CN111575224A - Single-spore propagation method for populus deciduous poplar and santa rust - Google Patents

Abstract

The invention discloses a single-spore propagation method for populus deciduous poplar and santa rust, belonging to the technical field of microbial culture. The method comprises the steps of seedling, collection of infected leaves, initial strain propagation, single spore line transfer, propagation expansion and the like, and comprises the following steps: two filter papers were placed on the bottom of the 90mm disposable petri dish, and 3mL of sterile water was added for moisturizing. Dispersing the single spore pile on the diseased leaf into 4 mu L agar water solution by using a liquid transfer gun, beating the solution mixed with the summer spore pile on young leaves of NL895 with disease-sensitive capability, placing the young leaves in a greenhouse with the temperature of 19 ℃ and the photoperiod of 16h/24h for culture after the inoculation is completed, further expanding the young leaves to a sufficient amount after the disease is developed, and storing the young leaves in a refrigerator at 4 ℃ for later use. The invention adopts an indoor inoculation method to culture rust fungi by a leaf disc method, can obtain separated spore piles, has simple and efficient method, and provides a single purified strain for better researching the rust resistance of poplar.

Description

Single-spore propagation method for populus deciduous poplar and santa rust

Technical Field

The invention belongs to the technical field of microbial culture, and particularly relates to a monospore propagation method of populus deciduous (Melampora larcipiperulina) psammophyte.

Background

The poplar is widely cultivated in the world, and becomes the main tree species of windbreak and sand fixation forests, farmland protection forests, water and soil conservation forests and timber forests due to fast growth, strong adaptability, multiple varieties and short breeding time. However, with the development of large-scale poplar artificial forests, a plurality of diseases are prevalent, and the growth of poplar is seriously hindered. Especially, the occurrence of the poplar leaf rust causes the diseases to spread rapidly in poplar forest lands, and brings huge loss to poplar plantation. One of the populus laris rust causing poplar leaf rust disease is a pathogenic bacterium seriously harmful to the populus crossbred in the world, and compared with the grown poplar, the seedling and young tree are seriously damaged, and the disease is that faint yellow powder appears on the back of the leaf, and the seedling is fallen in advance, so that the growth quantity of the seedling is seriously reduced, the great economic loss is caused, the tree vigor is weakened, and the capacity of the populus to resist other plant diseases and insect pests and natural disasters is weakened.

In recent years, the poplar rust disease is widely regarded and researched, when researching the pathogenic bacterium, namely, poplar, namely, largeleaf poplar rust disease, in order to obtain a sufficient amount of samples, researchers usually scrape a large amount of single spore piles from diseased poplar leaves directly, the mixed bacterial system is extremely unfavorable for researching the molecular inheritance of the poplar, particularly, when researching the group inheritance of the poplar, the used bacteria need to obtain a large amount of pure bacterial systems, and the prior art has no good method for artificially propagating a large amount of single spore systems of the pathogen, so that the method has great limitation and influence on many researches on the pathogen.

Many studies of poplar rust disease need to be carried out based on pure strains which are the dominant hosts, parasitize 0 and I stages on larch (Larix), parasitize II and III stages on populus variegates, populus nigra and hybrids taking the two as female parents. And the single spore line can only survive and propagate on the leaves of living poplar, the propagation of the single spore line is difficult, and the propagation of the single spore line of the santalin rust of populus largesii has not been reported at home and abroad. Therefore, a set of single-spore propagation system of the populus largesii rust is needed to obtain a purified strain.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a method for single-spore propagation of populus largesii rust, which is convenient, rapid and efficient.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a single-spore propagation method of poplar, folium pini aspen and rust fungus comprises the following steps: selecting single spore pile of the Daphne littoralis of the larch and the rust fungus on the infected poplar leaves, inoculating the single spore pile on the healthy poplar leaves, propagating the single spore pile by using new healthy poplar leaves, and finally obtaining a large amount of monosporal spore powder.

Further, the single-spore propagation method of the poplar, largeleaf poplar and rust fungus comprises the following specific steps:

1) seedling: taking three to four weeks of poplar tissue culture seedlings susceptible to poplar alternaria alternata rust fungus to acclimatize;

2) initial breeding of strains: picking healthy leaves of the poplar seedlings obtained in the step 1), making into leaf discs, cleaning the leaf discs with sterile water, selecting a monospore pile of the populus larcens rust on the infected poplar leaves, inoculating the monospore pile to the leaf discs for culturing, and inoculating only one monospore pile on each leaf disc;

3) single spore strain transfer: after the leaf discs are diseased, respectively picking spores growing on each leaf disc to a plurality of new leaf discs for culturing;

4) collecting spore powder according to a monospore line;

5) spray grafting and propagation of monospore series: and preparing the collected spore powder into spore suspension, inoculating the spore suspension onto a plurality of new leaf discs in a spraying mode, and continuously culturing to obtain enough spore powder.

Further, the preparation of the leaf disc and the culture method after inoculation are as follows: picking up leaves of the refined poplar seedlings, removing main veins, cutting into small squares of 2 x 2cm as leaf disks, washing with sterile water, placing the leaves in a culture dish with the back faces upward, adding filter paper and sterile water at the bottom of the culture dish in advance for moisturizing, and sealing the culture dish with a sealing film for culturing after inoculation.

Further, the specific operations of inoculation in the step 2) are as follows: 0.05% Tween 20 was uniformly sprayed on the washed leaf disks, then the monocystegium masses with clear upper edges of the diseased leaves were dispersed to 4. mu.L of 0.1g/L agar aqueous solution with a pipette, and the agar aqueous solution mixed with the summertime was sprayed on the prepared leaf disks with the pipette.

Further, the step 3) is specifically as follows: aligning the spore powder on the leaf discs with a needle tube of a 10mL disposable syringe, gently scraping the spore powder, collecting the spore powder in a centrifugal tube subjected to high-temperature sterilization, adding 1mL of 0.1g/L agar solution, uniformly mixing, then respectively transferring to a plurality of new leaf discs, and spraying 0.005% Tween 20 on the new leaf discs before inoculation.

Further, in the step 5), the spore suspension is prepared by the following method: suspending the spore powder collected in the step 4) in 0.1g/L agar solution containing 0.005% Tween 20, mixing to obtain summer spore suspension with concentration of 2mg/mL, and inoculating the spore suspension onto a healthy poplar leaf disc in a spraying manner by using a spraying pot to further expand the propagation of the monospore line to obtain enough spore powder.

Further, in steps 2), 3) and 5), leaf disks were inoculated and then placed in a greenhouse at a temperature of 19 ℃ and a photoperiod of 16h/24h for cultivation.

Further, the step 3) and the step 5) can be repeated for 1-5 generations when the obtained spore amount is not enough.

Further, the variety of the poplar tissue culture seedling susceptible to poplar, larch and rust in the step 1) is NL 895.

Compared with the prior art, the invention has the beneficial effects that:

the single cell system propagation system of the populus largeleaf rust provided by the invention has clear edges of grown single spores, the initial propagation survival rate is more than 90 percent under the condition of no other pollution, and the spraying success rate is 95 percent. The submerged culture period is only 6-7 days, the sporulation starts on the 7 th day of inoculation, and a large amount of spores are produced in 8-10 days. Therefore, the method has the characteristics of short time, high survival rate, pure bacterial line and high yield, and provides a bacterial propagation basis for the related research of the rust disease of populus largeleaf.

Drawings

FIG. 1 is a graph of three to four weeks old NL895 tissue culture seedlings;

FIG. 2 is a drawing of poplar leaves infected with Buxus sempervirens of poplar, the left side being the back side of the leaves and the right side being the front side of the leaves;

FIG. 3 is a diagram of initial propagation of bacterial species.

Detailed Description

The present invention will be further described with reference to the following specific examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Poplar seedling: nanlin 895(NL895, Populus delides × p. euramericica);

preparing an agar aqueous solution: weighing 0.1g of agar powder, adding 100mL of deionized water for dissolving, then fixing the volume to 1L, and placing in a sterilization pot for sterilization for later use.

Example 1: single spore propagation of Buxus lachryma

1. Seedling and seedling

1) Taking three to four-week NL895 tissue culture seedlings (figure 1), firstly opening a cover to culture for 3 days, and then adding water into a culture medium to submerge the culture medium to culture for 3 days;

2) selecting a flowerpot with the caliber of 10cm, and filling the flowerpot with nutrient soil, perlite and vermiculite in a ratio of 3: 1;

3) flattening the flowerpot filled with soil, soaking for half a day, selecting NL895 aseptic seedlings with good growth vigor to transplant into the soil for hardening the seedlings after the surface soil is slightly moist.

2. Collecting susceptible leaves:

selecting two to three complete infected leaves from populus tremuloides in Jiangsu Sixui Po with more infection (figure 2);

3. media preparation

Using a 90mm disposable culture dish, filling two pieces of filter paper with the same size at the bottom of the culture dish, adding 3mL of sterile water for moisturizing, selecting NL895 plants with the growth cycle of more than 3 weeks, selecting 3-7 leaves which are completely unfolded from top buds downwards, removing main veins, cutting into small squares of 2 x 2cm, cleaning with sterile water, placing the leaves in the culture dish with the back faces upwards, and spraying 0.005% of Tween 20;

4. initial breeding of strain

Two single sporangium masses with clear edges on the affected leaf discs were picked, and one sporangium mass was inoculated on each leaf disc. The method comprises the following specific steps:

1) the tip of a pipette tip (2.5. mu.L) for inoculation was sterilized and cooled.

2) Picking up a single spore pile on a diseased leaf by using a liquid transfer gun (Eppendorf company), repeatedly blowing and dispersing the single spore pile into a single row of pipes filled with 4 mu L of agar aqueous solution, sucking and spraying the solution mixed with the summer spore pile on prepared leaf discs by using the liquid transfer gun, inoculating spores of only one spore pile on each leaf disc, placing the leaf discs inoculated with one spore pile on each culture dish, covering the culture dish after inoculation, and sealing the culture dish by using a sealing film.

Since the summer sporangium is a process of asexual reproduction, we believe that a single sporangium is often reproduced from a single spore. The tips were changed each time a single spore line (single spore pile) was inoculated.

3) The inoculated leaf discs are placed in a greenhouse with the temperature of 19 ℃ and the photoperiod of 16h/24h for culture, and the separated spore masses can be seen by naked eyes after about 7-8 days (figure 3).

5. Monosporal system of transfer

After the initial breeding leaf discs are diseased, before spore powder begins to scatter, a needle tube part of a 10mL disposable injector is aligned to the spore powder on the two leaf discs, the spore powder is respectively and gently scraped, collected into a 1.5mL centrifugal tube which is sterilized at high temperature, then 1mL of 0.1g/L agar solution is added, after uniform mixing, the mixture is respectively transferred to 10 new leaf discs, and the numbers of the leaf discs are respectively A, B. Before inoculation, the new leaf discs are washed with sterile water and sprayed with 0.005% Tween 20. The culture conditions after inoculation are the same as the step 3) in the step 4 and the initial breeding of the strains.

6. Collecting spore powder

After the onset, collection of a single sporangium mass of the A and B discs began before the spore powder began to scatter. Collect a first, weigh the paper pad under the leaf, scrape the summer spores off the leaf gently. Before B is collected, water is sprayed into the air to settle spores, hands are disinfected again, and then bacteria are collected.

7. Spray grafting of monospore series

The collected spore powder of A and B are respectively prepared into 2mg/mL spore suspension: the collected spore powder of A and B were suspended in 0.005% Tween 20 agar solution (agar 0.1g/L) and mixed to a concentration of 2mg/mL of ureaba spore suspension. The resulting suspension was sprayed onto 5 new leaf disks, numbered A-1, A-2, A-3, A-4 and A-5, B-1, B-2, B-3, B-4 and B-5, respectively, using a mini-watering can (15 mL). The culture conditions after inoculation are the same as the step 3) in the step 4 and the initial breeding of the strains. Spores were collected after the onset, and a total of 0.5mg of summer spores was collected from each of the five leaf discs a and B.

8. Detection of

The collected spore powder is used for extracting DNA by using a glass bead method column type DNAout kit (Beijing Tianenzze Gene science and technology Co., Ltd.), an LSU sequence (SEQ ID NO.1) and an ITS sequence (SEQ ID NO.2) are obtained by amplifying the extracted DNA, and the obtained result is the agaricus lardii rust bacteria by comparing on NCBI.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims (9)

1. A single-spore propagation method of poplar, folium populi tremuloides and santalina rust is characterized by comprising the following steps: selecting single spore pile of the Daphne littoralis of the larch and the rust fungus on the infected poplar leaves, inoculating the single spore pile on the healthy poplar leaves, propagating the single spore pile by using new healthy poplar leaves, and finally obtaining a large amount of monosporal spore powder.

2. The single spore propagation method of Buxus lacryma-jobi as claimed in claim 1, which comprises the following steps:

1) seedling: hardening the tissue culture seedlings of the poplar, which is susceptible to the poplar, namely populus largeleaeus rust, of the poplar;

2) initial breeding of strains: picking healthy leaves of the poplar seedlings obtained in the step 1), making into leaf discs, cleaning the leaf discs with sterile water, selecting a monospore pile of the populus larcens rust on the infected poplar leaves, inoculating the monospore pile to the leaf discs for culturing, and inoculating only one monospore pile on each leaf disc;

3) single spore strain transfer: after the leaf discs are diseased, respectively picking spores growing on each leaf disc to a plurality of new leaf discs for culturing;

4) collecting spore powder according to a monospore line;

5) spray grafting and propagation of monospore series: and preparing the collected spore powder into spore suspension, inoculating the spore suspension onto a plurality of new leaf discs in a spraying mode, and continuously culturing to obtain enough spore powder.

3. The method for the single spore propagation of the rust fungus of populus deciduous in claim 2, wherein the leaf disc is prepared and cultured after inoculation by the following steps: picking up leaves of the refined poplar seedlings, removing main veins, cutting into small squares of 2 x 2cm as leaf disks, washing with sterile water, placing the leaves in a culture dish with the back faces upward, adding filter paper and sterile water at the bottom of the culture dish in advance for moisturizing, and sealing the culture dish with a sealing film for culturing after inoculation.

4. The method for the single-spore propagation of the rust fungus of populus deciduous in claim 2, wherein the inoculation in the step 2) comprises the following specific operations: 0.05% Tween 20 was uniformly sprayed on the washed leaf disks, then the monocystegium masses with clear upper edges of the diseased leaves were dispersed to 4. mu.L of 0.1g/L agar aqueous solution with a pipette, and the agar aqueous solution mixed with the summertime was sprayed on the prepared leaf disks with the pipette.

5. The single spore propagation method of Buxus lacryma populi var populi as claimed in claim 2, wherein said step 3) is specifically: aligning the spore powder on the leaf discs with a needle tube of a 10mL disposable syringe, gently scraping the spore powder, collecting the spore powder in a centrifugal tube subjected to high-temperature sterilization, adding 1mL of 0.1g/L agar solution, uniformly mixing, then respectively transferring to a plurality of new leaf discs, and spraying 0.005% Tween 20 on the new leaf discs before inoculation.

6. The method for the single-spore propagation of the rust fungus of populus deciduous in claim 2, wherein in the step 5), the spore suspension is prepared by the following steps: suspending the spore powder collected in the step 4) in 0.1g/L agar solution containing 0.005% Tween 20, mixing to obtain summer spore suspension with concentration of 2mg/mL, and inoculating the spore suspension onto a healthy poplar leaf disc in a spraying manner by using a spraying pot to further expand the propagation of the monospore line to obtain enough spore powder.

7. The method for the single spore propagation of the rust fungus of populus decipiens as claimed in claim 2, wherein in steps 2), 3) and 5), the leaf discs are inoculated and then cultured in a greenhouse at a temperature of 19 ℃ and a photoperiod of 16h/24 h.

8. The method for propagating Buxus lacryma populi according to claim 2, wherein said steps 3) and 5) are repeated for 1-5 generations when the obtained spore amount is insufficient.

9. The method for the monosporally propagating sarcandra populi of claim 2, wherein the tissue culture seedling of poplar susceptible to sarcandra populi in step 1) is NL 895.

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