CN103789215A - Artificial disease inducing method by inoculating tobacco damping off on in-vitro leaf vein - Google Patents
Artificial disease inducing method by inoculating tobacco damping off on in-vitro leaf vein Download PDFInfo
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Abstract
The invention discloses an artificial disease inducing method by inoculating tobacco damping off on in-vitro leaf veins. The method comprises the following steps: 1) preparing tools for inoculation, 2) culturing tobacco seedlings, 3) preparing inoculation mycelium, 4) collecting and treating in-vitro leaf veins, 5) putting the in-vitro leaf veins on the tools for inoculation, 6) inoculating germs, and 7) performing later treatment after inoculation. The method has the beneficial effects that firstly, the inoculation disease induction time is short, a small space is occupied, the method is simple, easy, rapid and convenient, and the working efficiency is high; secondly, large tobacco leaves are used in the method, so that the difference of result disease situations can be easily reflected, the obtaining of the tobacco leaves is ensured, the disease symptoms are clear and easy to identify, the reproducibility is good, the partial conditions for inoculation disease induction are easy to control, and the standardization of the technical method is easy to realize.
Description
Technical field
The present invention relates to agrotechnique and biotechnology.A kind of specifically artificial onset's method of utilizing in vitro vein sheet inoculation tobacco damping-off.
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 one of relevant basic means of studying of 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 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 a kind of artificial onset's method of utilizing in vitro vein sheet inoculation 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 of utilizing in vitro vein sheet inoculation 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) that retains main lobe arteries and veins, utilize a set of simple utensil inoculation tobacco rhizoctonia solani, cause in vitro vein sheet tissue morbidity and show symptom clearly, the step of artificial onset's method is as follows:
1. inoculation is prepared with utensil: the main utensil of inoculation use 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 according to a conventional method cigarette seedling, and conventional water and fertilizer management, tobacco leaf length is greater than 20cm and can uses.
3. the mycelial preparation of inoculation: rhizoctonia solani is transplanted to the dull and stereotyped center of the common fungi culture mediums such as 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 acquisition process of in vitro vein sheet: with the blade on clean scissors clip step 2 tobacco plant, reinstall indoor with clean container, under tap water, rinse gently blade surface well, the in vitro vein sheet that blade is cut into reservation main lobe arteries and veins with the clean instrument that cuts is 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 to the centre in large square plate, get 2 clean common bungees and be enclosed within respectively two up and down of working panel, working panel pad is leaned against on the 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,,, laid parallel is on working panel, and it is steady by the two ends folder of in vitro vein sheet to provoke bungee for cardinal extremity on top upward down; Then in primary side coil, inject clear water, make the base portion of the in vitro vein sheet of water surface submergence.
6. inoculation germ: the mycelia piece that picking step 3 is prepared, is placed with gently in step 5 and operates on complete vein sheet.
Inoculation after dispose: step 6 operate complete after, getting lens all covers on the working panel in primary side coil and dish and in vitro vein sheet on large square plate, and inject thin layer clear water in large square plate, make lens enclose inside and form stable high wet condition in cover; Then complete assembly is put in to the general room cultivation that possesses normal scattered light, culture temperature maintains 26～28 ℃.
Inoculation result causes vein sheet to occur the downright bad scab being perfectly clear, and generally inoculates latter 30 hours and starts to occur downright bad symptom, 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 for utilize blade quantity more, the extended period is longer;
2) actual enforcement only used vein bar but not whole lamina, is conducive to inoculate more host material in limited utensil space;
3) inoculation 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; Style and the postvaccinal state thereof of in vitro vein sheet 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 is the Symptoms gently weighing to the state of an illness from the state of an illness successively after morbidity.E and F portion are blank inoculation contrasts, 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 is the Symptoms gently weighing to the state of an illness from the state of an illness successively after morbidity.E and F portion are blank inoculation contrasts, wherein E portion and A portion same period, F portion and D portion same period.
Fig. 4 is that the present invention inoculates and uses utensil schematic diagram.
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 the each component assembling of utensil; B portion is that pattern card is seen in the side that A portion removes after lens c; C portion is the anterior view of B portion; D portion places in vitro vein sheet and covers the complete appliance anterior view after upper glass cover c at working panel d.
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 observe; 4) utensil is simple and to implement inoculation easy to operate, can keep again subenvironment in high wet condition.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, being very beneficial for implementing inoculation operation and pathogenic process observes, 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 that comprises main lobe arteries and veins and makes material, as shown in the B portion of Fig. 1, be conducive to like this inoculate more host material in limited utensil area, raise the efficiency; In fact, the vein sheet that cuts larger tobacco leaf also has 2 advantages as material, the one, the vein sheet of growing is from the disease that starts to serious morbidity, there is more state of an illness variation space, thereby be applicable to very much experimental study for carrying out state of an illness difference (as the size of comparative measurement different strains virulence etc.); The 2nd, in tobacco whole breeding time, blade the greater is in the great majority, and owing to can be applicable to use of the present invention after cutting, makes the tobacco leaf of the work of inoculating draw materials in time and quantitatively be not easy and is limited to.
After the in vitro vein sheet inoculation of tobacco rhizoctonia solani, under 25～28 ℃ of conditions, cultivate, 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 expands to the two ends of vein sheet, 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 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
Utilize artificial onset's method of in vitro vein sheet inoculation tobacco damping-off, on cigarette seedling kind cloud and mist 87 excised leafs, inoculate tobacco rhizoctonia solani bacterial strain Rs-1, as follows operation:
1. inoculation is prepared with utensil: the utensil 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 present embodiment utensil by material and size: the common square plate that large square plate a is length × wide × height=40 × 30 × 3cm; The common square plate that primary side coil b is length × wide × height=29 × 22 × 3cm; The plexiglass tent that lens c is length × wide × height=31 × 25 × 20cm; Working panel d is the simple glass plate of 24 × 24cm, the little sheet glass that supporting plate e is 24 × 9cm; Rack plate f is the foil of two ends folding hook, and folding hook after poppet sheet length is 15cm.With purificant by for subsequent use after these assembly washes clean.
2. cigarette seedling is cultivated: cultivate according to a conventional method cigarette seedling kind cloud and mist 87, and conventional water and fertilizer management, tobacco leaf length is greater than 20cm and can uses.
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 acquisition process of in vitro vein sheet: with the blade on clean scissors clip step 2 tobacco plant, reinstall indoor with clean container, under tap water, rinse gently blade surface well, the in vitro vein sheet that blade is cut into reservation main lobe arteries and veins with clean knife blade is 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 to the centre in large square plate, get 2 clean common bungees and be enclosed within respectively two up and down of working panel, in primary side coil, with 3 rack plates, supporting plate and the mutual hook of working panel are linked to be to a stable system, make working panel become heeling condition, as shown in the B of Fig. 4 and C portion.
2) place in vitro vein sheet: get the in vitro vein sheet that step 4 is got ready,, cardinal extremity down on top upward, laid parallel is on working panel, and provoke bungee the two ends of in vitro vein sheet are clamped fixing, then toward injecting clear water 800mL, the base portion of the in vitro vein sheet of water surface submergence in primary side coil.
6. inoculation germ: the mycelia piece that picking step 3 is prepared, be placed with gently in step 5 and operate on complete vein sheet, operate state shape after complete as shown in the B portion of Fig. 1;
Inoculation after dispose: step 6 operate complete after, getting lens all covers on the working panel in primary side coil and dish and in vitro vein sheet on large square plate, and inject thin layer clear water in large square plate, make lens enclose inside and form stable high wet condition in cover; Then complete assembly is put on the indoor cultivation frame that possesses normal scattered light and cultivates, culture temperature maintains 26～28 ℃.
The in vitro vein sheet of result shows downright bad scab clearly as the same with Fig. 3 in Fig. 2; Inoculate the width of latter 72 hours downright bad scabs from being 6.1cm.
Embodiment 2
A kind of artificial onset's method of utilizing in vitro vein sheet inoculation tobacco damping-off, on cigarette seedling kind cloud and mist 87 excised leafs, inoculate tobacco rhizoctonia solani bacterial strain Rs-1, pressing step 1 to step 7 operation of embodiment 1 implements, 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 26～28 ℃, and after the step 7) inoculation of the present embodiment, culture temperature maintains 20～22 ℃.The in vitro vein sheet of result also shows downright bad scab clearly as the same with Fig. 3 in Fig. 2, but that scab expands speed is slower than the speed of embodiment 1, inoculates the width of latter 120 hours downright bad scabs from being 5.9cm.
Embodiment 3
A kind of artificial onset's method of utilizing in vitro vein sheet inoculation tobacco damping-off, on cigarette seedling kind cloud and mist 87 excised leafs, inoculate tobacco rhizoctonia solani bacterial strain Rs-1, pressing step 1) to the step 7) operation of embodiment 1 implements, 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.The in vitro vein sheet of result also shows downright bad scab clearly as the same with Fig. 3 in Fig. 2, but that scab expands speed is slower than the speed of embodiment 1, inoculates the width of latter 72 hours downright bad scabs from being 2.8cm.
Embodiment 4
A kind of artificial onset's method of utilizing in vitro vein sheet inoculation tobacco damping-off, on cigarette seedling kind cloud and mist 87 excised leafs, inoculate tobacco rhizoctonia solani bacterial strain Rs-1, pressing step 1) to the step 3) operation of embodiment 1 implements, 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 20～22 ℃;
The in vitro vein sheet of result shows downright bad scab clearly as the same with Fig. 3 in Fig. 2 equally, but that scab expands speed is much slower than the speed of embodiment 1, inoculates the width of latter 120 hours downright bad scabs from being 2.1cm.
Claims (1)
1. one kind is utilized artificial onset's method of in vitro vein sheet inoculation 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) that retains 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 is prepared with utensil:
Inoculation comprises with utensil: 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 according to a conventional method cigarette seedling, 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 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) acquisition process of in vitro vein sheet:
By clean scissors clip step 2) blade on tobacco plant, reinstall indoorly with clean container, under tap water, rinse gently blade surface well, with the clean instrument that cuts, blade is cut into to retain the in vitro vein sheet of 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 to the centre in large square plate, get 2 clean common bungees and be enclosed within respectively two up and down of working panel, working panel pad is leaned against on the 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,,, laid parallel is on working panel, and it is steady by the two ends folder of in vitro vein sheet to provoke bungee for cardinal extremity on top upward down; Then in primary side coil, inject clear water, make the base portion of the in vitro vein sheet of water surface submergence;
6) inoculation germ: the mycelia piece that picking step 3) is prepared, is placed with gently in step 5) and operates on complete in vitro vein sheet;
7) after inoculation, dispose: after step 6) operation is complete, getting lens all covers on the working panel in primary side coil and dish and in vitro vein sheet on large square plate, and inject thin layer clear water in large square plate, make lens enclose inside and form stable high wet condition in cover; Then complete assembly is put in to the general room cultivation that possesses normal scattered light, culture temperature maintains 26～28 ℃;
Inoculation result causes vein sheet to occur the downright bad scab being perfectly clear, and generally inoculates latter 30 hours and starts to occur downright bad symptom, whole vein sheet total necrosis after 4～6 days.
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| CN107012197A (en) * | 2017-03-07 | 2017-08-04 | 广西壮族自治区农业科学院甘蔗研究所 | A kind of method for determining sugarcane top rot germ pathogenicity |
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| CN107012197A (en) * | 2017-03-07 | 2017-08-04 | 广西壮族自治区农业科学院甘蔗研究所 | A kind of method for determining sugarcane top rot germ pathogenicity |
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