CN103060207B - 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, be a kind ofly 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, one of Ye Shi China 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.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.), 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 forming between fungi and rhododendron, and it has the rhododendron of promotion to the absorption of nutritive element, 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 in phase of basic research, does not enter Application Areas always.

Summary of the invention

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

Technical solution of the present invention is: be a kind ofly 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 standby.

(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 after the root segment segment in step (1), be inoculated into media surface in culture dish, the biochemical cultivation case that is placed in 25 ± 2 ℃ is cultivated.

Treat that mycelia sends from root, picking square section hypha,hyphae, transfers and cultivates in upper continuation of new substratum (substratum in step (2)), purifying, and the inoculation after purifying is to invisible spectro slant medium, and the cyrogenic equipment that is placed in 3～5 ℃ is preserved.

The cultivation of liquid spawn mother liquor is the agar in substratum in step (2) to be removed to the liquid nutrient medium of rear configuration, by 6 bacterial classifications of purifying phialocephala fortinii, pezizilla ericace, oidiodendron maius, meliniomyces variabilis, cryptosporiopsis ercae, acaulospora lacunosabeing cultured to respectively mycelia in vitro covers with behind 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 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 tinfoil sealing, being placed in 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, liquid spawn mother liquor.

(4) bacterium solution preparation:

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

Advantage of the present invention is: the root system that 1, the present invention chooses 9 kind rhododendrons of China Changbaishan area Rhododendron has carried out separation, purifying, the preliminary evaluation of mycorrhizal fungi, and completed the female kind of liquid and produced the preparation with bacterium liquid, for Rhododendron in Changbai belongs to and the production application such as the introducing a fine variety of other kind of cuckoo, artificial breeding, cultivation and breeding both at home and abroad and fundamental research provide technological method, meanwhile, can promote the High-efficient Production of rhododendron and be applicable.Available this bacterium immersion bubble Rhododendron seeds 48 h, disseminate and in the matrix of having filled with bacterium liquid (matrix is vegetable mould and vermiculite, and ratio is 3:1), cover 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%.After rhododendron tissue cultured seedling bottle outlet, with after bacterium immersion bubble tissue cultured seedling root 18～24 h, tissue cultured seedling is planted in the matrix of having filled with bacterium liquid (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.More than can improving surviving rate to 99.5%.When rhododendron is transplanted, the thick root of rhododendron can be cut to 2/3, then steep 12～18 h by bacterium immersion, more than can improving surviving rate to 93.9%.

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

Embodiment

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.), felt cuckoo ( rhododendron confertissimumnakai) and Rhododendron redowskianum ( rhododendron redowskianumvery thin of the rhododendron of 9 kinds such as Maxim.).After breaking ground, the nearly thick root of clip place robust growth diameter is the ripe fibrous root below 1.0 mm, with banister brush, washes away gently earth, and flowing water is cut into 3～4 cm after rinsing and spending the night.Under Bechtop, with 70% alcohol (wherein containing 5% Streptomycin sulphate) sterilization, 20 s, again with the chlorine bleach liquor that mass concentration is 0.5% 8 min that sterilize, aseptic water washing 8 times, aseptic filter paper blots root surface-moisture, excision by virus killing disinfectant damaged portion after and to be cut into the root segment of approximately 2.0～3.0 cm left and right standby.

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

The separation of 2.2 fungies, purifying, preliminary evaluation, preservation, mother liquor and bacterium solution preparation

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

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

Adopt morphological method to carry out preliminary evaluation to the bacterial classification of purifying, by 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 agar in substratum in step 2.1 to be removed to the liquid nutrient medium of rear configuration, divide and install to (every bottle of 50～75 mL) in 250 mL triangular pyramidal bottles, a plurality of bacterial classifications of purifying are cultured to respectively to mycelia in vitro to be covered with behind 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, with the tinfoil of appropriate size, seal bottleneck and be placed on shaking culture 15～20 d on 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 tinfoil sealing, be placed in temperature for shaking 1～2 time every day during preserving 7～10 d(under (25 ± 2) ℃ condition, each 20～30 min) liquid spawn mother liquor.The anti-dry place, cool place that terminates in of liquid spawn mother liquor preserves.

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

result

The separation of 3.1 mycorrhizal fungis, purifying and preliminary evaluation

Ericoid mycorrhizal fungi growth and slow, need 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 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, in every kind of separated fungal colony form obtaining of rhododendron, 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.

Mycorrhizal fungi mother liquor and bacterium solution preparation after 3.2 purifying

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

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

Mycorrhizal fungi after 3.3 purifying, mother liquor and bacterium liquid are preserved

Inoculation after purifying is to invisible spectro slant medium, as for preserving in the cyrogenic equipment of 3～5 ℃.The anti-dry place, cool place that terminates in of liquid spawn mother liquor preserves.

The applicable cases of the mycorrhizal fungi bacterium liquid after 3.4 purifying

Apply 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 mycorhiza structure in rhododendron not of the same race, but have mycorhiza formation speed difference.

Method one: available bacterium immersion bubble Rhododendron seeds 48 h, disseminate and in the matrix of having filled with bacterium liquid (matrix is vegetable mould and vermiculite, and ratio is 3:1), cover the pine needle moisturizing of becoming thoroughly decomposed, 18 d can sprout, and germination rate is 92.1%, and transplanting survival rate is more than 90.0%.

Method two: after rhododendron tissue cultured seedling bottle outlet, with after bacterium immersion bubble tissue cultured seedling root 18～24 h, tissue cultured seedling is planted in the matrix of having filled with bacterium liquid (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.More than can improving surviving rate to 99.5%.

Method three: when rhododendron is transplanted, the thick root of rhododendron can be cut to 2/3, then steep 12～18 h by bacterium immersion, more than can improving surviving rate to 93.9%.

Claims (1)

1. be beneficial to the bacterium solution preparation method that rhododendron grows and mycorhiza forms, 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 standby;

(2) substratum and configuration:

The content of 1000 mL substratum is: KH ₂pO ₄380mg, MgSO ₄7H ₂o149mg, Catergen 6mg, calcium pantothenate 67mg, 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 after the root segment segment in step (1), be inoculated into media surface in culture dish, the biochemical cultivation case that is placed in 25 ± 2 ℃ is cultivated;

Treat that mycelia sends from root, picking square section hypha,hyphae, transfers and on new substratum, continues to cultivate, purifying, and the inoculation after purifying is to invisible spectro slant medium, and the cyrogenic equipment that is placed in 3～5 ℃ is preserved;

The cultivation of liquid spawn mother liquor is the agar in substratum in step (2) to be removed to the liquid nutrient medium of rear configuration, by 6 bacterial classifications of purifying phialocephala fortinii, pezizilla ericace, oidiodendron maius, meliniomyces variabilis, cryptosporiopsis ercae, acaulospora lacunosabeing cultured to respectively mycelia in vitro covers with behind 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 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 tinfoil sealing, being placed in 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, liquid spawn mother liquor;

(4) bacterium solution preparation:

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