CN103789215B - A kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off - Google Patents
A kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off Download PDFInfo
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Abstract
The invention discloses a kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off, the step of the method is as follows: 1) inoculation utensil prepares; 2) cigarette seedling is cultivated; 3) mycelial preparation is inoculated; 4) acquisition process of in vitro vein sheet; 5) on inoculation utensil, in vitro vein sheet is placed; 6) germ is inoculated; 7) dispose after inoculation.Advantage of the present invention is: 1. inoculate the pathogenic process time short, the place that takes up room is few, and method is simple and easy convenient, and working efficiency is high.2. the present invention utilizes larger tobacco leaf, and easily reflect the difference of the result state of an illness, drawing materials of tobacco leaf is also more guaranteed; Disease symptom is clear easy to identify, favorable reproducibility, and 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 kind of specifically artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off.
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 depend on the fundamental research strengthening tobacco damping-off, and one of artificial onset's relevant basic means studied that are this disease.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 kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
A kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off, to the effect that by larger tobacco leaf, cut into the lengthy motion picture bar (being called for short in vitro vein sheet) retaining main lobe arteries and veins, utilize a set of simple utensil inoculation tobacco rhizoctonia solani, cause in vitro vein sheet tissue fall ill and show symptom clearly, the step of artificial onset's method is as follows:
1. inoculation utensil prepares: the major appliances of inoculation comprises: large square plate, primary side coil, lens, working panel, supporting plate and rack plate etc.; With purificant by for subsequent use after these utensil washes clean.
2. cigarette seedling is cultivated: cultivate cigarette seedling according to a conventional method, conventional water and fertilizer management, tobacco leaf length is greater than 20cm and can uses.
3. inoculate mycelial preparation: rhizoctonia solani is transplanted to the common fungi culture medium plate center such as 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 acquisition process of in vitro vein sheet: with the blade on clean scissors clip step 2 tobacco plant, indoor are reinstalled with clean container, under tap water, rinse blade surface well gently, blade cut into the clean instrument that cuts the in vitro vein sheet retaining main lobe arteries and veins for subsequent use.
5. on inoculation utensil, place in vitro vein sheet:
1) assembling inoculation utensil, method is: take out the utensil that step 1 is got ready, primary side coil is placed in the centre in large square plate, get two up and down that 2 clean common bungees are enclosed within working panel respectively, working panel pad is leaned against on support that supporting plate and rack plate put up, make panel form stable heeling condition.
2) place in vitro vein sheet: get the in vitro vein sheet that step 4 is got ready, upward, down, laid parallel on working panel, and provokes bungee by steady for the two ends of in vitro vein sheet folder to cardinal extremity on top; Then toward injected clear water in primary side coil, the base portion of the in vitro vein sheet of water surface submergence is made.
6. inoculate germ: the mycelia block that picking step 3 prepares, be placed with gently and operate on complete vein sheet in step 5.
7. inoculation after dispose: step 6 operate complete after, getting lens all covers on large square plate by the working panel in primary side coil and dish and in vitro vein sheet, and thin layer clear water is injected in large square plate, make lens enclose inside and form high humidity state stable in cover; Then complete assembly is put in the general room possessing normal scattered light to cultivate, culture temperature maintains 26 ~ 28 DEG C.
Inoculation result causes vein sheet to occur the downright bad scab be perfectly clear, and general inoculation starts to occur downright bad symptom for latter 30 hours, whole vein sheet total necrosis after 4 ~ 6 days.
Advantage of the present invention is:
1) adopt larger tobacco leaf, the difference of morbidity result state of an illness weight is easily embodied; And tobacco plant can be more for the blade quantity utilized, the extended period is longer;
2) actual enforcement only uses vein bar but not whole lamina, is conducive to inoculating more host material in limited utensil space;
3) inoculate the pathogenic process time short, the place that takes up room is few; Implementation and operation is simple and easy convenient, and working efficiency is high;
4) disease symptom is clear easy to identify, favorable reproducibility;
5) inoculation morbidity local condition easily controls, and process management is simple, easily realizes the stdn of inoculation method.
Accompanying drawing explanation
Fig. 1 is the disposal options schematic diagram of in vitro vein sheet of the present invention.
In figure, A portion signal excised leaf full wafer is fixed on working panel and cardinal extremity inserts undersurface state; The style of in vitro vein sheet and postvaccinal state thereof are illustrated by B portion.
Fig. 2 is the morbidity performance process sample 1 of one group of in vitro vein sheet inoculation rhizoctonia solani of the present invention.
In figure, A ~ D portion be after morbidity successively from the state of an illness gently to the Symptoms that the state of an illness is heavy.E and F portion is blank inoculation contrast, wherein E portion and A portion same period, F portion and D portion same period.
Fig. 3 is the morbidity performance process sample 2 of one group of in vitro vein sheet inoculation rhizoctonia solani of the present invention.
In figure, A ~ D portion be after morbidity successively from the state of an illness gently to the Symptoms that the state of an illness is heavy.E and F portion is blank inoculation contrast, wherein E portion and A portion same period, F portion and D portion same period.
Fig. 4 is inoculation utensil schematic diagram of the present invention.
In figure, a is large square plate, and b is primary side coil, and c is lens, and d is working panel, and e is supporting plate, and f is rack plate, and g is bungee; A portion is the complete schematic diagram of each component assembling of utensil; B portion is that pattern card is seen in the side after A portion removes lens c; C portion is that the front in B portion is seen; D portion is that the complete appliance front on working panel d places in vitro vein sheet and covers after lens c is seen.
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, 3 again) utensil is transparent is convenient to blade daylighting and PD is observed; 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 a set of simple set of instruments to be made into inoculating appliance, can reach above-mentioned technical requirements.
Tobacco leaf is unfolded on the working panel of this cover utensil, as shown in the A portion of Fig. 1, be very beneficial for implementing inoculation operation and pathogenic process observation, because tobacco leaf is larger, footprint area is large, and the present invention is designed to cut the in vitro vein sheet of the tobacco comprising main lobe arteries and veins and makes material, as shown in the B portion of Fig. 1, be conducive to like this inoculating more host material in limited utensil area, raise the efficiency; In fact, the vein sheet cutting larger tobacco leaf also has 2 advantages as material, one is that longer vein sheet is from the disease that starts to serious morbidity, there is more state of an illness variation space, being thus applicable to very much the experimental study (size etc. as comparative measurement different strains virulence) for carrying out state of an illness difference; Two is that blade the greater is in the great majority, and owing to can be applicable to use of the present invention after cutting, the tobacco leaf of inoculation work is drawn materials in time and is quantitatively not easy to be limited in tobacco whole breeding time.
After tobacco in vitro vein sheet inoculation rhizoctonia solani, cultivate under 25 ~ 28 DEG C of conditions, after general 30 hours, the visible downright bad scab of excised leaf occurs, as shown in the A portion of Fig. 2 and Fig. 3, passing in time, scab constantly expands and two ends to vein sheet are expanded, and as shown in B ~ D portion of Fig. 2 and Fig. 3, finally can cause vein sheet total necrosis.From being inoculated into vein sheet total necrosis 4 ~ 6 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
Utilize in vitro vein sheet to inoculate artificial onset's method of tobacco damping-off, cigarette seedling kind cloud and mist 87 excised leaf inoculate tobacco rhizoctonia solani bacterial strain Rs-1, operate as follows:
1. inoculation utensil prepares: the utensil being used as inoculation comprises, large square plate a, primary side coil b, lens c, working panel d, supporting plate e and rack plate f as shown in Figure 4, the use material of the present embodiment utensil and size: large square plate a is the common square plate of length × wide × height=40 × 30 × 3cm; Primary side coil b is the common square plate of length × wide × height=29 × 22 × 3cm; Lens c is the plexiglass tent of length × wide × height=31 × 25 × 20cm; Working panel d is the simple glass plate of 24 × 24cm, and supporting plate e is the little sheet glass of 24 × 9cm; Rack plate f is the foil of two ends folding hook, and folding hook after poppet leaf length is 15cm.With purificant by for subsequent use after these assembly washes clean.
2. cigarette seedling is cultivated: cultivate cigarette seedling kind cloud and mist 87 according to a conventional method, conventional water and fertilizer management, tobacco leaf length is greater than 20cm and can uses.
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 acquisition process of in vitro vein sheet: with the blade on clean scissors clip step 2 tobacco plant, indoor are reinstalled with clean container, under tap water, rinse blade surface well gently, blade cut into clean knife blade the in vitro vein sheet retaining main lobe arteries and veins for subsequent use;
5. on inoculation utensil, place in vitro vein sheet:
1) assembling inoculation utensil, method is: take out the utensil that step 1 is got ready, primary side coil is placed in the centre in large square plate, get two up and down that 2 clean common bungees are enclosed within working panel respectively, in primary side coil, supporting plate and the mutual hook of working panel be linked to be a stable system with 3 rack plates, working panel is made to become heeling condition, as shown in B and the C portion of Fig. 4.
2) place in vitro vein sheet: get the in vitro vein sheet that step 4 is got ready, upward, cardinal extremity down on top, laid parallel is on working panel, and provoke bungee the two ends of in vitro vein sheet are clamped fixing, then toward injected clear water 800mL in primary side coil, the base portion of the in vitro vein sheet of water surface submergence.
6. inoculate germ: picking step 3 prepare mycelia block, be placed with gently and operate on complete vein sheet in step 5, operate complete after state shape as shown in the B portion of Fig. 1;
7. inoculation after dispose: step 6 operate complete after, getting lens all covers on large square plate by the working panel in primary side coil and dish and in vitro vein sheet, and thin layer clear water is injected in large square plate, make lens enclose inside and form high humidity state stable in cover; Then be put in by complete assembly on the indoor cultivation frame possessing normal scattered light and cultivate, culture temperature maintains 26 ~ 28 DEG C.
Result in vitro vein sheet shows downright bad scab clearly as the same in Fig. 2 with Fig. 3; Inoculate the latter 72 hours most widths of downright bad scab from being 6.1cm.
Embodiment 2
A kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off, cigarette seedling kind cloud and mist 87 excised leaf inoculates tobacco rhizoctonia solani bacterial strain Rs-1, enforcement is operated to step 7 by the step 1 of embodiment 1, 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 26 ~ 28 DEG C, and after the step 7) inoculation of the present embodiment, culture temperature maintains 20 ~ 22 DEG C.Result in vitro vein sheet also shows downright bad scab clearly as the same in Fig. 2 with Fig. 3, but scab to expand speed slower than the speed of embodiment 1, inoculate the most width of latter 120 hours downright bad scabs from being 5.9cm.
Embodiment 3
A kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off, cigarette seedling kind cloud and mist 87 excised leaf inoculates tobacco rhizoctonia solani bacterial strain Rs-1, implement 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 in vitro vein sheet also shows downright bad scab clearly as the same in Fig. 2 with Fig. 3, but scab to expand speed slower than the speed of embodiment 1, inoculate the most width of latter 72 hours downright bad scabs from being 2.8cm.
Embodiment 4
A kind of artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off, cigarette seedling kind cloud and mist 87 excised leaf inoculates tobacco rhizoctonia solani bacterial strain Rs-1, implement 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 20 ~ 22 DEG C;
Result in vitro vein sheet shows downright bad scab clearly as the same in Fig. 2 with Fig. 3 equally, but scab to expand speed much slower than the speed of embodiment 1, inoculate the most width of latter 120 hours downright bad scabs from being 2.1cm.
Claims (1)
1. the artificial onset's method utilizing in vitro vein sheet to inoculate tobacco damping-off, it is characterized in that, by larger tobacco leaf, cut into the lengthy motion picture bar (being called for short in vitro vein sheet) retaining main lobe arteries and veins, utilize a set of simple utensil inoculation tobacco rhizoctonia solani, cause in vitro vein sheet tissue necrosis and show disease symptom clearly;
The step of artificial onset's method is as follows:
1) inoculation utensil prepares:
Inoculation utensil comprises: large square plate, primary side coil, lens, working panel, supporting plate and rack plate; With purificant by for subsequent use after these utensil washes clean;
2) cigarette seedling is cultivated:
Cultivate cigarette seedling according to a conventional method, conventional water and fertilizer management, tobacco leaf length is greater than 20cm and can uses;
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) acquisition process of in vitro vein sheet:
By clean scissors clip step 2) blade on tobacco plant, reinstall indoor with clean container, under tap water, rinse blade surface well gently, blade cut into the clean instrument that cuts the in vitro vein sheet retaining main lobe arteries and veins for subsequent use;
5) on inoculation utensil, in vitro vein sheet is placed:
1. assembling inoculation utensil, method is: take out the utensil that step 1) is got ready, primary side coil is placed in the centre in large square plate, get two up and down that 2 clean common bungees are enclosed within working panel respectively, working panel pad is leaned against on support that supporting plate and rack plate put up, make panel form stable heeling condition;
2. place in vitro vein sheet: get the in vitro vein sheet that step 4) is got ready, upward, down, laid parallel on working panel, and provokes bungee by steady for the two ends of in vitro vein sheet folder to cardinal extremity on top; Then toward injected clear water in primary side coil, the base portion of the in vitro vein sheet of water surface submergence is made;
6) inoculate germ: the mycelia block that picking step 3) prepares, be placed with gently and operate on complete in vitro vein sheet in step 5);
7) dispose after inoculation: after step 6) operation is complete, getting lens all covers on large square plate by the working panel in primary side coil and dish and in vitro vein sheet, and thin layer clear water is injected in large square plate, make lens enclose inside and form high humidity state stable in cover; Then complete assembly is put in the general room possessing normal scattered light to cultivate, culture temperature maintains 26 ~ 28 DEG C;
Inoculation result causes vein sheet to occur the downright bad scab be perfectly clear, and general inoculation starts to occur downright bad symptom for latter 30 hours, whole vein sheet total necrosis after 4 ~ 6 days.
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| 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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|---|---|---|---|---|
| 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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