CN110551674A - Inoculation and propagation method of maize puccinia polycarya - Google Patents
Inoculation and propagation method of maize puccinia polycarya Download PDFInfo
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Abstract
The invention relates to a propagation method of phytopathogen, in particular to an inoculation propagation method of maize puccinia polycephala. The invention provides an inoculation and propagation method of maize puccinia polycephala, which comprises the following steps: spraying Tween-20 solution on diseased leaves, eluting spores on the diseased leaves in the Tween-20 solution to prepare spore suspension, inoculating the spore suspension to healthy corn seedlings, culturing, harvesting the spores obtained by propagation, and repeatedly transferring by using the healthy corn seedlings. The method has the advantages of fast propagation speed and high efficiency of the puccinia polycephala, can rapidly obtain a large amount of high-activity puccinia polycephala spores, greatly shortens the propagation period of the puccinia polycephala, and provides an efficient strain propagation method and technical support for researching southern rust of corn.
Description
Technical Field
The invention relates to a propagation method of phytopathogen, in particular to an inoculation propagation method of maize puccinia polycephala.
Background
Southern corn rust is an air-borne disease caused by Puccinia polysora Underw, which occurs in many areas. In recent years, southern rust of corn has become one of the main diseases affecting corn production, and therefore, attention is increasingly paid to the research and control of corn puccinia polycarpa. The study of maize puccinia polyclona and maize southern rust is not open to artificial inoculation, which typically requires large numbers of highly active spores. Because the puccinia polycephala is obligate parasitic fungus and can only survive and propagate on a live corn plant, the method for obtaining a large number of spores by propagating the puccinia polycephala can only be carried out by co-culturing with the corn plant, so that the propagation speed of the puccinia polycephala and a large number of sources of the spores are limited.
Chinese patent 201510765142.1 discloses a single-spore propagation method for maize puccinia polycephala, which aims to obtain pure lines of maize puccinia polycephala, but the propagation efficiency of the method still cannot meet the large demand of artificial inoculation on spores. Therefore, the development of an inoculation propagation method which has high propagation efficiency and can rapidly obtain a large amount of maize puccinia sorghi spores is urgently needed.
Disclosure of Invention
in order to solve the technical problems in the prior art, the invention aims to provide a method for inoculating and propagating puccinia polycephala of corn with high propagation efficiency.
In order to achieve the purpose, the technical scheme of the invention is as follows:
The invention provides an inoculation and propagation method of maize puccinia polycephala, which comprises the following steps: eluting spores on diseased leaves, preparing a spore suspension, inoculating the spore suspension to healthy corn seedlings, culturing, harvesting and propagating the spores, and repeatedly transferring the healthy corn seedlings; wherein the eluting spores on diseased leaves comprises: spraying Tween-20 solution on the diseased leaves, and eluting spores on the diseased leaves in the Tween-20 solution.
According to the invention, the tween-20 solution is sprayed on the diseased leaves before the spores on the diseased leaves are eluted, so that the spores can be better adhered to the leaves, the spores are effectively prevented from falling off, and the loss of the spores is reduced.
Preferably, the concentration of the Tween-20 solution for spraying is 0.02-0.05%. The invention discovers that spraying the diseased leaves with the Tween-20 solution with the concentration can connect water beads, effectively prevent spores of multi-stack puccinia to fall off, and simultaneously better keep the activity of the spores of multi-stack puccinia.
Preferably, the spraying dosage of the Tween-20 solution is 1-2 mL per diseased leaf.
More preferably, the elution of spores from diseased leaves to prepare a spore suspension is specifically: spraying Tween-20 solution on the diseased leaves, cutting the diseased leaves, adding Tween-20 solution, soaking the diseased leaves, shaking, and eluting spores on the diseased leaves in the Tween-20 solution to obtain spore suspension.
Preferably, the concentration of the Tween-20 solution for elution is 0.02-0.05%. The elution and the preparation of the spore suspension by using the Tween-20 solution with the concentration range can better improve the inoculation success rate and the propagation expansion efficiency of the spores.
more preferably, the concentration of the tween-20 solution for spraying and eluting is 0.05% each.
When spore harvest is carried out, the traditional needle scraping blade fungus collecting method can damage the blades to a certain extent, the repeated fungus collecting times of the blades are reduced, and the spore harvest efficiency of the needle scraping blade fungus collecting is lower. According to the invention, the spore is harvested by adopting a method of shaking the stem of a corn plant to make the spore fall off in the culture process after the beginning of sporulation, and the repeated fungus harvesting times of the diseased leaves can be obviously increased by combining the method of eluting the spores on the diseased leaves by adopting the Tween-20 solution when one round of propagation expanding culture is finished, so that the spore harvesting efficiency is effectively improved. By using the method, in the culture process, each diseased leaf can be repeatedly harvested at least twice by shaking the corn plant, and when the culture is finished, the spores on the diseased leaves are eluted finally, so that the harvested spore amount can be effectively ensured.
Preferably, the harvesting and expanding the obtained spores comprise: after the spore production is started, the spores on the leaves are shed by shaking the stem of the corn plant; further comprising: and when one round of propagation is finished, spraying a Tween-20 solution on the diseased leaves, cutting the diseased leaves, adding the Tween-20 solution, soaking the diseased leaves, shaking, and eluting spores on the diseased leaves in the Tween-20 solution. Further preferably, shake maize plant stem portion for adopt glass stick or glass chi strike maize plant stem portion.
Preferably, the inoculation is spraying the spore suspension on the leaves of healthy corn seedlings.
Further preferably, the healthy corn seedlings are corn seedlings in a three-leaf one-heart seedling stage; the healthy corn seedlings were dewaxed prior to inoculation.
As a preferable scheme of the invention, the inoculation and propagation method of the maize puccinia polycephala comprises the following steps:
(1) Seedling: sowing corn seeds to obtain healthy corn seedlings in a three-leaf one-heart seedling stage;
(2) spraying 0.02-0.05% Tween-20 solution on the diseased leaves, cutting the diseased leaves into small pieces of 2-3cm 2, placing the small pieces in a container, adding 0.02-0.05% Tween-20 solution, soaking the diseased leaves, shaking, and eluting the spores on the diseased leaves in the Tween-20 solution to obtain spore suspension;
(3) Inoculating bacteria: spraying the spore suspension prepared in the step (2) onto the healthy corn seedlings in the trilobal single-heart seedling stage obtained in the step (1) after dewaxing treatment, and culturing the inoculated healthy corn seedlings;
(4) Harvesting spores: after sporulation has begun, the spores are harvested by knocking the stem of the maize plant to shed the sporophyte on the paper.
(5) And (4) repeating the steps (2) to (4) 18-25 days after inoculation, and carrying out a new round of inoculation and propagation.
Preferably, in the step (1), before sowing, the corn seeds are subjected to pregermination, and after 1-2mm radicles grow at the front ends of the corn seeds, the corn seeds are sown, wherein the radicles face upwards and face to the center of the planting container during sowing.
Further preferably, the method for accelerating germination of corn seeds specifically comprises the following steps: selecting plump corn seeds, soaking the seeds for 24-48h at room temperature by using 0.1% hydrogen peroxide, pouring off liquid or taking out the seeds, covering wet paper towels or wet cloth on the seeds, and accelerating germination at a ventilated place.
preferably, in the step (2), the container is a large-size centrifuge tube or a conical flask.
Preferably, in the step (3), after inoculation, the healthy maize seedlings are kept wet for 24-48h in the dark at the temperature of 20-30 ℃ and then are transferred to the environment with sufficient illumination, the relative air humidity of more than 80% and the temperature of 20-30 ℃ for continuous culture.
Preferably, in step (4) above, the spores are harvested every 3-5 days.
Preferably, in the step (4), the stem is knocked by a glass rod or a glass ruler to make the spore pile fall off on the paper.
The invention has the beneficial effects that:
The inoculation propagation method of the maize multi-stack puccinia has the advantages of saving labor and time for harvesting the fungi (compared with the traditional needle grafting, the method for preparing the spore suspension and then carrying out the spray grafting of multiple pots of maize seedlings simultaneously), saving a large amount of time and labor for harvesting the spores by oscillating the maize plant and combining the spore harvesting with the method for eluting the diseased leaves compared with the traditional method for harvesting the spores by needle scraping), reducing the loss amount of the spores and improving the utilization rate of the spores (compared with the traditional method for harvesting the spores by needle scraping, the method for preparing the spore suspension by eluting the diseased leaves has the advantages of less loss amount of the spores and higher utilization rate), can realize higher inoculation success rate, has high propagation speed and high efficiency, can rapidly obtain a large amount of high-activity spores (the spores begin to produce the spores after 6-10 days of inoculation and can be harvested once every 3-5 days, the strain can be repeatedly collected for at least 2 times, the diseased leaves can be cut after 18-25 days after inoculation, spores are eluted, a spore suspension is prepared, a new round of inoculation and propagation is started, and the harvested spores can be inoculated to obtain at least 12 pots of 8 three-leaf and one-heart corn seedlings with good morbidity, so that the propagation period of the multi-stack puccinia striiformis is greatly shortened, and an effective strain propagation method and technical support are provided for researching the southern rust disease of the corn.
Drawings
FIG. 1 is a photograph of a maize seedling which begins to produce spores after inoculation of a healthy maize seedling in example 1 of the present invention.
FIG. 2 is a photograph of a maize seedling after spraying spore suspension in example 2 of the present invention.
FIG. 3 is a photograph of a maize seedling after inoculation with spores in comparative example 1 of the present invention.
FIG. 4 is a photograph showing the onset of disease in corn seedlings after harvesting spores by needle scraping, eluting the diseased leaves, harvesting spores, and spraying healthy corn seedlings in comparative example 1.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
example 1 inoculation and propagation method of Puccinia polycarpa of corn (1)
The embodiment provides an inoculation and propagation method of puccinia polycephala of corn, which specifically comprises the following steps:
(1) Seedling: selecting plump corn seeds, soaking the seeds for 24-48h at room temperature by using 0.1% hydrogen peroxide, pouring liquid or taking out the seeds, covering wet paper towels or wet cloth on the seeds, and accelerating germination at a ventilated place; sowing after 1-2mm radicles grow out from the front ends of the corn seeds, wherein the radicles face upwards and towards the center of the planting container during sowing, and culturing to obtain healthy corn seedlings in a three-leaf one-heart seedling stage;
(2) Spraying 0.05% Tween-20 solution (about 2 mL/diseased leaf), cutting diseased leaf into small pieces of 2-3cm 2, placing in a large centrifuge tube, adding 0.05% Tween-20 solution, soaking diseased leaf, shaking, and eluting spore in Tween-20 solution to obtain spore suspension;
(3) inoculating bacteria: spraying the spore suspension prepared in the step (2) to the healthy maize seedlings in the three-leaf one-heart seedling stage obtained in the step (1) after dewaxing treatment; keeping moisture of the inoculated healthy maize seedlings in the dark at 20-30 ℃ for 24-48h, and then transferring to the environment conditions of sufficient illumination, relative air humidity of more than 80% and temperature of 20-30 ℃ for continuous culture;
(4) Harvesting spores: knocking the stem of the corn plant by a glass ruler after the spore production begins to fall off the spore pile on paper, and harvesting the spores once every 3-5 days;
(5) And (3) spraying 0.05% Tween-20 solution with the concentration of about 2 mL/diseased leaf 20 days after inoculation, cutting the diseased leaf into small blocks with the concentration of 2-3cm 2, placing the small blocks into a large centrifugal tube, adding 0.05% Tween-20 solution, soaking the diseased leaf, shaking, eluting spores on the diseased leaf into the Tween-20 solution to obtain spore suspension, and repeating the steps (3) to (4) for 2 times.
By using the method of the embodiment, after the spore suspension is sprayed and inoculated, the spore piles on the leaf are uniformly distributed; maize shoots that have just begun to sporulate are shown in FIG. 1. The harvested spores can obtain 12 pots of corn seedlings with good disease (8 corn seedlings with three leaves and one heart in each pot), 30ng of spores are harvested in each pot, and 360ng of spores are obtained.
Example 2 inoculation propagation method of Puccinia polycarpa of corn (2)
this example provides a method for inoculating and propagating Puccinia sorghi of maize, which is different from example 1 only in that, in step (5), steps (3) to (4) are repeated 1 time.
By using the method of the embodiment, after the spore suspension is sprayed, the spore piles on the leaf are uniformly distributed (as shown in figure 2); the harvested spores can obtain 3 pots of corn seedlings with good morbidity, 30ng of spores are harvested in each pot, and 100ng of spores are obtained.
Comparative example 1
the comparative example provides an inoculation and propagation method of maize puccinia polycephala, which adopts the traditional needle scraping to harvest spores and a needle inoculation method to inoculate the fungi, and comprises the following specific steps:
(1) Seedling: selecting plump corn seeds, soaking the seeds for 24-48h at room temperature by using 0.1% hydrogen peroxide, pouring liquid or taking out the seeds, covering wet paper towels or wet cloth on the seeds, and accelerating germination at a ventilated place; sowing after 1-2mm radicles grow out from the front ends of the corn seeds, wherein the radicles face upwards and towards the center of the planting container during sowing, and culturing to obtain healthy corn seedlings in a three-leaf one-heart seedling stage;
(2) inoculating bacteria: dewaxing the leaves of the healthy corn seedlings in the three-leaf one-heart-seedling stage obtained in the step (1), spraying 0.05% of Tween-20 solution on the leaves, needling the sporophores obtained by needle scraping to the leaves of the healthy corn seedlings, preserving moisture for 24-48h in the dark at the temperature of 20-30 ℃, and transferring to the environment conditions of sufficient illumination, over 80% of air relative humidity and 20-30 ℃ for continuous culture;
(3) Harvesting spores: after the start of sporulation, spores were harvested 1 time per diseased leaf using a needle-scrape method.
With the method of the present comparative example, after inoculating spores, the distribution of the sporophyte on the leaves was uneven (as shown in FIG. 3); when spores are harvested, because the blades are damaged due to needle scraping, each diseased leaf can only be harvested for 1 time of spores, and the spores cannot be harvested repeatedly; by using the method, 2 pots of corn seedlings with good morbidity can be obtained, 15ng of spores are harvested in each pot, and 30ng of spores are obtained.
In order to verify that the spore utilization rate is low by using the method of comparative example 1, the sporule-scraped leaves are subjected to the steps (2) and (3) of example 1, namely 0.05% of Tween-20 solution is sprayed on diseased leaves, about 2 mL/diseased leaf is cut into 2-3cm 2 small blocks, the small blocks are placed in a large-size centrifuge tube, 0.05% of Tween-20 solution is added to soak the diseased leaves and shake the diseased leaves, spores on the diseased leaves are eluted into the Tween-20 solution to obtain spore suspension, the spore suspension is sprayed on the dewaxed healthy corn seedlings in the three-leaf one-heart seedling stage, the inoculated healthy corn seedlings are kept wet for 24-48h in the dark at the temperature of 20-30 ℃, the seedlings are transferred to the environment conditions of sufficient illumination, the relative air humidity is more than 80%, and the temperature is 20-30 ℃ for continuous culture, the results show that the sporule-scraped leaves after the sporule-scraped leaves are subjected to spore harvesting by blade elution and are sprayed on the healthy corn seedlings, the spore utilization rate can still be well reduced (as shown in a graph 4).
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for inoculating and propagating puccinia sorghi in multiple piles of corns is characterized in that spores on diseased leaves are eluted and prepared into spore suspension, the spore suspension is inoculated on healthy corn seedlings, the spores obtained by propagation are harvested after culture, and the healthy corn seedlings are used for repeated transfer;
The eluting spores on diseased leaves comprises: spraying Tween-20 solution on the diseased leaves, and eluting spores on the diseased leaves in the Tween-20 solution.
2. The inoculation and propagation method of Puccinia polystachya of corn according to claim 1, wherein the concentration of the sprayed Tween-20 solution is 0.02-0.05%.
3. the inoculation and propagation method of Puccinia polystachya of corn according to claim 1 or 2, wherein the spraying amount of the Tween-20 solution is 1-2 mL/diseased leaf.
4. the inoculation propagation method of Puccinia polystachya of corn according to any one of claims 1 to 3, wherein the method comprises spraying Tween-20 solution on diseased leaves, cutting the diseased leaves, adding Tween-20 solution, soaking the diseased leaves and shaking, and eluting spores on the diseased leaves in Tween-20 solution to obtain spore suspension.
5. The inoculation and propagation method of Puccinia polystachya of corn according to any one of claims 1 to 4, wherein the concentration of the eluted Tween-20 solution is 0.02 to 0.05%.
6. The inoculation and propagation method of Puccinia polystachya of corn according to any one of claims 1 to 5, wherein the harvesting and propagation of the spores comprises: after the spore production is started, the spores on the leaves are shed by shaking the stem of the corn plant; further comprising: and when one round of propagation is finished, spraying a Tween-20 solution on the diseased leaves, cutting the diseased leaves, adding the Tween-20 solution, soaking the diseased leaves, shaking, and eluting spores on the diseased leaves in the Tween-20 solution.
7. The inoculation and propagation method of Puccinia polystachya of corn according to any one of claims 1 to 6, wherein the inoculation is spraying the spore suspension on leaves of healthy corn seedlings;
Preferably, the healthy corn seedlings are corn seedlings in a three-leaf one-heart seedling stage; the healthy corn seedlings were dewaxed prior to inoculation.
8. the inoculation propagation method of puccinia polycephala of corn according to any one of claims 1 to 7, comprising the steps of:
(1) Seedling: sowing corn seeds to obtain healthy corn seedlings in a three-leaf one-heart seedling stage;
(2) Spraying 0.02-0.05% Tween-20 solution on the diseased leaves, cutting the diseased leaves into small pieces of 2-3cm 2, placing the small pieces in a container, adding 0.02-0.05% Tween-20 solution, soaking the diseased leaves, shaking, and eluting the spores on the diseased leaves in the Tween-20 solution to obtain the spore suspension;
(3) Inoculating bacteria: spraying the spore suspension prepared in the step (2) to the healthy maize seedlings with three leaves and one heart seedling stage obtained in the step (1) after dewaxing treatment, and culturing the inoculated healthy maize seedlings;
(4) harvesting spores: after the spore production is started, knocking the stem of the corn plant to enable the spore pile to fall off on paper, and harvesting the spores;
(5) Repeating the steps (2) to (4) 18-25 days after inoculation, and carrying out a new round of inoculation and propagation;
preferably, in step (4), the spores are harvested every 3-5 days.
9. The method for inoculating and propagating Puccinia polystachya of corn of claim 8, wherein in the step (3), after inoculation, the healthy corn seedlings are kept moist for 24-48h in dark under the condition of 20-30 ℃ and then are transferred to the environment condition of sufficient illumination, above 80% of air relative humidity and 20-30 ℃ for continuous culture.
10. The inoculation and propagation method of Puccinia polystachya of corn according to claim 8 or 9, wherein in step (1), before sowing, the corn seeds are subjected to germination acceleration, and after 1-2mm radicles grow from the front ends of the corn seeds, the corn seeds are sown with the radicles facing upwards and towards the center of the planting container.
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| CN114214210A (en) * | 2021-12-27 | 2022-03-22 | 中国农业大学 | Method for rapidly preparing maize puccinia polycephala bacterial liquid in batches |
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