CN103782816B - One quick method growing tobacco damping-off artificial onset - Google Patents
One quick method growing tobacco damping-off artificial onset Download PDFInfo
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- CN103782816B CN103782816B CN201410006422.XA CN201410006422A CN103782816B CN 103782816 B CN103782816 B CN 103782816B CN 201410006422 A CN201410006422 A CN 201410006422A CN 103782816 B CN103782816 B CN 103782816B
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- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 37
- 241000208125 Nicotiana Species 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 24
- 241000918585 Pythium aphanidermatum Species 0.000 title claims abstract description 12
- 238000011081 inoculation Methods 0.000 claims abstract description 30
- 241000813090 Rhizoctonia solani Species 0.000 claims abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 235000019504 cigarettes Nutrition 0.000 claims description 11
- 230000017074 necrotic cell death Effects 0.000 claims description 11
- 239000002689 soil Substances 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 6
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 claims description 6
- 229920005372 Plexiglas® Polymers 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 5
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- 241001582888 Lobus Species 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
- 230000035558 fertility Effects 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
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- 230000008901 benefit Effects 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
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- 230000001717 pathogenic effect Effects 0.000 description 3
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- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 240000001131 Nostoc commune Species 0.000 description 1
- 235000013817 Nostoc commune Nutrition 0.000 description 1
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- 238000003892 spreading Methods 0.000 description 1
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Abstract
The invention discloses a quick method growing tobacco damping-off artificial onset, the step of the method is as follows: 1. tobacco seedling is cultivated and prepared; 2. inoculate mycelial preparation; 3. dispose before tobacco seedling inoculation; 4. inoculate rhizoctonia solani; 5. dispose after inoculation.Advantage of the present invention is: 1) inoculation technique system is microminiaturized, uses small seedling, and in very little space, inoculation morbidity, consumes the very short test period.2) implementation process is simple, and easy to operate efficiency is high.3) whole process completes in routine experimentation indoor, and morbidity local condition easily controls, and is disturbed little, easy actualizing technology methodological standardization.
Description
Technical field
The present invention relates to agrotechnique and biotechnology.A specifically quick method growing tobacco damping-off artificial onset.
Background technology
Infecting by pathogenic fungi Rhizoctoniasolani 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.Due to weak to the research of this disease for a long time, also do not form specification and ripe artificial onset's method so far.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 a quick method growing tobacco damping-off artificial onset, complete artificial onset's work in a short time.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
One quick method growing tobacco damping-off artificial onset is utilize rhizoctonia solani to infect characteristic that tobacco leaf causes tissue necrosis, inoculates immature cigarette seedling, cause cigarette seedling leaf to be fallen ill fast, show clear symptom in culture dish.The step of artificial onset's method is as follows:
1. tobacco seedling is cultivated and is prepared:
The soil better and loose by common tray splendid attire fertility, by ordinary method at indoor culture cigarette seedling, for subsequent use after growing to 3 ~ 4 vanelets.
2. 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 under 28 DEG C of conditions 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.
3. dispose before tobacco seedling inoculation:
Get the applicable seedling that step 1 cultivates, first water before inoculation moistening softening nursery soil, so that lift seedlings.Take out clean culture dish to be placed in common shallow square plate, and add thin layer clear water in culture dish; Choose the seedling having 3 ~ 4 vanelets, gently pull up with have gentle hands and forward in the clear water of other container splendid attire, rock the soil in wash-out seedlings root gently, seedling is forwarded in the above-mentioned culture dish got ready, leaflet horse is put on the ware edge of culture dish, root system leans against at the bottom of ware, makes seedling be upright state, and makes clear water submergence root system.
4. inoculate rhizoctonia solani:
The mycelia block that picking step 2 prepares, is placed with gently on the lobus cardiacus that step 3 disposes complete seedling or on heart bud.
5. dispose after inoculation:
Step 4 operate complete after, culture dish splendid attire being inoculated seedling is placed in shallow square plate, and is covered on by plexiglass tent on shallow square plate, injects thin layer clear water toward in shallow square plate, makes plexiglass tent enclose inside and forms high humidity state stable in cover; Then complete assembly is put in the general room possessing daily scattered light to cultivate, culture temperature maintains 25 ~ 30 DEG C.Note avoiding direct sunlight according to shining.
Inoculation result causes seedling leaves to occur the necrosis of water stain shape, and last whole seedling total necrosis is rotted.Under normal circumstances, visible obviously necrosis afterwards shows to inoculate 1 day, and after 3 ~ 4 days, complete stool is rotted.
Advantage of the present invention is:
1) inoculation technique system is microminiaturized, is mainly manifested in small seedling, and in very little space, inoculation morbidity, consumes the very short test period.
2) implementation process is simple, and easy to operate efficiency is high.
3) whole process completes in routine experimentation indoor, and morbidity local condition easily controls, and is disturbed little, easy actualizing technology methodological standardization.
Accompanying drawing explanation
Fig. 1 is the tobacco seedling schematic diagram of the present invention in the indoor cultivation of routine experimentation.
In figure, A cultivates with common enamel square plate; B cultivates with culture dish.
Fig. 2 is that frame of the present invention is placed on the seedling on culture dish ware edge and the schematic diagram after inoculation mycelia block thereof.
Fig. 3 is that postvaccinal seedling is covered on the schematic diagram in shallow square plate by the present invention's plexiglass tent.
Fig. 4 is that the present invention inoculates the performance of the morbidity after 2 days.
In figure, A ~ E is the performance of different seedling inoculation germ; F portion is blank inoculation contrast.
Fig. 5 is that the present invention inoculates the performance of the morbidity after 3 days.
In figure, A ~ E is the morbidity performance of inoculation germ, and wherein A illustrates the state of an illness of many strains seedling, and B ~ E illustrates the performance of individual plant seedling; F is blank inoculation contrast.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
It is omnidistance all in the indoor enforcement of routine experimentation that cigarette seedling of the present invention is cultivated and inoculation is fallen ill, and it is to the south that laboratory only needs to possess window, and printing opacity is good, and is equipped with common air-conditioning at once.In indoor, nearly window is other according to a conventional method cultivates cigarette seedling, and the present invention's design mainly adopts the unified a large amount of nursery of larger seedling pan, and as shown in the A of Fig. 1, this can make nursery nursing (nursing of mainly watering) relatively simple; After seedling size is applicable to using, seedling is passed on other utensil and implements inoculation operation; Adopt this mode nursery to also have a benefit, be exactly that normally size is uneven for the general tobacco seedling growth cultivated, inoculation elsewhere of transplanting seedlings operation can select seedling of the same size to test; In addition, the seedling operating and can obtain without earthy state of transplanting seedlings, needs the test getting rid of soil interference favourable for some.Practical application also can adopt the utensil nursery such as culture dish, as shown in the B of Fig. 1;
What inoculation adopted is the small cigarette seedling growing to 3 ~ 4 vanelets, as shown in the B portion of Fig. 1, usually only needs cultivation 3 ~ 4 weeks.Inoculation operation implements in Nostoc commune Vanch ware, the vanelets center of seedling verticillate shape inoculation germ, as shown in Figure 2.
The key condition that tobacco rhizoctonia solani infects tobacco pathogenesis needs environment to be in high humidity state, and the present invention adopts self-control lens to cover the mode of seedling, as shown in Figure 3, provides stable super-humid conditions, is also convenient to pathogenic process simultaneously and observes.
Morbidity starts in vaccination usually, shows as water stain shape downright bad, and as shown in Figure 4, spreading with mycelia, necrotic plaque is expanded fast towards periphery, and last whole seedling is all downright bad rots, as shown in Figure 5.Under normal circumstances, inoculate 1 day afterwards visible necrotic plaque occur, after 3 ~ 4 days, complete stool necrosis is rotted.
Inoculation causes speed or the weight of PD, to inoculate mycelium cell age state relevant with rhizoctonia solani, also relevant with postvaccinal culture temperature, state of an illness weight can be weighed by the distance of the forefront point of necrosis and vaccination or the index such as downright bad lesion area or strain sickness rate.
Embodiment 1
One quick method growing tobacco damping-off artificial onset, the small seedling of tobacco bred cloud and mist 87 inoculates tobacco rhizoctonia solani bacterial strain Rs-1, operates as follows:
1. tobacco seedling is cultivated and is prepared: the soil better and loose by splendid attire fertility in common enamel square tray, by ordinary method at indoor culture cigarette seedling, for subsequent use after growing to 3 ~ 4 vanelets as shown in the B of Fig. 1.
2. inoculate mycelial preparation: rhizoctonia solani is transplanted to PSA culture medium flat plate center, PSA medium component is: potato 200 grams, sucrose 20 grams, 18 grams, agar, water 1000mL, cultivate at 28 DEG C 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.
3. dispose before tobacco seedling inoculation: get the applicable seedling that step 1 cultivates, inoculate first 0.5 ~ 1 hour and first water moistening softening nursery soil, so that lift seedlings.Take out clean 9cm culture dish to be placed in common shallow square plate, and add 20ml clear water in culture dish; Choose the seedling having 3 ~ 4 vanelets, gently pull up with have gentle hands and forward in the clear water of other container splendid attire, rock the soil in wash-out seedlings root gently, seedling is forwarded in the above-mentioned 9cm culture dish got ready, leaflet horse is put on the ware edge of culture dish, root system leans against at the bottom of ware, makes seedling be upright state, and clear water submergence root system in ware.
4. inoculate rhizoctonia solani: the mycelia block that picking step 2 prepares, be placed with gently on the lobus cardiacus that step 3 disposes complete seedling or on heart bud, after complete as shown in Figure 2.
5. dispose after inoculation: step 4 operate complete after, take from the culture dish that splendid attire inoculated seedling by plexiglass tent processed and cover on shallow square plate, in shallow square plate, inject thin layer clear water, make lens enclose inside and form the interior stable high humidity state of cover, as shown in Figure 3; Then complete assembly is put in the general room possessing daily scattered light to cultivate, culture temperature maintains 25 ~ 27 DEG C.Note avoiding direct sunlight according to shining.
Result shows the downright bad symptom of blade clearly as the same in Fig. 3 with Fig. 4; Inoculate forefront point downright bad after 48 hours and the distance 2.1cm of vaccination.
Embodiment 2
One quick method growing tobacco damping-off artificial onset, the small seedling of tobacco bred cloud and mist 87 inoculates tobacco rhizoctonia solani bacterial strain Rs-1, to operate implement by the step 1 of embodiment 1 to step 5.Wherein in the changing with the operation of embodiment 1 step 5 of operation of step 5, after the step 5) inoculation of embodiment 1, culture temperature maintains 25 ~ 27 DEG C, and after the inoculation of the step 5) of the present embodiment, culture temperature maintains 19 ~ 20 DEG C.Result also shows the downright bad symptom of blade clearly as the same in Fig. 3 with Fig. 4; Inoculate forefront point downright bad after 48 hours and the distance 0.6cm of vaccination.
Embodiment 3
One quick method growing tobacco damping-off artificial onset, the small seedling of tobacco bred cloud and mist 87 inoculates tobacco rhizoctonia solani bacterial strain Rs-1, to operate implement by the step 1 of embodiment 1 to step 5.Wherein in step 2) operation with embodiment 1 step 2) operation change, the step 2 of embodiment 1) the cultivation duration of inoculum mycelia is 48 hours, the step 2 of the present embodiment) the cultivation duration of inoculum mycelia is 96 hours.Result shows the symptom of blade necrosis clearly as Fig. 3 ~ Fig. 5 equally, but scab expansion speed is slower than the speed of embodiment 1, inoculates forefront point downright bad after 48 hours and the distance 0.8cm of vaccination.
Claims (1)
1. a quick method growing tobacco damping-off artificial onset, is characterized in that, utilize rhizoctonia solani to infect characteristic that tobacco leaf causes tissue necrosis, inoculates immature cigarette seedling, cause cigarette seedling leaf to be fallen ill fast, show clear symptom in culture dish;
The step of method is as follows:
1) tobacco seedling is cultivated and is prepared:
The soil better and loose by common tray splendid attire fertility, by ordinary method at indoor culture cigarette seedling, for subsequent use after growing to 3 ~ 4 vanelets;
2) 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 under 28 DEG C of conditions 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;
3) dispose before tobacco seedling inoculation:
Get step 1) the applicable seedling that cultivates, first water before inoculation moistening softening nursery soil, so that lift seedlings, takes out clean culture dish and be placed in common shallow square plate, and add thin layer clear water in culture dish; Choose the seedling having 3 ~ 4 vanelets, gently pull up with have gentle hands and forward in the clear water of other container splendid attire, rock the soil in wash-out seedlings root gently, seedling is forwarded in the above-mentioned culture dish got ready, leaflet horse is put on the ware edge of culture dish, root system leans against at the bottom of ware, makes seedling be upright state, and makes clear water submergence root system;
4) rhizoctonia solani is inoculated:
Picking step 2) the mycelia block for preparing, be placed with gently in step 3) on the lobus cardiacus of disposing complete seedling or on heart bud;
5) dispose after inoculation:
Step 4) operate complete after, culture dish splendid attire being inoculated seedling is placed in shallow square plate, and is covered on by plexiglass tent on shallow square plate, injects thin layer clear water toward in shallow square plate, makes plexiglass tent enclose inside and forms high humidity state stable in cover; Then complete assembly is put in the general room possessing daily scattered light to cultivate, culture temperature maintains 25 ~ 30 DEG C, notes avoiding direct sunlight according to shining;
Inoculating result causes seedling leaves to occur the necrosis of water stain shape, and last whole seedling total necrosis is rotted, and under normal circumstances, visible obviously necrosis afterwards shows to inoculate 1 day, and after 3 ~ 4 days, complete stool is rotted.
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| CN103782816B true CN103782816B (en) | 2015-12-09 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1287175A (en) * | 2000-09-08 | 2001-03-14 | 广东省农业科学院蔬菜研究所 | Water culture bacterination process to determine bacterial wilt resistance of plant |
| CN1709030A (en) * | 2005-07-27 | 2005-12-21 | 中国农业科学院蔬菜花卉研究所 | Method for identifying bacterial wilt resistivity of solanaceous crops |
| CN101496481A (en) * | 2009-03-09 | 2009-08-05 | 云南省烟草科学研究所 | Method for identifying resistance to tobacco bacterial wilt during seedling stage |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1287175A (en) * | 2000-09-08 | 2001-03-14 | 广东省农业科学院蔬菜研究所 | Water culture bacterination process to determine bacterial wilt resistance of plant |
| CN1709030A (en) * | 2005-07-27 | 2005-12-21 | 中国农业科学院蔬菜花卉研究所 | Method for identifying bacterial wilt resistivity of solanaceous crops |
| CN101496481A (en) * | 2009-03-09 | 2009-08-05 | 云南省烟草科学研究所 | Method for identifying resistance to tobacco bacterial wilt during seedling stage |
Non-Patent Citations (2)
| Title |
|---|
| Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn;MARLENY GONZALEZ等;《Molecular Plant Pathology》;20110630;第12卷(第3期);209-216 * |
| 烟草靶斑病原鉴定及生物学特性研究;吴元华等;《沈阳农业大学学报》;20121031;第43卷(第5期);521-527 * |
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