CN112048542A

CN112048542A - Inoculation method of puccinia micrantha

Info

Publication number: CN112048542A
Application number: CN202010697897.3A
Authority: CN
Inventors: 刘博�; 钱万强; 任行海; 张付斗; 万方浩
Original assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Current assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2020-12-08

Abstract

The invention discloses an inoculation method of mikania micrantha puccinia, which comprises the following steps: placing a fresh mikania micrantha plant to be inoculated in an openable box, wherein water is filled at the bottom of the box; horizontally installing a screen plate in the box, wherein the screen plate is positioned above the mikania micrantha plants; placing a fungus-carrying leaf with mature puccinia micrantha spores on the mesh plate, wherein the side of the leaf with the spores faces downwards; sealing the box, placing in a constant temperature incubator or room at 18-25 deg.C, culturing in dark for 48 hr, taking out, and culturing in room. The invention has the beneficial effects that: through the simple device, the leaves with the bacteria and the healthy plants are reasonably arranged, the humidity in the box is kept, stable conditions are created for inoculation of the bacteria, and the effect of the method is greatly improved compared with that of the existing inoculation method.

Description

Inoculation method of puccinia micrantha

Technical Field

The invention belongs to the field of a microbial culture method of plant parasitism, and particularly relates to an inoculation method of mikania micrantha dersoni.

Background

In recent years, biological control has been the subject of research with increasing drug resistance and pesticide residue. The mikania micrantha puccinia is a pathogenic fungus parasitic on mikania micrantha, and does not cause harm to plants except hosts. Has the potential of becoming a biological prevention preparation of mikania micrantha. The mikania micrantha Puccinia (Puccinia spegazzini) belongs to pucciniales (Uredinales) Puccinia (Puccinia), is a fungus with short life cycle and strong host specificity, cannot be cultured in vitro at present, and can only continuously survive on living plants. The propagation of the puccinia micrantha is to open the spore apparatus of mature winter spores under proper environmental conditions to release basidiospores. Basidiospores are one of 5 types of spores of rust fungi, and are called basidiospores because they are born on basidiomycetes. In the prior art, healthy mikania micrantha plants and fungi-carrying mikania micrantha plants with mature mikania micrantha stalk rust fungi are generally put together for cultivation. When the mikania micrantha puccinia is inoculated, the release amount of basidiospores is low, and the inoculation effect is poor. In addition, the strain has higher requirements on the temperature and the humidity in the environment during inoculation, so that 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 price of the related instrument is expensive, and on the other hand, when the amount of the plant to be tested is large, the constant temperature incubator is adopted for inoculation, so that the requirement cannot be met. Therefore, it is highly desirable to improve the inoculation apparatus and the inoculation method to improve the inoculation effect.

Disclosure of Invention

In view of the above, the invention provides an inoculation method of puccinia micrantha.

The technical scheme is as follows:

the inoculation method of the puccinia micrantha is characterized by comprising the following steps of:

placing a fresh mikania micrantha plant to be inoculated in an openable box, wherein the bottom of the box is filled with water, and a layer of water mist is sprayed on the surface of the mikania micrantha plant;

step two, horizontally installing a screen plate in the box, wherein the screen plate is positioned above the mikania micrantha plants;

placing the leaves with the mature puccinia micrantha spores on the screen plate, wherein the side of the leaves with the spores faces downwards;

and step four, sealing the box, placing the box in a constant-temperature incubator or a room at the temperature of 18-25 ℃, culturing for 48 hours under the dark condition, taking out, and culturing indoors.

As a preferable technical scheme, in the second step, the mesh plate is positioned 5-10cm above the mikania micrantha plant.

As a preferred technical scheme, in the third step, a layer of water mist is sprayed on the leaf surfaces and leaf backs of the leaves with the bacteria, and then the leaves are placed on the screen plate.

As a preferable technical scheme, in the fourth step, the inner wall of the box is sprayed with a layer of water mist and then is closed.

Preferably, in the fourth step, the temperature in the constant temperature incubator or the room is 20 ℃.

According to a preferable technical scheme, the box comprises a water tank, a barrel is vertically arranged on the water tank, a cover plate is covered at the upper end of the barrel in a buckling manner, and a lifting adjusting mechanism is arranged between the barrel wall of the barrel and the screen plate;

in the second step, the screen plate is connected with the lifting adjusting mechanism, and then the distance from the screen plate to the mikania micrantha plants is adjusted.

As a preferred technical scheme, 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 wall;

the lifting adjusting mechanism comprises a hanging strip and a fastening mechanism arranged on the wall of the cylinder body;

at least two positioning holes are formed in the hanging strip along the length direction of the hanging strip;

in the second 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 and the hanging strip.

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 second 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 tightly on the inner wall of the cylinder body, so that the hanging strip is connected with the cylinder body in a positioning mode.

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;

in the second step, the butterfly nut is screwed to enable the large-diameter section to penetrate through the corresponding positioning hole.

Drawings

FIG. 1 is a schematic diagram of the construction of an inoculating device for use in the present invention;

FIG. 2 is an enlarged view of the portion m1 in FIG. 1;

FIG. 3 is a cross-sectional view taken along line A-A of FIG. 1;

FIG. 4 is an enlarged view of the portion m2 in FIG. 3;

FIG. 5 is a schematic diagram of inoculation.

Detailed Description

The present invention will be further described with reference to the following examples and the accompanying drawings.

(I) inoculation device

Example one

The utility model provides an inoculation device of mikania micrantha handle rust fungus, includes the combination formula case, this case internal seal, but this case bottom water greatly, this case upper portion is equipped with

otter board

4, 4 levels of this otter board set up in the case, this otter board 4 with be equipped with lifting adjusting mechanism between the case.

As shown in fig. 1 and 3, the box comprises a water tank 1 located at the bottom, a cylinder 2 is vertically arranged on the water tank 1, the lower end of the cylinder 2 falls on the bottom of the water tank 1, a cover plate 3 is covered at the upper end of the cylinder 2, and the water tank 1, the cover plate 3 and the cylinder 2 are combined into a closed box. The cylindrical body 2 may be cylindrical or prismatic.

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. The lifting adjusting mechanism comprises a hanging strip 5 and a fastening mechanism arranged on the wall of the barrel body 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. 2 and 4, 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, 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-falling head 6c which are sequentially connected, 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. And 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.

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 screen 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 5 is positioned again. With the adoption of the design, after the fastening structure is conveniently unlocked, the position of the hanging bar 5 is adjusted through the adjusting slot 5b, and the hanging rod is not required to be taken out.

The water tank 1, the cylinder 2 and the cover plate 3 can be made of PVC plastic, stainless steel, wood and other materials. 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 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 on the stems, leafstalks and leaves for later use.

Inoculation with puccinia micrantha was performed using the apparatus of example 1. The method comprises the following steps:

step one, injecting clear water with the depth of about 3-5 cm into a water tank 1, placing a cylinder body 2 into the water tank 1, immersing the lower end of the cylinder body below the water surface of the water tank 1 to realize sealing, then placing a healthy fresh mikania micrantha plant to be inoculated into the water tank 1, and spraying a layer of water mist on the surface of the mikania micrantha plant;

step two, horizontally installing a screen plate 4 in the box, and adjusting the screen plate 4 to enable the screen plate 4 to be located 5-10cm above the healthy plants;

spraying a layer of water mist on the leaf surface and the leaf back of a fungus-carrying leaf with mature mikania micrantha puccinia spores, and then placing the leaf on the screen plate 4, wherein the side of the leaf with the spores faces downwards;

and fourthly, after a layer of water mist is sprayed on the inner wall of the cylinder body 2, in order to further ensure the box to be wet, a layer of preservative film sprayed with water mist can be covered on the upper end of the cylinder body 2, wherein the water mist is positioned on the lower surface of the preservative film, finally, the cover plate 3 is covered on the upper end of the cylinder body 2 in a buckling manner, as shown in figure 5, the box is placed in a constant-temperature incubator or a room with the temperature of 18-25 ℃ and the humidity of 100%, and is taken out after being cultured for 48 hours under the dark condition, and is cultured indoors.

The invention has no special requirements on the species of the mikania micrantha, is limited to the size of the culture device, the distance between the bacteria source and the mikania micrantha to be inoculated and the moisturizing effect, and is suitable for the plant height of the mikania micrantha plant not exceeding 20 cm. The inoculation box has high tightness, the bacterium-carrying blades spray a layer of water mist during inoculation, and the surface of the blades needs to be kept to have a layer of water mist during the whole inoculation process, so that the environment condition suitable for spore release is ensured.

Taking a group of plants which are cultured in a constant temperature incubator at 20 ℃ as an example, taking out the plants, and then culturing the plants indoors at the indoor temperature of 25-35 ℃, wherein the green fading disease spots can be seen in 4-6 days, and the rust winter spore pile can be seen on the leaf back in 19-22 days.

At least 10 parallel samples are designed by taking a basin as an independent sample. And after 15 days, counting the morbidity and the illness index.

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

Inoculating and culturing by adopting a conventional method:

putting healthy mikania micrantha plants and mikania micrantha plants with mature mikania micrantha puccinia into a constant-temperature incubator with the temperature of 20 ℃, the humidity of 100% and the dark condition, and spraying a layer of water mist on the surfaces of the plants; checking the humidity of the surface of the plant in the morning and afternoon every day, spraying a layer of water mist on the surface of the plant in time to ensure the humidity, taking out after 48 hours, and culturing indoors. The leaf infection with Acremonium mii mikanoi was counted as in example 2.

(III) analysis of Fulvia micrantha inoculation experiment result

In terms of moisturizing effect, in the method of example 2 of the present invention, the inoculated plant was taken out from the time when the inoculation apparatus was placed in a closed incubator or room to the end of inoculation, and the leaf surface of the plant had a large amount of water droplets. In the method of comparative example 1, the inoculated plants were taken out from the time of their placement in the incubator until the end of inoculation, and the surfaces of the plants were humidified by spraying water 4 times during this period, and it was found that there were few, if any, water droplets on the leaf surfaces of the plants when the water was sprayed again.

The superiority of the experimental method is judged from two aspects of morbidity and disease index. As shown in table 1, the method of example 2 had an incidence of 40.65% and a disease index of 26.63 after inoculation compared to the conventional method. The incidence rate after inoculation by the conventional method is 30.24 percent, and the disease index is 20.02. It is clear that the method of example 2 is superior to the inoculation effect of the conventional method. The reason for this analysis may be that: (1) the bacterial source, namely the leaves with the bacteria are placed right above the healthy plants to be inoculated, and the spores are easy to fall on the plant leaves after being released; (2) the inoculation device of example 1 has good sealing performance, and maintains a stable high-humidity environment inside; (3) before the box is sealed, a layer of water beads is sprayed on the inner wall of the box and the surface of the leaf with bacteria, and the winter spores of the puccinia micrantha are opened as much as possible in a humid environment to release basidiospores.

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 simple structure, with low costs, help keeping the humidity in the case after the flourishing water of case bottom, for the inoculation of fungus creates stable condition, the winterspore of mikania micrantha handle rust fungus is opened as far as, releases the basidiospore, reduces the condition emergence that the basidiospore can't be released because of inoculation environment humidity is not enough to the winterspore. Compared with other inoculation methods, the method has the advantages of large release amount of basidiospores, high inoculation efficiency and adjustable height of the screen plate, and is suitable for plants with different heights.

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

1. An inoculation method of puccinia micrantha is characterized by comprising the following steps:

step two, horizontally installing a screen plate (4) in the box, wherein the screen plate (4) is positioned above the mikania micrantha plants;

placing the leaves with the mature puccinia micrantha spores on the screen plate (4), wherein the side of the leaves with the spores faces downwards;

2. The inoculation method of puccinia micrantha according to claim 1, characterized in that: in the second step, the mesh plate (4) is positioned 5-10cm above the mikania micrantha plant.

3. The inoculation method of puccinia micrantha according to claim 1, characterized in that: and in the third step, spraying a layer of water mist on the leaf surface and the leaf back of the leaf with the bacteria, and then placing the leaf surface and the leaf back on the screen plate (4).

4. The inoculation method of puccinia micrantha according to claim 1, characterized in that: and in the fourth step, the inner wall of the box is sprayed with a layer of water mist and then is closed.

5. The inoculation method of puccinia micrantha according to claim 1, characterized in that: in the fourth step, the temperature in the constant temperature incubator or the room is 20 ℃.

6. The inoculation method of puccinia micrantha according to any one of claims 1 to 5, characterized in that: the box comprises a water tank (1), a barrel body (2) is vertically arranged on the water tank (1), a cover plate (3) is covered at the upper end of the barrel body (2), and a lifting adjusting mechanism is arranged between the barrel wall of the barrel body (2) and the screen plate (4);

in the second step, the screen plate (4) is connected with the lifting adjusting mechanism, and then the distance from the screen plate (4) to the mikania micrantha plants is adjusted.

7. The inoculation method of puccinia micrantha according to claim 6, characterized in that: 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 wall of the cylinder body (2);

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 second step, the hanging strip (5) is hung on the fastening mechanism through the positioning hole (5a) at a proper position, and the lower end of the hanging strip (5) is connected with the screen plate (4) and the hanging strip (5).

8. The inoculation method of puccinia micrantha according to claim 7, 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 cylinder wall of the cylinder body (2) corresponding to each fastening mechanism;

in the second 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) tightly presses the hanging strip (5) on the inner wall of the cylinder body (2), so that the hanging strip (5) is connected with the cylinder body (2) in a positioning mode.

9. The inoculation method of puccinia micrantha according to claim 8, characterized in that: the suspension bar (5) comprises a suspension bar body, the positioning holes (5a) are distributed on the suspension bar body along a straight line, a long-strip-shaped adjusting seam (5b) is further formed in the suspension bar body, 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 larger than the outer diameter of the screw section (6a) and smaller than the aperture of the positioning holes (5a), and the adjusting seam (5b) is used for communicating all the positioning holes (5 a);

a large-diameter section (6b) is connected between the screw section (6a) and the anti-drop head (6c), and the outer diameter of the large-diameter section (6b) is consistent with the inner diameter of the positioning hole (5 a);

in the second step, the butterfly nut (7) is screwed to enable the large-diameter section (6b) to penetrate through the corresponding positioning hole (5 a).