CN103060207A - Preparation method for bacteria liquid beneficial for growth-development and mycorrhiza formation of rhododendron plants - Google Patents

Abstract

The invention relates to a bacteria liquid for growth and development of plants, and in particular relates to a preparation method for bacteria liquid beneficial for growth-development and mycorrhiza formation of rhododendron plants. The method comprises the following steps: (1) preparation and treatment of materials: collecting and disinfecting mature fibrous roots of rhododendron plants, and cutting the treated mature fibrous roots to root segments for later use; (2) culture medium configuration: the content of 1000mL of culture medium comprises the following components of 380mg of KH2PO4, 149mg of MgSO4.7H2O, 26mg of vitamin C, 67mg of calcium pantothenate, 1.4mg of nicotinic acid, 3.0g of peptone, 16.5g of glucose, 8.5g of agar powder, 4mL of 1.5% of sodium deoxycholate liquid and 0.05mg of indoleacetic acid; (3) preparation of mother liquid; and (4) preparation of the bacteria liquid. The preparation method provided by the invention provides a technical method for production, application and fundamental research on introduction, artificial breeding, cultivation, breeding and the like of the rhododendron plants in Changbai Mountain and other rhododendron plants at home and abroad.

Description

A kind ofly be beneficial to the bacterium solution preparation method that rhododendron grows and mycorhiza forms

Technical field

The present invention relates to a plant development bacterium liquid, namely a kind ofly be beneficial to the bacterium solution preparation method that rhododendron grows and mycorhiza forms.

Background technology

In the prior art, cuckoo is world-renowned ornamental plant, also is one of China's ten great tradition famous flowers.China's azalea resource is very abundant, is valuable source and the regional distribution center of rhododendron.But both at home and abroad the researchs such as the introducing a fine variety of wild-type azalea, artificial breeding, cultivation and breeding are carried out slowly, traced it to its cause, because of rhododendron artificial propagation, the larger difficult point of link existence of cultivating and grow etc., particularly wild alpine rose is particularly evident mostly.The Rhododendron in Changbai platymiscium comprise Rhododendron aureum ( Rhododendron chrysanthumPall.), short fruit cuckoo ( Rhododendron brachycarpumD. Don), according to white cuckoo ( Rhododendron micranthumTurcz.), Twig and leaf of Capitate Rhododendron ( Rhododendron parvifoliumAdams.), large word cuckoo ( Rhododendron schlippenbachiiMaxim.), Folium Cuculus polioephalus ( Rhododendron dauricumL.), korean rhododendron ( Rhododendron mucronulatumTurcz.), the felt cuckoo ( Rhododendron confertissimumNakai) and Rhododendron redowskianum ( Rhododendron redowskianumMaxim.) etc. have 9 kinds, be Jilin Province and lay special stress on protecting plant.

Rhododendron class mycorhiza is a kind of symbiote that forms between fungi and the rhododendron, and it has the rhododendron of promotion to nutrient uptake, improvement rhizosphere soil, strengthens the effects such as growing way, transplanting survival rate, drought resisting and disease resistance of plant.Domestic rhododendron mycorrhizal fungi research is in the starting stage, and relevant report is less, mostly is in phase of basic research, does not enter Application Areas always.

Summary of the invention

The objective of the invention is provides a kind of bacterium solution preparation method that rhododendron grows and mycorhiza forms that is beneficial to for above-mentioned deficiency, and this bacterium liquid is used for soaking Rhododendron seeds, soaks the tissue cultured seedling root, soaks the rhododendron root of transplanting.

Technical solution of the present invention is: a kind ofly be beneficial to the bacterium solution preparation method that rhododendron grows and mycorhiza forms, its step is as follows:

(1) material and processing:

Gather the ripe fibrous root of rhododendron, sterilization is cut into root segment for subsequent use.

(2) substratum and configuration:

The content of 1000 mL substratum is: KH ₂PO ₄380 mg, MgSO ₄7H ₂O149 mg, Catergen 6 mg, calcium pantothenate 67 mg, nicotinic acid 1.4 mg, peptone 3.0 g, glucose 16.5 g, agar powder 8.5 g, 1.5% Septochol sodium solution, 4 mL and 0.05 mg indolylacetic acid.

(3) mother liquor preparation:

To be inoculated into media surface in the culture dish after the root segment segment in the step (1), place 25 ± 2 ℃ biochemical cultivation case to cultivate.

Treat that mycelia sends from root, picking square section hypha,hyphae is transferred and is cultivated in upper continuation of new substratum (substratum in the step (2)), purifying, and the inoculation behind the purifying places 3～5 ℃ cyrogenic equipment to preserve to invisible spectro slant medium.

The cultivation of liquid spawn mother liquor is the liquid nutrient medium that the agar in the substratum in the step (2) is removed rear configuration, with 6 bacterial classifications of purifying Phialocephala fortinii, Pezizilla ericace, Oidiodendron maius, Meliniomyces variabilis, Cryptosporiopsis ercae, Acaulospora lacunosaAfter being cultured to respectively mycelia in vitro and covering with the inclined-plane, get respectively the agar block that contains mycelia and put into the triangular pyramidal bottle that liquid nutrient medium is housed, tinfoil seals bottleneck and is placed on shaking culture 15～20 d on the constant-temperature table, collect 500 mL nutrient solutions and be settled to 1000 mL with sterilized water, and be sub-packed in the dark brown container of 500 mL with shaking all after the tinfoil sealing, placing temperature is to preserve 7～10 d under 25 ± 2 ℃ of conditions, shake every day during this time 1～2 time, each 20～30 min, the liquid spawn mother liquor.

(4) bacterium solution preparation:

Producing with bacterium liquid collocation method is liquid spawn mother liquor 500 mL, after the brown sugar 500 g dissolving, adds water to 50 L, 7～10 d that ferment, and can use after smelling fragrance.

Advantage of the present invention is: 1, the present invention's root system of choosing 9 kind rhododendrons of China Changbaishan area Rhododendron has carried out separation, purifying, the preliminary evaluation of mycorrhizal fungi, and finished the female kind of liquid and produced the preparation of using bacterium liquid, for Rhododendron in Changbai belongs to and the production application such as the introducing a fine variety of other kind cuckoo, artificial breeding, cultivation and breeding both at home and abroad and fundamental research provide technological method, simultaneously, can promote rhododendron High-efficient Production and be applicable.Available this bacterium liquid soaks Rhododendron seeds 48 h, disseminates in the saturating matrix (matrix is vegetable mould and vermiculite, and ratio is 3:1) of bacterium liquid filling and covers the pine needle moisturizing of becoming thoroughly decomposed, and 18 d can sprout, and germination rate is 92.1%, and transplanting survival rate is more than 90.0%.Behind the rhododendron tissue cultured seedling bottle outlet, behind bacterium liquid immersion tissue cultured seedling root 18～24 h, tissue cultured seedling is planted in filling with bacterium liquid in the saturating matrix (matrix is vegetable mould, become thoroughly decomposed pine needle and vermiculite, and ratio is 3:2:1), cover the good plastics film heat and moisture preserving (hardening) of light transmission.Can improve more than the surviving rate to 99.5%.When rhododendron is transplanted, the thick root of rhododendron can be cut 2/3, soak 12～18 h with bacterium liquid again, can improve more than the surviving rate to 93.9%.

Below in conjunction with embodiment embodiments of the present invention are described in further detail.

Embodiment

A kind ofly be beneficial to the bacterium solution preparation method that rhododendron grows and mycorhiza forms, its step is as follows:

1 material and processing

In Changbaishan area gather Rhododendron aureum ( Rhododendron chrysanthumPall.), short fruit cuckoo ( Rhododendron brachycarpumD. Don), according to white cuckoo ( Rhododendron micranthumTurcz.), Twig and leaf of Capitate Rhododendron ( Rhododendron parvifoliumAdams.), large word cuckoo ( Rhododendron schlippenbachiiMaxim.), Folium Cuculus polioephalus ( Rhododendron dauricumL.), korean rhododendron ( Rhododendron mucronulatumTurcz.), the felt cuckoo ( Rhododendron confertissimumNakai) and Rhododendron redowskianum ( Rhododendron redowskianumVery thin of the rhododendron of 9 kinds such as Maxim.).The nearly thick root place of the rear clip that breaks ground robust growth diameter is the following ripe fibrous roots of 1.0 mm, with banister brush flush away earth gently, is cut into 3～4 cm after the flowing water flushing is spent the night.Under Bechtop, with 70% alcohol (wherein containing 5% Streptomycin sulphate) sterilization, 20 s, be 0.5% chlorine bleach liquor, 8 min that sterilize with mass concentration again, aseptic water washing 8 times, aseptic filter paper blots the root surface-moisture, excision by virus killing disinfectant damaged portion after and be cut into that approximately the root segment about 2.0～3.0 cm is for subsequent use.

2 methods

2.1 substratum and configuration

Medium component and content (content of 1000 mL substratum) are: KH ₂PO ₄380 mg, MgSO ₄7H ₂O149 mg, Catergen 6 mg, calcium pantothenate 67 mg, nicotinic acid 1.4 mg, peptone 3.0 g, glucose 16.5 g, agar powder 8.5 g.

Above component mixed and dissolve after be sub-packed in the culture dish, 121 ℃ of moist heat sterilization 20～25 min take out, treat that solution does not solidify before, by adding 1.5% Septochol sodium solution, 4 mL and 0.05 mg indolylacetic acid in each culture dish of filtration sterilization.

2.2 the separation of fungi, purifying, preliminary evaluation, preservation, mother liquor and bacterium solution preparation

The root segment of processing in the step 1 is cut into 0.5 cm(cut sides large as far as possible) after be inoculated into media surface in the culture dish, every ware connects 3 sections, totally 10 wares place the biochemical cultivation case of (25 ± 2) ℃ to cultivate.

Treat that mycelia sends from root, picking square section hypha,hyphae is transferred and is cultivated in upper continuation of new substratum (substratum in the step 2.1), and every switching is cultivated all needs for 1 time microscopically to observe bacterial strain purity, until obtain purifying bacterial strain (form is consistent).Inoculation behind the purifying is to invisible spectro slant medium, as for preserving in 3～5 ℃ the cyrogenic equipment.

Adopt morphological method that the bacterial classification of purifying is carried out preliminary evaluation, with inoculation in substratum, as for cultivating under (25 ± 2) ℃ condition.And record strain growth speed, observe colony characteristics.

The female cultivation of planting of liquid is the liquid nutrient medium that the agar in the substratum in the step 2.1 is removed rear configuration, divide and install to (every bottle of 50～75 mL) in the 250 mL triangular pyramidal bottles, after a plurality of bacterial classifications of purifying are cultured to respectively mycelia in vitro and cover with the inclined-plane, get respectively the agar block that contains mycelia of 1.0 cm sizes and put into the triangular pyramidal bottle (putting 1 for every kind) that liquid nutrient medium is housed, seal bottleneck with the tinfoil of appropriate size and be placed on shaking culture 15～20 d on the constant-temperature table, collect 500 mL nutrient solutions and be settled to 1000 mL with sterilized water, and be sub-packed in the dark brown container of 500 mL with shaking all after the tinfoil sealing, place temperature be during preserving 7～10 d(under (25 ± 2) ℃ condition every day shake each 20～30 min 1～2 time) the liquid spawn mother liquor.The liquid spawn mother liquor is prevented terminating in shady and cool dry place preservation and is got final product.

Producing with bacterium liquid collocation method is liquid spawn mother liquor 500 mL, after the brown sugar 500 g dissolving, adds water to 50 L, 7～10 d that ferment, and smells and can use behind the fragrance (during stir 2 times every day with clean wooden stick).

The result

3.1 the separation of mycorrhizal fungi, purifying and preliminary evaluation

Ericoid mycorrhizal fungi growth and slow needs to cultivate 20～30 days, and the slightly fast colony diameter of growth reaches 5～7 cm, and slower only 0.5～2.5 cm grows.

By separation and Culture, purifying and the preliminary evaluation to 9 kinds of wild-type azalea section plant root mycorrhizal fungis, be divided into from obtaining more than 70 strains of root endogenetic fungus, every kind of rhododendron separates on the fungal colony form that obtains and can roughly be divided into more than 3～8 kinds.

Utilize morphological method to identify the sociales of 6 kinds of ericoid mycorrhizal fungis that obtain 9 kinds of cuckoos, be respectively Phialocephala fortinii, Pezizilla ericace, Oidiodendron maius, Meliniomyces variabilis, Cryptosporiopsis ercae, Acaulospora lacunosa

3.2 mycorrhizal fungi mother liquor and bacterium solution preparation behind the purifying

According to method in the step 2.2, the agar block that will contain 6 bacterial classification mycelia is put into the triangular pyramidal bottle that liquid nutrient medium is housed and is carried out mixed culture, obtains the liquid spawn mother liquor.

Producing with bacterium liquid collocation method is liquid spawn mother liquor 500 mL, after the brown sugar 500 g dissolving, adds water to 50 L, 7～10 d that ferment, and can use after smelling fragrance.

3.3 the mycorrhizal fungi behind the purifying, mother liquor and bacterium liquid are preserved

Inoculation behind the purifying is to invisible spectro slant medium, as for preserving in 3～5 ℃ the cyrogenic equipment.The liquid spawn mother liquor is prevented terminating in shady and cool dry place preservation and is got final product.

3.4 the applicable cases of the mycorrhizal fungi bacterium liquid behind the purifying

Use the test of above fungi tieback and bacterium liquid and infect that spring cuckoo, Xia Juan, Savoury Rhododendron Leaf, deer horn cuckoo, Chinese azalea, Ledum palustreL .subsp .decumdens, Vaccinium uliginosum, various Rhododendron potted and alpine rose etc. more than 120 are planted all in various degree or at least a kind of fungi forms the mycorhiza structure in rhododendron not of the same race, but have mycorhiza formation speed difference.

Method one: available bacterium liquid soaks Rhododendron seeds 48 h, disseminates in the saturating matrix (matrix is vegetable mould and vermiculite, and ratio is 3:1) of bacterium liquid filling and covers the pine needle moisturizing of becoming thoroughly decomposed, and 18 d can sprout, and germination rate is 92.1%, and transplanting survival rate is more than 90.0%.

Method two: behind the rhododendron tissue cultured seedling bottle outlet, behind bacterium liquid immersion tissue cultured seedling root 18～24 h, tissue cultured seedling is planted in filling with bacterium liquid in the saturating matrix (matrix is vegetable mould, become thoroughly decomposed pine needle and vermiculite, and ratio is 3:2:1), cover the good plastics film heat and moisture preserving (hardening) of light transmission.Can improve more than the surviving rate to 99.5%.

Method three: when rhododendron is transplanted, the thick root of rhododendron can be cut 2/3, soak 12～18 h with bacterium liquid again, can improve more than the surviving rate to 93.9%.

Claims (1)

1. one kind is beneficial to the bacterium solution preparation method that rhododendron grows and mycorhiza forms, and it is characterized in that step is as follows:

(1) material and processing:

Gather the ripe fibrous root of rhododendron, sterilization is cut into root segment for subsequent use;

(2) substratum and configuration:

The content of 1000 mL substratum is: KH ₂PO ₄380 mg, MgSO ₄7H ₂O149 mg, Catergen 6 mg, calcium pantothenate 67 mg, nicotinic acid 1.4 mg, peptone 3.0 g, glucose 16.5 g, agar powder 8.5 g, 1.5% Septochol sodium solution, 4 mL and 0.05 mg indolylacetic acid;

(3) mother liquor preparation:

To be inoculated into media surface in the culture dish after the root segment segment in the step (1), place 25 ± 2 ℃ biochemical cultivation case to cultivate;

Treat that mycelia sends from root, picking square section hypha,hyphae is transferred and is continued to cultivate on new substratum, purifying, and the inoculation behind the purifying places 3～5 ℃ cyrogenic equipment to preserve to invisible spectro slant medium;

The cultivation of liquid spawn mother liquor is the liquid nutrient medium that the agar in the substratum in the step (2) is removed rear configuration, with 6 bacterial classifications of purifying Phialocephala fortinii, Pezizilla ericace, Oidiodendron maius, Meliniomyces variabilis, Cryptosporiopsis ercae, Acaulospora lacunosaAfter being cultured to respectively mycelia in vitro and covering with the inclined-plane, get respectively the agar block that contains mycelia and put into the triangular pyramidal bottle that liquid nutrient medium is housed, tinfoil seals bottleneck and is placed on shaking culture 15～20 d on the constant-temperature table, collect 500 mL nutrient solutions and be settled to 1000 mL with sterilized water, and be sub-packed in the dark brown container of 500 mL with shaking all after the tinfoil sealing, placing temperature is to preserve 7～10 d under 25 ± 2 ℃ of conditions, shake every day during this time 1～2 time, each 20～30 min, the liquid spawn mother liquor;

(4) bacterium solution preparation:

Producing with bacterium liquid collocation method is liquid spawn mother liquor 500 mL, after the brown sugar 500 g dissolving, adds water to 50 L, 7～10 d that ferment, and can use after smelling fragrance.