keeps with contact with moisture but avoids water immersion to steep whole blade again; 3) utensil is transparent is convenient to blade daylighting and PD observation; 4) utensil is simple and to implement inoculation easy to operate, and subenvironment can be kept again to be in high humidity state.Contriver utilizes the simple operations added water in slant setting culture dish, reaches above-mentioned technical requirements dexterously.The present invention for core, sets up a kind of matching method inoculating damping-off on tobacco tooth in vitro with this technical essential, namely with tilted-putted culture dish for major appliances, as shown in Figure 1, in culture dish, place excised leaf and inoculate germ, as shown in Figure 2.After tobacco Isolated leaf inoculation rhizoctonia solani, cultivate under 25 ~ 28 DEG C of conditions, the 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, the necrosis of whole blade finally can be caused to rot, as shown in Fig. 3 ~ Fig. 5.From being inoculated into whole blade necrosis 4 ~ 5 days consuming time usually.
Inoculation causes speed or the weight of PD, and to inoculate mycelium cell age state relevant with rhizoctonia solani, and 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 growing tobacco damping-off in vitro inoculation, cigarette seedling kind cloud and mist 87 excised leaf inoculates tobacco rhizoctonia solani bacterial strain Rs-1, operates as follows:
1. major appliances prepares: employing diameter is that inoculation utensil made by the Nostoc commune Vanch ware of 15cm, for subsequent use after cleaning-sterilizing.
2. cigarette seedling is cultivated: cultivate cigarette seedling kind cloud and mist 87 according to a conventional method, conventional water and fertilizer management, until when cigarette seedling leaf length is consistent with the culture dish diameter of step 1 (blade length is about 14cm), can use.
3. inoculate mycelial preparation: bacterial strain Rs-1 is transplanted to PSA culture medium flat plate center, PSA nutrient media components is: potato 200 grams, sucrose 20 grams, 18 grams, agar, water 1000mL; Cultivate at 28 DEG C of temperature and grow comparatively macrocolony in 48 hours, cut-off footpath is that 6mm punch tool is beaten in flat-plate bacterial colony periphery and got mycelia agar nahlock of the same size, makes inoculum with this mycelia block.
4. the collection of excised leaf: use clean scissors, the blade length on clip step 2 tobacco plant is about the blade of 14cm, reinstalls indoor with clean container, stand-by rinse blade surface well gently under tap water after.
5. the disposal of excised leaf: the culture dish slant setting as shown in Figure 1 step 1 got ready; Open ware lid, the excised leaf a slice of getting step 4 preparation is launched 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 the clean clear water of 30mL and inject culture dish, clear water submergence blade base, as shown in Figure 2.
6. inoculate germ: the mycelia block that picking step 3 prepares, be placed with gently and operate on complete blade in step 5, as shown in Figure 2, then cover back culture dish lid, make the little space forming a stable high humidity in culture dish.
7. inoculation after dispose: step 6 is loaded inoculation blade culture dish be placed in possess normal scattered light general room culturing rack on cultivate, culture temperature maintains 25 ~ 27 DEG C.
Result shows the symptom of blade necrosis clearly as Fig. 3 ~ Fig. 5; Inoculating latter 72 hours downright bad lesion diameter is 4.5cm.
Embodiment 2
The one artificial onset's method growing tobacco damping-off in vitro inoculation, cigarette seedling kind cloud and mist 87 excised leaf inoculates tobacco rhizoctonia solani bacterial strain Rs-1, operation steps operates enforcement by the step 1 of embodiment 1 to step 7, wherein in the changing with the operation of embodiment 1 step 7 of operation of step 7, after the step 7) inoculation of embodiment 1, culture temperature maintains 25 ~ 27 DEG C, and after the step 7) inoculation of the present embodiment, culture temperature maintains 19 ~ 21 DEG C.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 lesion diameter is 1.7cm.
Embodiment 3
The present invention one is adopted to grow tobacco artificial onset's method of damping-off in vitro inoculation, cigarette seedling kind cloud and mist 87 excised leaf inoculates tobacco rhizoctonia solani bacterial strain Rs-1, operation steps is implemented to step 7) operation by the step 1) of embodiment 1, wherein the changing with the operation of embodiment 1 step 3) of 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 lesion diameter is 1.9cm.
Embodiment 4
The present invention one is adopted to grow tobacco artificial onset's method of damping-off in vitro inoculation, cigarette seedling kind cloud and mist 87 excised leaf inoculates tobacco rhizoctonia solani bacterial strain Rs-1, operation steps is implemented to step 3) operation by the step 1) of embodiment 1, but in step 3), the inoculum mycelium culture duration of the present embodiment is 96 hours; Implement to step 7) operation by the step 4) of embodiment 1, but in step 7), the postvaccinal culture temperature of the present embodiment maintains 19 ~ 21 DEG C; 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 lesion diameter is 0.9cm.
Claims (1)
1. an artificial onset's method growing tobacco damping-off in vitro inoculation, is characterized in that, by tobacco Isolated leaf inoculation tobacco rhizoctonia solani in the culture dish tilted, causes leaf tissue downright bad and shows disease symptom clearly;
Operation steps is as follows:
1) inoculation major appliances prepares: make inoculation major appliances with Nostoc commune Vanch ware, for subsequent use after cleaning-sterilizing;
2) cigarette seedling is cultivated: cultivate cigarette seedling according to a conventional method, conventional water and fertilizer management, until during the culture dish similar diameters of cigarette seedling leaf length and step 1), can use;
3) mycelial preparation is inoculated: rhizoctonia solani is transplanted to the common fungi culture medium plate center of PSA, PSA medium component is: potato 200 grams, sucrose 20 grams, 18 grams, agar, water 1000mL, be cultured at 28 DEG C and grow comparatively macrocolony, cut-off footpath is that 6mm punch tool is beaten in flat-plate bacterial colony periphery and got mycelia agar nahlock of the same size, makes inoculum with this mycelia block;
4) collection of excised leaf: use clean scissors, clip step 2) blade of culture dish similar diameters of blade length on tobacco plant and step 1), reinstall indoor with clean container, 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, the excised leaf a slice of getting step 4) preparation is launched 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) inoculate germ: the mycelia block that picking step 3) prepares, be placed with gently and operate on complete blade in step 5), cover back culture dish lid;
7) dispose after inoculation: culture dish step 6) being loaded inoculation blade is placed in the general room possessing normal scattered light and cultivates, and culture temperature maintains 26 ~ 28 DEG C;
The downright bad symptom of the blade that inoculation result causes blade to occur being perfectly clear, general inoculation starts to occur downright bad symptom for latter 30 hours, whole blade total necrosis after 4 ~ 6 days.
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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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN102876767A (en) * | 2012-10-11 | 2013-01-16 | 中国农业科学院烟草研究所 | Method for screening alternaria alternata resistant germplasm material from petri dish |
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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 |
| CA2378107A1 (en) * | 1999-07-23 | 2001-02-01 | Wisconsin Alumni Research Foundation | <i>arabidopsis thaliana</i> cyclic nucleotide-gated ion channel/<i>dnd</i> genes; regulators of plant disease resistance and cell death |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN102876767A (en) * | 2012-10-11 | 2013-01-16 | 中国农业科学院烟草研究所 | Method for screening alternaria alternata resistant germplasm material from petri dish |
Non-Patent Citations (1)
| Title |
|---|
| 新型杀菌剂对立枯丝核菌的室内毒力测定;蒋家珍 等;《江苏农业学报》;20041228;第20卷(第4期);第271-272页 * |
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