CN108370808B

CN108370808B - Pinus massoniana mycorrhizal seedling raising method

Info

Publication number: CN108370808B
Application number: CN201810260003.7A
Authority: CN
Inventors: 丁贵杰; 陈后英; 刘海燕; 李快芬
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2020-06-12
Anticipated expiration: 2038-03-27
Also published as: CN108370808A

Abstract

The invention discloses a pinus massoniana mycorrhizal seedling raising method which comprises five steps of seed disinfection and germination acceleration, seedling raising matrix disinfection, strain acquisition and bacterial liquid preparation, seedling root cutting treatment, bacterial liquid inoculation and transplantation management. The method is easy to obtain mycorrhizal seedlings of the target strains, ensures the infection rate of the mycorrhizal seedlings, is simple and convenient in operation process, has ideal inoculation effect and has the infection rate as high as more than 95%; the root cutting process is simple, convenient and quick, and the damage to the seedlings is small; the germination box and the net separator adopted by the invention can be recycled, thereby saving and protecting the environment.

Description

Pinus massoniana mycorrhizal seedling raising method

Technical Field

The invention relates to a mycorrhizal seedling raising method for pinus massoniana, and belongs to the technical field of mycorrhizal seedling raising.

Background

Pinus massoniana Lamb is a native tree species with the largest planting area, the widest distribution and the highest comprehensive utilization degree of the whole tree in conifer species in China, is also one of the most main industrial wood tree species in south, has important economic and ecological values, and belongs to typical ectomycorrhizal tree species. The ectomycorrhiza can promote the absorption of mineral nutrition of plants, enhance the stress resistance of the plants and the like, and further promote the growth of the plants. However, in the actual seedling culture work, obtaining mycorrhizal pinus massoniana seedlings of target strains is very difficult.

The ectomycorrhiza is a symbiont formed by ectomycorrhizal fungi and young roots of the pinus massoniana, has structures such as epitaxial hypha, fungus sheaths, Harti's nets and the like, and can promote the pinus massoniana to absorb nutrient substances and enhance the stress resistance of the pinus massoniana. The conventional mycorrhization seedling cultivation adopts modes of seed dressing, soil mixing, root dipping and the like to cultivate mycorrhization seedlings, and in the practical process, a technology for cultivating the mycorrhization seedlings of the pinus massoniana by a root cutting method is formed. The technology is mainly carried out in three steps, firstly, pinus massoniana seeds are sown in a matrix, after pinus massoniana seedlings grow for 15 days, the seedlings are sequentially moved out, 1-2 cm of young roots are cut off, and finally, the cut-root seedlings are dipped in an ectomycorrhizal fungi culture solution and then are moved into soil cavities. However, the process is tedious and time-consuming, the seedling transfer process has great damage to the seedlings, and further development of the seedlings is affected. Therefore, it is necessary to research a convenient and fast cultivation method for mycorrhizal seedlings of pinus massoniana so as to improve the seedling cultivation efficiency.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for cultivating seedlings by mycorrhizal transformation of pinus massoniana, which enables the seedling cultivation by mycorrhizal transformation of the pinus massoniana to be more convenient and simpler, and solves the problems of complicated root cutting process, low survival rate of mycorrhizal seedlings, low infection rate and the like in the process of cultivating the mycorrhizal seedlings of a target strain of pinus massoniana.

The technical scheme of the invention is as follows: a pinus massoniana mycorrhizal seedling raising method comprises the following steps:

(1) seed disinfection and germination acceleration: soaking the seeds subjected to water separation in 0.5% potassium permanganate for 2h, taking out, placing in a beaker, covering with a film, sealing, standing for 30 minutes, and washing with sterile water for 3-4 times; placing the disinfected seeds in sterile water with the initial temperature of 45 ℃, placing the seeds in a 25 ℃ artificial climate box for soaking for 24 hours, changing water every 6-8 hours midway, wrapping the seeds with wet gauze after soaking, placing the seeds in the artificial climate box with the temperature of 25 ℃ for accelerating germination, and sowing the seeds after the seeds are exposed to the white;

(2) seedling culture substrate disinfection: sterilizing the seedling culture substrate for 2 hours at the temperature of 121 ℃ by using a high-pressure steam sterilization pot, and then cooling, so that seeding and seedling culture can be carried out;

(3) strain acquisition and bacterial liquid preparation: taking the ectomycorrhizal fungi out of the solid culture medium, inoculating the ectomycorrhizal fungi into the liquid culture medium, and culturing the ectomycorrhizal fungi in an artificial climate box for 15 days for later use;

(4) root cutting treatment of seedlings and inoculation of bacterial liquid: placing the seedling substrate into a germination box to a height of 1cm, placing the seedling substrate into a net for spacing, continuously adding the seedling substrate to a position 1cm away from the edge of the germination box, watering the seedling substrate thoroughly, sowing the seedling substrate, covering the seeds with the substrate, wherein the thickness of the seedling substrate is not too thick, placing the seedling substrate into a climatic chamber after sowing, giving appropriate illumination and humidity, replenishing water in time, and removing a box cover after the seeds come out of the soil; after 15 days of sowing, lifting the net spacer together with the matrix and the seedling integrally, cutting off roots exposed out of the net spacer parallel to the bottom, pouring ectomycorrhizal fungi liquid on the root cutting surface of the bottom of the net spacer in the germination box and the contact surface of the seedling matrix, putting the net spacer in the germination box, continuously culturing in a phytotron for 2 weeks, and then transplanting;

(5) transplanting management: and (3) pulling out the inoculated seedlings from the germination box, dipping the inoculated seedlings in bacterial liquid, and transferring the inoculated seedlings into a nursery cave tray, keeping the moisture and humidity of the soil, and giving proper illumination to prevent weed breeding and prevent damage of plant diseases and insect pests.

In the method, the ectomycorrhizal fungi is obtained by separating fruiting bodies collected from pinus massoniana forest.

In the above method, the solid culture medium is prepared from ammonium tartrate 0.5g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, glucose 20g/L, and vitamin B10.1 × 10^-3g/L, agar 20g/L, boric acid 8.45mg/L, manganese sulfate 5mg/L, copper sulfate 0.625mg/L, ferrous sulfate 6mg/L, zinc chloride 2.77mg/L and ammonium molybdate 0.27mg/L, wherein the formula of the liquid culture medium is the same as that of the solid culture medium, but the agar is not added.

Due to the adoption of the technical scheme, the invention has the advantages that:

firstly, the method can easily obtain the mycorrhizal seedlings of the target strains, and ensures the infection rate of the mycorrhizal seedlings. The operation process of the invention is simple and convenient, the inoculation effect is very ideal, and the infection rate is as high as more than 95 percent;

secondly, the root cutting process is simple, convenient and quick, and the seedling is less damaged;

thirdly, the germination box and the net adopted by the invention can be recycled, thereby saving and protecting the environment.

Drawings

FIG. 1 is a schematic view of the construction of the germination box according to the invention;

fig. 2 is a schematic view of the structure of the screen in the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and examples.

The embodiment of the invention comprises the following steps: a pinus massoniana mycorrhizal seedling raising method comprises the following steps:

(1) selecting seeds with better quality, performing water selection, sterilizing the seeds for 2 hours by using 5 percent potassium permanganate, taking the seeds out, placing the seeds in a beaker, covering a film, sealing and standing the seeds for 30 minutes, washing the seeds for 3 times by using sterile water, placing the seeds in sterile water with the initial temperature of 45 ℃, then placing the seeds in a climatic chamber with the temperature of about 25 ℃ for soaking for 24 hours, changing the water every 6 hours midway, wrapping the seeds by using wet gauze after soaking, placing the seeds in the climatic chamber with the temperature of 25 ℃ for germination acceleration, and sowing the seeds after the seeds are exposed to the white.

(2) Sterilizing the seedling culture substrate for 2h at 121 ℃ by using a high-pressure steam sterilization pot, and cooling for later use. The structural schematic diagram of the germination box is shown in fig. 1 and fig. 2, and the germination box comprises a germination box 1 and a box cover 2, wherein the germination box 1 is an inverted trapezoidal box body with a wide upper part and a narrow lower part, a net partition 3 is arranged in the germination box 1, the shape of the net partition 3 is the same as that of the germination box 1, and the volume of the net partition 3 is smaller than that of the germination box 1. When the seed box is used, after a seedling substrate with the thickness of 1cm is filled in the germination box 1, the net partition 3 is placed in the germination box 1, the seedling substrate is continuously added to the position 1cm away from the edge of the germination box 1, the substrate is watered thoroughly, and then the seeds are sowed, wherein 200-300 seeds are planted in each box. After the sowing is finished, the box cover 2 is covered, the germination box 1 is placed in an artificial climate box, appropriate illumination and humidity are given, moisture is supplemented in time, and the box cover 2 is removed after the seeds come out of the soil.

(3) The ectomycorrhizal fungi collected and separated from the masson pine forest is activated and propagated. Firstly, the ectomycorrhizal fungi are inoculated on a Pachlewski solid culture medium and are cultured in an artificial climate box for 20 days for standby. The solid culture medium is prepared from ammonium tartrate 0.5g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, glucose 20g/L, and vitamin B10.1 × 10^- ³g/L, agar 20g/L, boric acid 8.45mg/L, manganese sulfate 5mg/L, copper sulfate 0.625mg/L, ferrous sulfate 6mg/L, zinc chloride 2.77mg/L and ammonium molybdate 0.27 mg/L. Wherein the liquid culture medium has the same formula as the solid culture medium, but no agar is added. The bacterial liquid preparation process comprises the following steps: placing 250ml triangular flask into 150ml liquid culture medium, sterilizing with high pressure steam autoclave at 121 deg.C for 30 min, cooling, collecting fungus block from solid culture medium, inoculating into liquid culture mediumThe inoculation process is completed on a clean bench. And (3) placing the triangular flask in an artificial climate box for culturing for 20-25 days for later use, wherein the temperature is about 25 ℃, and the humidity is adaptive.

(4) After 15 days of sowing, the net partition 3 and the seedling substrate are integrally lifted up, and the radicle of the pinus massoniana seedling exposed out of the net partition 3 is cut off in parallel with the bottom of the net partition 3.

(5) 100ml of ectomycorrhizal fungi liquid is poured into the contact surface of the root cutting surface at the bottom of the net partition 3 and the seedling culture substrate in the germination box, the net partition 3 containing the substrate and the seedlings is integrally placed in the germination box and is continuously cultured in a climatic chamber for 2 weeks, and then transplanting is carried out. During transplanting, the seedlings are carefully pulled out, dipped with the same bacterial liquid and then transferred into a plug tray.

(6) The management process mainly keeps the moisture and humidity of soil, prevents weeds from breeding, prevents and treats the damage of plant diseases and insect pests in time, and can obtain mycorrhizal seedlings of target strains after the whole process is completed, wherein the whole process is carried out in the beginning of 3 months.

Claims

1. A method for cultivating seedlings of pinus massoniana by mycorrhization is characterized by comprising the following steps:

(4) root cutting treatment of seedlings and inoculation of bacterial liquid: placing the seedling substrate into a germination box to a height of 1cm, placing the seedling substrate into a net for spacing, continuously adding the seedling substrate to a position 1cm away from the edge of the germination box, watering the seedling thoroughly, sowing the seedling, covering the seedling with the substrate to cover the seedling, ensuring that the seedling is not too thick, placing the seedling in a climatic chamber after sowing, giving illumination and humidity, replenishing water in time, and removing a box cover after the seedling is unearthed; after 15 days of sowing, lifting the net spacer together with the matrix and the seedling integrally, cutting off roots exposed out of the net spacer parallel to the bottom, pouring ectomycorrhizal fungi liquid on the root cutting surface of the bottom of the net spacer in the germination box and the contact surface of the seedling matrix, putting the net spacer in the germination box, continuously culturing in a phytotron for 2 weeks, and then transplanting;

(5) transplanting management: and (3) pulling out the inoculated seedlings from the germination box, dipping the inoculated seedlings in bacterial liquid, and transferring the inoculated seedlings into a nursery cave tray, wherein the moisture and humidity of the soil are kept, and illumination is given to prevent weed breeding and prevent damage of plant diseases and insect pests.

2. The method for cultivating mycorrhizal seedlings of pinus massoniana according to claim 1, wherein the method comprises the following steps: the ectomycorrhizal fungi is obtained by separating fruiting bodies collected from pinus massoniana forest.

3. The method for cultivating mycorrhizal seedlings of pinus massoniana according to claim 1, wherein the method comprises the following steps: the solid culture medium is prepared from ammonium tartrate 0.5g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, glucose 20g/L, and vitamin B10.1 × 10^- ³g/L, agar 20g/L, boric acid 8.45mg/L, manganese sulfate 5mg/L, copper sulfate 0.625mg/L, ferrous sulfate 6mg/L, zinc chloride 2.77mg/L and ammonium molybdate 0.27mg/L, wherein the formula of the liquid culture medium is the same as that of the solid culture medium, but the agar is not added.