CN111972265A - Mikania micrantha puccinia expanding propagation method - Google Patents
Mikania micrantha puccinia expanding propagation method Download PDFInfo
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- CN111972265A CN111972265A CN202010698803.4A CN202010698803A CN111972265A CN 111972265 A CN111972265 A CN 111972265A CN 202010698803 A CN202010698803 A CN 202010698803A CN 111972265 A CN111972265 A CN 111972265A
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- 238000000034 method Methods 0.000 title claims abstract description 54
- 241001440840 Mikania micrantha Species 0.000 title claims abstract description 32
- 241000221300 Puccinia Species 0.000 title claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 100
- 241000196324 Embryophyta Species 0.000 claims abstract description 58
- 241000653900 Micrantha Species 0.000 claims abstract description 24
- 230000001902 propagating effect Effects 0.000 claims abstract description 14
- 230000007246 mechanism Effects 0.000 claims description 32
- 239000003595 mist Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 13
- 239000007921 spray Substances 0.000 claims description 6
- 230000007226 seed germination Effects 0.000 claims 1
- 238000011081 inoculation Methods 0.000 abstract description 22
- 238000004321 preservation Methods 0.000 abstract description 7
- 241001233242 Lontra Species 0.000 abstract description 5
- 241000233866 Fungi Species 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000009434 installation Methods 0.000 abstract 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 abstract 1
- 239000000779 smoke Substances 0.000 abstract 1
- 201000010099 disease Diseases 0.000 description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 13
- 239000000725 suspension Substances 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 7
- 238000005507 spraying Methods 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 230000003902 lesion Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 238000012136 culture method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003020 moisturizing effect Effects 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 241001600161 Fulvia Species 0.000 description 1
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- 235000021307 Triticum Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
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- 239000011159 matrix material Substances 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
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- 229910001220 stainless steel Inorganic materials 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
- A01G31/02—Special apparatus therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Abstract
The invention discloses a method for propagating puccinia micrantha by using puccinia micrantha, which comprises the following steps: in cultivateing indoorly, place a plurality of boxes in the basin, box lower part opening, the lower part of the body top is equipped with the apron that can open, sets up atomizing water jet equipment in every box respectively, will treat the healthy plant of mikania micrantha of inoculation to place respectively in each box, to basin injected water in order to seal box lower part and basin junction, again with otter board difference horizontal installation in healthy plant top, will have the area fungus plant of ripe mikania micrantha handle rust fungus spore to place on the otter board, last to spout one deck water smoke on box inner wall and the plant, seal the box, take out after cultivateing 48h under the dark condition, place in indoor cultivation. The invention has the beneficial effects that: the method has the same inoculation effect as a small-scale seed preservation method adopting a constant-temperature incubator in a laboratory, and is suitable for large-scale propagation of the puccinia micrantha.
Description
Technical Field
The invention belongs to the field of microbial culture methods of plant parasites, and particularly relates to a method for propagating stalactite from mikania micrantha.
Background
The mikania micrantha puccinia is a fungus with strong host specificity, and does not cause harm to plants except the host. In a series of studies on puccinia micrantha, it is particularly important to maintain continuous culture of the strain. Because the strain has higher requirements on the temperature and the humidity of the environment during inoculation, the strain is mostly inoculated in a constant temperature incubator in a small scale, and the reproduction quantity of the strain is small. On one hand, the related instruments are expensive; on the other hand, when the amount of the plants to be tested is large, the constant temperature incubator is adopted for inoculation, so that the requirement cannot be met; in addition, the existing culture method has low inoculation rate of bacteria by putting the bacteria-carrying plants and healthy plants together for culture. Therefore, the invention provides a large-scale and efficient propagation method for the puccinia micrantha.
Disclosure of Invention
In view of the above, the invention provides a method for propagating puccinia micrantha.
The technical scheme is as follows:
the key point of the method for propagating the puccinia micrantha is that the method is carried out according to the following steps:
step one, arranging a culture device: placing at least two boxes in a water tank in a culture room at 18-25 ℃, wherein each box comprises a barrel, the barrel is vertically arranged, the lower end of the barrel falls on the bottom of the water tank, the upper end of the barrel is covered with a cover plate in an openable and closable manner, a water inlet pipe is respectively arranged in each barrel, the water inlet pipe is arranged at the upper part of the barrel, at least one atomizing nozzle is connected to the water inlet pipe, and the water inlet pipe penetrates out of the barrel and is connected with a water supply device;
step two, respectively placing the healthy mikania micrantha plants to be inoculated in each cylinder, and injecting water into the water tank to seal the joint of the lower end of each cylinder and the water tank;
step three, horizontally installing a screen plate in each cylinder, wherein the screen plate is positioned above the healthy plants;
step four, placing a bacteria-carrying plant with mature mikania micrantha puccinia spores on the screen plate;
fifthly, the water supply device works to enable the atomizing nozzle to spray a layer of water mist on the inner wall of the box body and the plants;
and step six, covering the cover plate on the cylinder body in a buckling manner to seal the cylinder body, culturing for 48 hours under a dark condition, taking out, and placing in a room for culturing.
By adopting the method, the culture of the mikania micrantha can be simultaneously carried out on a large scale, the large-scale propagation of the mikania micrantha puccinia is realized, the plants with bacteria are prevented above the healthy plants, the atomizing spray heads are arranged in each box body and connected with the same water supply device, the inside of each box body is artificially and controllably atomized and watered, the humidity inside each box body is maintained, meanwhile, the air flow in each box body is increased, mature spores on the cultured mikania micrantha leaves are more easily released, and the healthy mikania micrantha plant leaves are more easily infected by the spores.
As a preferred technical scheme, in the third step, the net plate is positioned 5-10cm above the healthy plants.
By adopting the method, after the strain-carrying plant is placed on the screen plate, the distance between the strain-carrying plant and the healthy plant is proper.
Preferably, in the fourth step, the plants with bacteria are horizontally laid down, and the back surfaces of the leaves with bacteria are downward as many as possible.
By adopting the method, the spores are convenient to fall after being released.
According to a preferable technical scheme, the water supply device comprises a water pump, a water inlet of the water pump is communicated with the inner cavity of the water tank, a water outlet of the water pump is connected with a water distribution pipe, the water distribution pipe is connected with all the water inlet pipes, and water mist is sprayed into the tank body by opening the water pump.
By adopting the method, the water mist is synchronously and controllably sprayed into all the boxes, the water in the water tank is pumped into the boxes by the water pump, the water falls down after being sprayed and then converges into the water tank, the circulation is repeated, the humidity in the boxes is maintained, too much water is not accumulated in the water tank, and the spores falling into the water can be sprayed onto healthy plants along with the water mist again, so that the inoculation rate is improved.
And as a preferred technical scheme, in the process of culturing the plants in the box body, repeating the step five at intervals so as to spray water mist into the box body for multiple times.
By adopting the method, the humidity in the box body is kept, the air in the box body is disturbed, and the release of spores is facilitated.
Preferably, the temperature in the culture chamber is 20 ℃.
With the above method, the temperature is suitable for mature spore release.
As a preferred technical scheme, lifting adjusting mechanisms are arranged between the screen plate and the cylinder body, at least two sets of lifting adjusting mechanisms are arranged, and all the lifting adjusting mechanisms are distributed along the circumferential direction of the cylinder body;
and in the fourth step, the screen plate is connected with the lifting adjusting mechanism, and then the distance from the screen plate to the healthy plants is adjusted.
By adopting the method, the height of the screen plate is adjustable, and the method is suitable for plants with different heights.
As a preferred technical scheme, the lifting adjusting mechanism comprises a suspension bar and a fastening mechanism arranged on the wall of the cylinder;
at least two positioning holes are formed in the hanging strip along the length direction of the hanging strip;
and in the fourth step, the hanging strip is hung on the fastening mechanism through the positioning hole at a proper position, and the lower end of the hanging strip is connected with the screen plate.
By adopting the method, the screen plate can be adjusted up and down by matching different positioning holes on the hanging strip with the fastening mechanism.
As a preferred technical scheme, the fastening mechanism comprises a hanging rod and a butterfly nut;
the hanging rod comprises a screw section, and any end of the screw section is connected with an anti-drop head;
mounting holes are respectively penetrated through the cylinder wall of the cylinder body corresponding to each fastening mechanism;
in the fourth step, the screw end of the hanging rod sequentially penetrates through the positioning hole and the mounting hole and then extends out of the cylinder wall of the cylinder body, then is in threaded fit with the butterfly nut, and the anti-drop head presses the hanging strip onto the inner wall of the cylinder body so that the hanging strip is connected with the cylinder body in a positioning mode.
By adopting the method, the fastening structure can be conveniently locked or unlocked outside the cylinder body.
As a preferred technical scheme, the suspension bar comprises a suspension bar body, wherein the suspension bar body is provided with positioning holes along a straight line, the suspension bar body is also provided with a strip-shaped adjusting seam, the adjusting seam is arranged along the central connecting line of all the positioning holes, the width of the adjusting seam is larger than the outer diameter of the screw section and smaller than the aperture of the positioning holes, and the adjusting seam connects all the positioning holes;
a large-diameter section is connected between the screw section and the anti-drop head, and the outer diameter of the large-diameter section is consistent with the inner diameter of the positioning hole;
and in the fourth step, the butterfly nut is screwed to enable the large-diameter section to penetrate through the corresponding positioning hole.
By adopting the method, after the fastening structure is unlocked, the position of the hanging strip can be conveniently adjusted by adjusting the seam without taking out the hanging rod.
As a preferred technical scheme, a ring gasket is arranged on the outer wall of the cylinder body at the mounting hole, the ring gasket is attached to the outer wall of the cylinder body, and an inner hole of the ring gasket is communicated with the mounting hole;
the hanging rod penetrates through the ring pad outwards and then is connected with the butterfly nut.
By adopting the method, the stability of the fastening mechanism during locking is improved.
As a preferred technical scheme, the mikania micrantha plant is prepared by germinating and culturing seeds for about 60 days or putting stem nodes in water for rooting and then performing cutting culture for about 30 days for standby application, and the plant height of the healthy plant to be inoculated is controlled and tillering is promoted by shearing growth points in the culture process, so that the healthy plant has a plurality of growth points and the number of tender leaves is increased.
By adopting the method, the plant to be inoculated has a plurality of growing points and a plurality of tender leaves, and is easy to be infected by spores.
Drawings
FIG. 1 is a schematic diagram of the apparatus of the present invention;
FIG. 2 is a schematic view showing the mesh plate and the water inlet pipe installed in the barrel;
FIG. 3 is a right side view of FIG. 2;
FIG. 4 is an enlarged view of the portion m1 in FIG. 3;
FIG. 5 is a cross-sectional view taken along line A-A of FIG. 2;
FIG. 6 is an enlarged view of the portion m2 in FIG. 5;
fig. 7 is a schematic structural diagram of a latch.
Detailed Description
The present invention will be further described with reference to the following examples and the accompanying drawings.
(I) inoculation device
Example one
As shown in fig. 1, 2 and 3, a puccinia micrantha expands numerous device, including locating basin 1 in the blake room, be provided with two at least boxes in this basin 1, the bottom opening of box, the box upper portion is equipped with otter board 4, this otter board 4 level set up in the box. Each box still is equipped with inlet tube 10 respectively, inlet tube 10 is located otter board 4 upper portion, be connected with at least one atomizer 9 on the inlet tube 10. All inlet tubes 10 are connected with the same water supply device, the water supply device is located outside the box body, the water supply device comprises a water pump 12, a water inlet of the water pump 12 is communicated with an inner cavity of the water tank 1, a water outlet of the water pump 12 is connected with a water distribution pipe 11, and the water distribution pipe 11 is connected with all inlet tubes 10.
The box includes barrel 2, and this barrel 2 is vertical to be set up, and 2 lower extremes of this barrel fall on 1 tank bottoms of basin, and 2 upper ends of this barrel are the buckle closure has apron 3, basin 1 with apron 3 will 2 inner chambers of barrel seal. A pipe orifice is formed at the upper edge of the cylinder 2, a clamping block 13 is arranged at the pipe orifice, as shown in fig. 7, a pipe hole 13a penetrates through the clamping block 13, and continuous clamping grooves 13b are formed in the side surface and the lower surface of the clamping block 13. The clamping groove 13b is buckled on the inner edge of the pipe orifice, so that the clamping block 13 is fixed on the pipe orifice. The fixture block 13 is further provided with a pipe hole slit 13c, and the pipe hole slit 13c extends out of the surface of the fixture block 13 from the pipe hole to form an opening of the pipe hole. The pipe hole slit 13c is separated to fit the water inlet pipe 10 into the pipe hole, and then the latch 13 is fitted at the pipe orifice. The latch 13 may be a rubber block. The upper surface of the clamping block 13 is level with the upper edge of the cylinder body 2, and when the cover plate 3 is buckled on the cylinder body 2, the pipe hole seam 13c is pressed to be closed.
And a lifting adjusting mechanism is arranged between the screen plate 4 and the cylinder body 2.
At least two sets of lifting adjusting mechanisms are arranged, and all the lifting adjusting mechanisms are uniformly distributed along the circumferential direction of the barrel body 2. In this embodiment, as shown in fig. 2 and 3, the barrel 2 is rectangular, and a set of the lifting adjusting mechanism is provided near each of four edges of the barrel. As shown in fig. 5, the lifting adjusting mechanism includes a suspension bar 5 and a fastening mechanism provided on the wall of the cylinder 2. The hanging strip 5 is connected with the cylinder 2 through the fastening mechanism, and the lower end of the hanging strip 5 is connected with the screen plate 4 through a rope with two separated ends.
As shown in fig. 5 and 6, the suspension bar 5 includes a suspension bar body, and at least a plurality of positioning holes 5a are linearly distributed on the suspension bar body along the length direction thereof. The hanging strip body is further provided with a long strip-shaped adjusting seam 5b, the adjusting seam 5b is arranged along the central connecting line of all the positioning holes 5a, the width of the adjusting seam 5b is smaller than the aperture of the positioning holes 5a, and the adjusting seam 5b is used for communicating all the positioning holes 5 a. The suspension bar 5 is made of a rigid metal or plastic sheet.
As shown in fig. 4 and 5, the fastening mechanism includes a hanging rod 6 and a wing nut 7. The hanging rod 6 comprises a screw section 6a, a large-diameter section 6b and an anti-drop head 6c which are connected in sequence, the outer diameter of the screw section 6a is not larger than the width of the adjusting seam 5b, and the outer diameter of the large-diameter section 6b is consistent with the inner diameter of the positioning hole 5 a.
And mounting holes 2a are respectively penetrated through the wall of the cylinder body 2 corresponding to each fastening mechanism. The screw end of the hanging rod 6 sequentially penetrates through the positioning hole 5a and the mounting hole 2a and then extends out of the cylinder wall of the cylinder 2, and then is in threaded fit with the butterfly nut 7, and the anti-drop head 6c presses the hanging strip 5 tightly on the inner wall of the cylinder 2.
In order to improve the stability of the fastening mechanism, a ring gasket 8 is arranged on the outer wall of the cylinder body 2 at the position of the mounting hole 2a, the ring gasket 8 is attached to the outer wall of the cylinder body 2, and an inner hole of the ring gasket 8 is communicated with the mounting hole 2 a. The hanging rod 6 penetrates through the ring pad 8 outwards and then is connected with the butterfly nut 7, one surface, facing the butterfly nut 7, of the ring pad 8 is a plane, and when the ring pad is locked, the butterfly nut 7 abuts against the ring pad 8. The ring pad 8 may be made of a rubber block.
The process of adjusting the height of the screen plate 4 by the lifting adjusting mechanism comprises the following steps: when the large-diameter section 6b of the hanging rod 6 penetrates into the positioning hole 5a and is locked with the butterfly nut 7, the hanging strip 5 is stably positioned; when the mesh plate 4 needs to move up or down, the butterfly nut 7 is unscrewed, the hanging rod 6 moves inwards until the screw section 6a penetrates through the positioning hole 5a, the screw section 6a can move freely in the adjusting slot 5b due to the small outer diameter, the hanging strip 5 moves up and down to a proper height, the positioning hole 5a in a proper position is opposite to the mounting hole 2a, then the butterfly nut 7 is screwed, and the hanging strip is positioned again.
The water tank 1 may be formed by pouring concrete, and the cylinder 2 and the cover plate 3 may be made of PVC plastic, stainless steel, wood, or the like. The net plate 4 can be a plastic net or an iron wire net and has the characteristics of light weight and high porosity.
(II) inoculation experiment of puccinia micrantha
Example 2
Experimental materials: the experimental strain is Puccinia spezazinii, which is preserved in the plant protection research institute of academy of agricultural sciences in China, and the strain preservation number is as follows: IMI 3939075; the inoculation material is mikania micrantha: the matrix material is purchased from Guangzhou Sheng agriculture GmbH, and the mikania micrantha is obtained by cutting and culturing the stem nodes of wild mikania micrantha for about 30 days to obtain plants. Specifically, mikania micrantha stem nodes are placed in water, planted in a seedling raising pot with the caliber of 10cm after rooting, and reserved after about 30 days. Inoculating healthy mikania micrantha by adopting a natural inoculation method, culturing in a room with the temperature of 25 ℃ and the humidity of 70% for about 25 days after inoculation, and completely maturing (the life history of puccinia micrantha is 19-22 days) the stems, leafstalks and leaves of the successfully inoculated mikania micrantha growing visible wintersporidium piles for later use. And for the plant to be inoculated, the plant height of the healthy plant to be inoculated is controlled and tillering of the healthy plant is promoted by cutting the growth points in the culture process, so that the healthy plant has a plurality of growth points and the number of tender leaves is increased, and the plant height of the mikania micrantha plant is not more than 40 cm.
The apparatus of example 1 was used for the propagation of puccinia micrantha. The method comprises the following steps:
step one, arranging a culture device, setting the temperature in a culture room at 20 ℃, placing a box body in a water tank 1, arranging a water inlet pipe 10 in each barrel 2 respectively, wherein the water inlet pipe 10 is arranged at the upper part of the barrel 2, and the water inlet pipe 10 penetrates out of the barrel 2 and then is connected with a water supply device;
secondly, respectively placing the healthy mikania micrantha plants to be inoculated in each cylinder body 2, and injecting 3-5cm of clear water into the water tank 1 to seal the joint of the lower end of the cylinder body 2 and the water tank 1;
thirdly, horizontally installing the screen plates 4 in the cylinder bodies 2 respectively, and adjusting the screen plates 4 to be positioned 5-10cm above the healthy plants through the lifting adjusting mechanism;
placing the bacteria-carrying plant with the mature puccinia micrantha puccinia spores on the screen plate 4, horizontally laying the bacteria-carrying plant, and enabling the back surfaces of the bacteria-carrying leaves to be downward as much as possible to be positioned above the growth point of the healthy plant;
fifthly, the water pump 12 works to enable the atomizing nozzle 9 to spray a layer of water mist on the inner wall of the box body and plants;
step six, covering the cover plate 3 on the cylinder body 2 in a buckling manner to seal the cylinder body, culturing under the dark condition, repeating the step five every 2 hours in the culturing process, spraying water mist into the box body for multiple times, 3min each time, taking out after culturing for 48 hours, and then placing the box body in a room for culturing at the indoor temperature of 25-35 ℃.
The culture method uses a pot as an independent sample, and each method has at least 10 repeated samples. After 15 days, the incidence and disease index were counted.
The method for calculating the morbidity comprises the following steps:
the incidence rate is the number of the affected leaves/the total number of the leaves of the sample multiplied by 100 percent;
the disease index calculation method comprises the following steps:
the disease grade judgment standard refers to the wheat disease grade classification standard of Lifuning (1991) and is slightly modified:
level 0: no infection;
level 1: the relative area of the lesion spots is less than 5 percent;
and 2, stage: the relative area of the scab is 5-20 percent;
and 3, level: the relative lesion area is 20 to 40 percent;
4, level: the relative lesion area is 40-60 percent;
and 5, stage: the relative lesion area is more than 60 percent;
none of the above selection ranges includes an upper limit;
the disease index ∑ (number of diseased plants at each stage × the disease grade value)/(total number of investigated plants × highest grade value) × 100.
Comparative example 1
The conventional experimental seed conservation method adopts a constant-temperature incubator for inoculation culture, and comprises the following steps:
1) putting mikania micrantha stem nodes in water, planting the mikania micrantha stem nodes in a seedling raising pot with the caliber of 10cm after rooting, and reserving the mikania micrantha stem nodes after 30 days per month;
2) putting healthy mikania micrantha plants into the bottom of a moisture preservation box for inoculation, injecting 3-5cm of water into the bottom, and spraying a layer of water mist on the plant leaf surfaces;
3) suspending the wire netting in the air and placing the wire netting in a moisture preserving box at a height of 5-10cm away from the mikania micrantha;
4) cutting off leaves with mature puccinia micrantha stipe spores, spraying a layer of water mist on the surfaces of the leaves, putting the leaves on an iron wire net, and enabling each leaf with the bacteria to be above a healthy plant growing point;
5) spraying a layer of water mist on the inner wall and the cover of the moisture preservation box, covering the moisture preservation box with the cover, and placing the moisture preservation box in a constant-temperature incubator under dark environment conditions with the temperature of 20 ℃ and the humidity of 100%;
6) and taking out the mikania micrantha after 48 hours, and culturing in a room.
Statistical analysis of morbidity and disease was performed as in example 2.
(III) analysis of Fulvia micrantha inoculation experiment result
Two methods are evaluated from the moisture retention, incidence and disease index. In terms of moisturizing effect, in example 2, inoculated plants were taken from inoculation to completion of inoculation, and the leaf surfaces of the plants had a large amount of water droplets. In comparative example 1, the inoculated plants were placed in a stable incubator until the inoculation was completed, and the leaves on the leaf surface of the plants had a large amount of water drops, and the moisturizing effects of the two methods were similar.
In example 2, the incidence of disease after inoculation in example 2 was 41.03% and the disease index was 29.53, as compared to control 1, as shown in table 1. The incidence of disease after inoculation in comparative example 1 was 40.65%, and the disease index was 26.63. The incidence of vaccination was close to that of both methods, whereas the disease index of example 2 was higher, indicating that the vaccination effect of the present invention was comparable or better than that of the conservation method. This may be due to: the two methods can maintain the humidity stability of the culture environment, are beneficial to opening the winter spores of the puccinia micrantha as far as possible and releasing basidiospores; in the embodiment 2, the strain with the bacteria is taken as a bacteria source, spores are continuously matured in the culture process, and the density of sporophytes in a unit area on a horizontal plane is higher; in example 2, the air flow in the box body is increased by spraying water mist into the box body, so that the release of spores is promoted.
TABLE 1 comparison of the inoculation results of the two inoculation methods
Compared with the prior art, the invention has the beneficial effects that: the device has simple structure and low cost, can simultaneously carry out batch culture of the mikania micrantha, has the inoculation effect equivalent to that of a small-scale seed preservation method adopting a constant-temperature incubator in a laboratory, and is suitable for large-scale propagation of the mikania micrantha puccinia.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and that those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Claims (10)
1. A method for propagating puccinia micrantha is characterized by comprising the following steps:
step one, arranging a culture device: placing at least two boxes in a water tank (1) in a culture room at 18-25 ℃, wherein each box comprises a barrel (2), the barrels (2) are vertically arranged, the lower ends of the barrels (2) fall on the bottom of the water tank (1), a cover plate (3) is covered at the upper end of each barrel (2) in a buckling manner in an openable manner, a water inlet pipe (10) is respectively arranged in each barrel (2), the water inlet pipe (10) is arranged at the upper part of each barrel (2), at least one atomizing nozzle (9) is connected onto the water inlet pipe (10), and the water inlet pipe (10) penetrates out of the barrels (2) and then is connected with a water supply device;
secondly, placing healthy mikania micrantha plants to be inoculated in each cylinder body (2) respectively, and injecting water into the water tank (1) to seal the joint of the lower end of each cylinder body (2) and the water tank (1);
step three, horizontally installing a screen plate (4) in each cylinder body (2) respectively, wherein the screen plate (4) is positioned above the healthy plants;
step four, placing a bacteria-carrying plant with mature mikania micrantha puccinia spores on the screen plate (4);
fifthly, the water supply device works to enable the atomizing nozzle (9) to spray a layer of water mist on the inner wall of the box body and plants;
and step six, covering the cover plate (3) on the cylinder body (2) in a buckling manner to seal the cylinder body, culturing for 48 hours under a dark condition, taking out, and placing in a room for culturing.
2. The method for propagating puccinia micrantha according to claim 1, characterized in that: in the third step, the mesh plate (4) is positioned 5-10cm above the healthy plants.
3. The method for propagating puccinia micrantha according to claim 1, characterized in that: in the fourth step, the strain-carrying plants are horizontally laid down, so that the back surfaces of the strain-carrying leaves are downward as much as possible.
4. The method for propagating puccinia micrantha according to claim 1, characterized in that: the water supply device comprises a water pump (12), a water inlet of the water pump (12) is communicated with an inner cavity of the water tank (1), a water outlet of the water pump (12) is connected with a water distribution pipe (11), the water distribution pipe (11) is connected with all the water inlet pipes (10), and water mist is sprayed into the tank body by opening the water pump (12).
5. The method for propagating puccinia micrantha according to claim 4, characterized in that: and repeating the fifth step at intervals in the cultivation process of the plants in the box body, so as to spray water mist into the box body for multiple times.
6. The method for propagating puccinia micrantha according to claim 1, characterized in that: the temperature in the culture chamber was 20 ℃.
7. The method for propagating puccinia micrantha according to claim 1, characterized in that: lifting adjusting mechanisms are arranged between the screen plate (4) and the barrel body (2), at least two sets of lifting adjusting mechanisms are arranged, and all the lifting adjusting mechanisms are distributed along the circumferential direction of the barrel body (2);
in the fourth step, the screen plate (4) is connected with the lifting adjusting mechanism, and then the distance from the screen plate (4) to the healthy plants is adjusted.
8. The method for propagating puccinia micrantha according to claim 7, characterized in that: the lifting adjusting mechanism comprises a hanging strip (5) and a fastening mechanism arranged on the wall of the barrel body (2);
at least two positioning holes (5a) are formed in the hanging strip (5) along the length direction of the hanging strip;
in the fourth step, the hanging strip (5) is hung on the fastening mechanism through the positioning hole (5a) in a proper position, and the lower end of the hanging strip (5) is connected with the screen plate (4).
9. The method for propagating puccinia micrantha according to claim 8, characterized in that: the fastening mechanism comprises a hanging rod (6) and a butterfly nut (7);
the hanging rod (6) comprises a screw section (6a), and any end of the screw section (6a) is connected with an anti-drop head (6 c);
mounting holes (2a) are respectively penetrated through the wall of the barrel body (2) corresponding to each fastening mechanism;
in the fourth step, the screw end of the hanging rod (6) sequentially penetrates through the positioning hole (5a) and the mounting hole (2a) and then extends out of the cylinder wall of the cylinder body (2), and then is in threaded fit with the butterfly nut (7), and the anti-drop head (6c) presses the hanging strip (5) on the inner wall of the cylinder body (2) so that the hanging strip (5) is in positioning connection with the cylinder body (2).
10. The method for propagating puccinia micrantha according to claim 1, characterized in that: the mikania micrantha plant is cultured for about 60 days by seed germination or is cultured for about 30 days by cuttage after stem nodes are put in water for rooting, the plant height of the healthy plant to be inoculated is controlled and tillering is promoted by cutting growth points in the culture process, so that the healthy plant has a plurality of growth points and the number of tender leaves is increased.
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| CN111165266A (en) * | 2020-01-08 | 2020-05-19 | 华南农业大学 | Method for continuously expanding propagation of arbuscular mycorrhizal fungal spores through compartment culture |
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|---|---|---|---|---|
| CN101608159A (en) * | 2008-06-20 | 2009-12-23 | 钟明基 | A kind of cultivation from method and the device of the inoculation bacterium of mycorrhizal fungi |
| CN103320329A (en) * | 2013-05-20 | 2013-09-25 | 兰州大学 | Apocynum venetum melampsora propagation method |
| CN106834087A (en) * | 2017-03-01 | 2017-06-13 | 厦门大学 | A kind of AM fungies of timing cycle expand numerous and functional experiment device and application method |
| CN208454982U (en) * | 2018-06-01 | 2019-02-01 | 青岛万图明生物制品有限公司 | A kind of water isolation type constant incubator |
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