CN103314943B - Soil sterilization method for outdoor large-scale propagation of mycorrhizal fungi - Google Patents
Soil sterilization method for outdoor large-scale propagation of mycorrhizal fungi Download PDFInfo
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Abstract
The invention discloses a soil sterilization method for outdoor large-scale propagation of mycorrhizal fungi. The method provided by the invention comprises the following steps: (1) spraying spirit with alcoholic strength of over 56 degrees to the surface of soil to be sterilized, wherein the using amount of the spirit is that 0.8-1.5 kg of spirit is used for every cubic meter of the soil to be sterilized; (2) sealing the soil to be sterilized on which the spirit is sprayed for 2-7 days; (3) removing the sealing and drying the soil to be sterilized to finish soil sterilization. Experiments prove that the numbers of pathogenic fungi, bacteria and actinomycetes in the soil can be effectively reduced and the quality of the mycorrhizal fungi can also be improved by performing soil sterilization with the method for propagation of the mycorrhizal fungi. According to the method, the spirit is used as a sterilizer, so that the defect that alcohol is difficult to buy at a mining area is overcome, the requirements of ecological agriculture are also met and the method is innocuous and non-toxic. The method has wide adaptability, so that a feasible new method is provided by applying mycorrhiza to ecological reconstruction and soil remediation of the mining area.
Description
Technical field
The present invention relates to a kind of soil disinfection method expanding numerous mycorrhizal fungi for outdoor large-scale.
Background technology
Mycorrhizal fungi (mycorrhizal fungi) refers to and can form the fungi of symbiont with plant nutrition root, mycorhiza is divided into 7 types, i.e. arbuscular mycorrhiza, ectotrophic mycorrhiza, ectendotrophic mycorrhiza, berry cuckoo class mycorhiza, pinesap class mycorhiza, brier class mycorhiza and Orchid Mycorrhizae.Wherein, arbuscular mycorrhizal fungi is the ubiquitous a kind of edaphon of occurring in nature, and the flowering plant on land more than 90% can both form mycorrhizas homobium with it.Arbuscular mycorrhiza can expand root absorbing area, accelerates the absorption of Plant To Nutrient element N, P, K and organic matter, improves disease resistance of plant and resistance, improvement soil structure, strengthen soil fertility, promote revegetation, the restoration of the ecosystem of mycorhiza to coal field has obvious ecological effect.
Arbuscular mycorrhizal fungi is obligate biotroph microorganism, and vegetative propagation has abundant genetic diversity, still completely in vitro pure culture, can not can only rely on live plant and breed it up to now.Biological technology of mycorrhiza is as a kind of method being widely used in coal field ecological management at present, and the expansion of mycorhiza is numerous becomes a part indispensable in this technology.
The expanding propagation method of current mycorrhizal fungi is numerous, as live body earth culture method, Solution culture method, in vitro dual cultivation etc.Live body land for growing field crops earth culture method wherein after improvement is comparatively suitable for the application of coal field.Culture matrix selected by the method mostly is local Farmland Soil, must carry out soil disinfection, the seed of host plant and Inoculant could be sowed simultaneously, not only can prevent damage by disease and insect, can also improve the quality of microbial inoculum.
Soil disinfection is by using chemosterilant in soil, with the method for killing pathogenic bacteria, nematode and other pest.
Summary of the invention
An object of the present invention is to provide a kind of soil Portable safety sterilization method expanding numerous mycorrhizal fungi for outdoor large-scale.
Soil disinfection method for expanding numerous mycorrhizal fungi provided by the present invention, can comprise the steps:
(1) by alcoholic strength, more than 56 degree, the white wine of (as 56 degree to 68 degree) is sprayed on soil surface to be sterilized, and the consumption of described white wine is described white wine 0.8-1.5 kilogram, every cubic metre of described soil to be sterilized (as 0.8 kilogram or 1.47 kilograms);
(2) will spray the soil sealing described to be sterilized of described white wine, the time of described sealing is 2-7 days;
(3) remove sealing, described soil to be sterilized is dried, namely completes soil disinfection.
In the step (1) of said method, before described white wine is sprayed on described soil surface to be sterilized, as required, the step of being dried by described soil to be sterilized is also comprised; After described white wine is sprayed on described soil surface to be sterilized, also comprise the step both stirred.In an embodiment of the present invention, described white wine is 56 degree of white wine, 65 degree of white wine or 68 degree of white wine.More concrete, described 56 degree of white wine for " 56 ° special herd ox vexed fall donkey "; Described 65 degree of white wine are " 65 ° of vexed donkeys of Mongolian spirits "; Described 68 degree of white wine are " put horsewhip for 68 ° and burn cutter grassland spirits ".
In the step (2) of said method, carrying out sealing is in order to avoid alcohol volatilization, makes it permeate in soil layer.The material carrying out described sealing can be plastic film or plastics valve bag.In an embodiment of the present invention, the time of described sealing is specially 2 days or 3 days or 7 days.
In the step (3) of said method, be to make alcohol thoroughly volatilize by the object that described soil to be sterilized dries, thus avoid it to produce the harmful effects such as Developing restraint to the plant of plantation or bacterial strain.In the present invention, be 7-10 days by the time that described soil to be sterilized dries.
Further, dried by described soil to be sterilized, the soil tiling 7-10 days described to be sterilized specifically will sealed, frequently stirs soil up and down, soil moisture and alcohol is thoroughly volatilized as early as possible.
In the present invention, described white wine is specially at least one as follows:
1) 56 degree of white wine: 56 ° special herd ox vexed fall donkey, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type;
2) 65 degree of white wine: 65 ° of vexed donkeys of Mongolian spirits, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type;
3) 68 degree of white wine: put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine factory in Ningcheng County, the Inner Mongol, delicate fragrance type.
Another object of the present invention is to provide a kind of method expanding numerous mycorrhizal fungi.
The method of the numerous mycorrhizal fungi of expansion provided by the present invention, can comprise the steps:
A () is according to the method described above to disinfecting soil;
B () to be seeded in step (a) disinfection soil with wait the plant seed expanding numerous mycorrhizal fungi symbiosis with described after expanding numerous mycorrhizal fungi and mixing, thus wait that the expansion of expanding numerous mycorrhizal fungi is numerous described in realizing.
In the above-mentioned methods, described mycorrhizal fungi can be arbuscular mycorrhizal fungi.
In the present invention, described arbuscular mycorrhizal fungi is specially Glomus mosseae (Glomus mosseae) or Glomus intraradices (Glomus intraradices).
More concrete, described Glomus mosseae (Glomus mosseae) for Glomus mosseae (Glomus mosseae) BGC XJ01(be documented in " Cui Weidong; dragon a surname Qi; Hou Xinqiang etc. the former bacterium of verticillium wilt is coerced arbuscular mycorrhiza cotton seedling root protective ferment and Ultrastructural impact. Xinjiang Agricultural Sciences; 2009,46 (6): 1235-1244 " one literary composition); Described Glomus intraradices (Glomus intraradices) for Glomus intraradices (Glomus intraradices) BGC AH01(be documented in " Xiao Jiaxin; Yang Hui; Zhang Shaoling. arbuscular mycorrhizal fungi is on the impact [J] of trifoliate orange seedling mineral nutrition under the clean ion current of trifoliate orange root and zinc pollution. Acta Ecologica Sinica; 2012,32(7): 2127-2134 " one literary composition).
In the method expanding numerous mycorrhizal fungi, the described plant seed with waiting to expand numerous mycorrhizal fungi symbiosis specifically can be the seed of alfalfa.In the present invention, the kind of described alfalfa is specially Algonquin.
In said method step (b), described " can wait to expand numerous mycorrhizal fungi mix with described with the plant seed waiting to expand numerous mycorrhizal fungi symbiosis ", be by described plant seed with contain described in wait that the microbial inoculum expanding numerous mycorrhizal fungi mixes, the mixed volume of described plant seed and described microbial inoculum is than being 1:1.
Described microbial inoculum is specially following microbial inoculum A or microbial inoculum B:
Microbial inoculum A: the microbial inoculum containing described Glomus mosseae (Glomus mosseae) BGC XJ01, is numbered BGC XJ01 at Chinese arbuscular mycorrhiza germplasm resource bank, and its spore density is 126/gram of microbial inoculums.
Microbial inoculum B: the microbial inoculum containing described Glomus intraradices (Glomus intraradices) BGC AH01, is numbered BGC AH01 at Chinese arbuscular mycorrhiza germplasm resource bank, and its spore density is 158/gram of microbial inoculums.
In above-mentioned soil disinfection and expand in the method for numerous mycorrhizal fungi, described soil available phosphorus content is lower than the soil of 10 mg/kg.Described available phosphorus is the phosphorus that directly can be absorbed by plant.
In the present invention, described soil is the soil of the sinking land in coalmining areas, is specially Shaanxi Province Yulin City great Liu tower---the soil that live chickens rabbit mining collapse is located in, in soil, available phosphorus (phosphorus that directly can be absorbed by plant) content is lower than 10 mg/kg.
Experiment proves, adopt method provided by the present invention to carry out soil disinfection, expand numerous mycorrhizal fungi, not only effectively can reduce disease fungus in soil, bacterium and actinomycetic quantity, the quality of mycorrhizal fungi can also be improved, as increased Mycorrhizal Infection Incidence, mycelial density and spore density.The present invention is using white wine as disinfectant, and its main component of white wine is alcohol and water (accounting for the 98%-99% of total amount), and is dissolved in the organic trace compounds (only accounting for the 1%-2% of total amount) of the huge number such as acid, ester, alcohol, aldehyde wherein.Easily can buy white wine at coal field, its Disinfection Effect reached is better than formaldehyde equally, solves the defect that alcohol is difficult to buy, also meets the requirement of the ecological agriculture, nontoxic.Method of the present invention is easy and simple to handle, with low cost, remarkable in economical benefits.The method wide adaptability, makes mycorhiza in coal field ecological reconstruction, soil remediation, become a kind of feasible new method.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
White wine involved in following embodiment has several as follows:
56 degree of white wine: 56 ° special herd ox vexed fall donkey, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type;
65 degree of white wine: 65 ° of vexed donkeys of Mongolian spirits, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type;
68 degree of white wine: put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine factory in Ningcheng County, the Inner Mongol, delicate fragrance type.
Arbuscular mycorrhizal fungi involved in following embodiment and microbial inoculum thereof:
Glomus mosseae (Glomus mosseae) BGC XJ01 and microbial inoculum thereof: be numbered BGC XJ01 at Chinese arbuscular mycorrhiza germplasm resource bank, in microbial inoculum containing Glomus mosseae (Glomus mosseae) BGC XJ01, the spore density of Glomus mosseae (Glomus mosseae) BGC XJ01 is 126/gram of microbial inoculums." Cui Weidong; dragon a surname Qi; Hou Xinqiang etc. the former bacterium of verticillium wilt is coerced arbuscular mycorrhiza cotton seedling root protective ferment and Ultrastructural impact. Xinjiang Agricultural Sciences; 2009,46 (6): 1235-1244 " record Glomus mosseae (Glomus mosseae) BGC XJ01 in a literary composition.The public can buy from Chinese arbuscular mycorrhiza germplasm resource bank.
Glomus intraradices (Glomus intraradices) BGC AH01 and microbial inoculum thereof: be numbered BGC AH01 at Chinese arbuscular mycorrhiza germplasm resource bank, in microbial inoculum containing Glomus intraradices (Glomus intraradices) BGC AH01, the spore density of Glomus intraradices (Glomus intraradices) BGC AH01 is 158/gram of microbial inoculums." Xiao Jiaxin; Yang Hui; Zhang Shaoling. arbuscular mycorrhizal fungi is on the impact [J] of trifoliate orange seedling mineral nutrition under the clean ion current of trifoliate orange root and zinc pollution. Acta Ecologica Sinica, 2012,32(7): 2127-2134 " record Glomus intraradices (Glomus intraradices) BGC AH01 in a literary composition.The public can buy from Chinese arbuscular mycorrhiza germplasm resource bank.
Plant that is involved and arbuscular mycorrhizal fungi symbiosis in following embodiment:
Alfalfa: kind is Algonquin, buys from Beijing Zhong Zhong grass cultivation Co., Ltd (middle kind of grass cultivation).
Embodiment 1, laboratory soil disinfection
Soil sample used in the present embodiment takes from lawn in China Mining Univ. (Beijing) campus, after measured in soil available phosphorus (phosphorus that directly can be absorbed by plant) content lower than 10 mg/kg (adopt the method for available phosphorus content in existing conventional determining soil---sodium bicarbonate lixiviate, molybdenum antimony resistance colorimetric method measure).
One, fungi, bacterium and actinomycetic detection analysis in soil before and after the inventive method sterilization
As follows soil sample is carried out disinfection, fungi, bacterium and actinomycetic detection are carried out to the soil sample before and after sterilization simultaneously, thus the Disinfection Effect of Analysis and Identification the method.
1, soil disinfection
(1) get high 11 centimetres, basin mouth diameter 13 centimetres, the little red basin of redness of diameter at the bottom of basin 9 centimetres fills 0.0034 cubic metre of soil (taking from lawn in China Mining Univ. (Beijing) campus), and is laid on newspaper by soil and dries.
(2) 65 degree of white wine (65 ° of vexed donkeys of Mongolian spirits, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type) are sprayed on soil surface, white wine consumption is 0.8 kilogram every cubic metre, i.e. every 2.72 grams, basin, and mixes.
(3) with plastic film sealing, continue after 3 days, open plastic film, tiled dry spraying the soil after white wine (alcohol thoroughly volatilizees) for 7 days, and namely completed sterilization, can be used for connecing bacterium and sowing.
2, fungi, bacterium and actinomycetic detection in soil sample
Adopt dilution-plate method to detect the fungi in soil sample (before white wine sterilization soil and the rear soil of white wine sterilization), bacterium and actinomycetes, operating procedure is as follows:
(1) preparation of medium
Bacteria culture media (beef extract-peptone agar, for measuring total number of bacterial colonies): beef extract 3g, peptone 10g, distilled water 1000ml, pH7.0-7.2(NaOH or HCl regulate pH), agar 18g(adds after having surveyed pH).Note: the easy moisture absorption of peptone, weighs and wants rapidly.
Actinomycetes medium (improvement Gao Shi No. 1 medium, for measuring actinomycetes total plate count), for measuring total number of bacteria: KNO
31g, FeSO
47H
2o0.01g, K
2hPO
40.5g, starch 20g, MgSO
47H
2o0.5g, NaCl0.5g, distilled water 1000ml, pH7.4-7.6, agar 18g(adds after having surveyed pH).Face the used time adds potassium bichromate solution in the Gao Shi melted No. 1 medium, with the growth of anti-bacteria and mould.3%(3g/100ml is added in every 300ml medium) potassium bichromate 1ml.
Fungi culture medium (brave red agar, for measuring fungus colony sum): add the red agar of 35g tiger in 1000ml distilled water.
(2) sterilizing
Culture dish: two is 1 cover, 6 cover culture dish newspapers are wrapped, and are fastened with rubber band.Each sample needs a bag culture dish.
Medium: load in 500ml triangular flask by the medium prepared, after stopper sealing, wraps with newspaper.
Test tube: inject 9ml distilled water in test tube, with stopper sealing, puts into large beaker and fixes, wrap with newspaper.
100ml triangular flask: load 50ml distilled water in each triangular flask, be stoppered with tampon, and wrap with newspaper.Each sample needs a triangular flask.
1ml pipette: wrap every root pipette with newspaper.
Note: for preventing accident, needs the container of sterilizing and medium all to need certain surplus.
Above thing subject to sterilization is loaded pressure cooker and carries out high temperature and high pressure steam sterilizing (121 DEG C, 20min).
(3) a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices
Pour the medium of sterilized heat into culture dish, general each culture dish 20ml, spends the night, treats medium cooled and solidified, for subsequent use.Note: culture dish pours medium into after needing finish-drying again, otherwise medium not easily solidifies, cannot scraper plate.
(4) detect
1. get 5g soil sample to be measured to add and fill in the 100ml triangular flask of 50ml sterile water.
2. this triangular flask is placed on 10min that oscillator vibrates, forms soil suspension.
3. draw soil supension 1ml in the test tube that 9ml sterile water is housed, successively by 10 times of method dilutions, be diluted to required gradient.Pipette used each draw suspension time, pressure-vaccum 3-5 time repeatedly in dilution, to reduce error.
4. according to each quasi-microorganism extension rate that how many selections of quantity are suitable in soil.The dilution factor that general fungi adopts is 10
-1-10
-3, actinomycetes 10
-3-10
-5, bacterium 10
-4-10
-6.
5. the inoculation of soil supension: adopt and scrape the skill in using a kitchen knife in cookery, specific as follows:
Drawing the soil supension of gradient needed for 0.1ml drips in culture dish, then immediately with glass slicker by suspension uniform application in media surface.Note: inoculate the different dilution suspension of same sample when the culture dish with same pipette, from high dilution, then should inoculate lower dilution suspension successively.
6. be vaccinated with the culture dish of soil supension, be inverted in 28-30 DEG C of insulating box and cultivate certain hour (fungi 2 days, bacterium 3-4 days, actinomycetes 4-5 days) and take out afterwards, find clump count.
7. result calculates
Clump count (CFU/ml)=bacterium colony mean (CFU/0.1ml) in every ml soil bacteria suspension × extension rate × 10 is tested in triplicate, the detection that each soil sample of each experiment often plants bacterium all arrange 2 parallel, result gets three mean values repeated.
3, result
According to the method for step 1 to before and after soil sample carries out disinfection, in soil, fungi, bacterium and actinomycetic number change are as shown in table 1.As can be seen from the table, the method white wine of employing described in step 1 is to the fungi that can significantly reduce after disinfecting soil in soil, bacterium and Population of Actinomycetes.
Table 1 alcohol disinfecting soil is on the impact of edaphon
Note: the different lowercase alphabet of same column shows significant difference (p<0.05).
Two, the difference on effect of distinct methods to soil disinfection is compared
Get three parts of soil (taking from lawn in China Mining Univ. (Beijing) campus), 0.0034 cubic metre every part, be laid on newspaper and dry.Afterwards three parts of soil samples are handled as follows respectively:
Process one (56 degree white wine sterilization): (56 ° special herds the vexed donkey of ox by 56 degree of white wine with sprayer, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type) spray soil surface (white wine consumption is 5 grams), stir with glass bar, immediately soil is put into No. 8 plastics valve bags (240mm*170mm) and carry out sealing sterilization.After 2 days, soil is taken out tiling, dries for subsequent use.
Process two (0.1% formaldehyde sterilization): be that the formalin spray of 0.1% is soil surface (0.1% formaldehyde consumption is 5 grams) by mass fraction with sprayer, stir with glass bar, immediately soil is put into No. 8 plastics valve bags (240mm*170mm) and carry out sealing sterilization.After 2 days, soil is taken out tiling, dries for subsequent use.
Process three (blanks): with sprayer, 5 grams of distilled water are sprayed at soil surface, stir with glass bar, immediately soil is put into No. 8 plastics valve bags (240mm*170mm) and seal.After 2 days, soil is taken out tiling, dries for subsequent use.
After having sterilized, detect fungi, bacterium and actinomycetic quantity (detection method and result data analysis are with step one) in the soil of three process, and analysis is compared to result data.
Result is as shown in table 2, and as can be seen from the table, 56 degree of white wine are best to the Disinfection Effect of soil, and compared with blank, after sterilization, in soil, fungi, bacterium and Population of Actinomycetes all significantly reduce.
The different sterilization method of table 2 is on the impact of edaphon
Note: the different lowercase alphabet of same column shows significant difference (p<0.05).
The expansion of embodiment 2, mining collapse area soil disinfection and arbuscular mycorrhizal fungi is numerous
Soil sample used in the present embodiment is mining collapse area soil, is specially Shaanxi Province Yulin City great Liu tower---the soil that live chickens rabbit mining collapse is located in.After measured in soil available phosphorus (phosphorus that directly can be absorbed by plant) content lower than 10 mg/kg (adopt the method for available phosphorus content in existing conventional determining soil---sodium bicarbonate lixiviate, molybdenum antimony resistance colorimetric method measure).
One, soil disinfection and expand numerous Glomus mosseae (Glomus mosseae)
1, experiment grouping and process
Experimental group (white wine sterilization): Shaanxi Province Yulin City great Liu tower---live chickens rabbit mining collapse is located in, long 3m is set up in the vacant lot selecting a place not to be interfered, wide 2m, the culture pond of high 0.5m, sandy soil are dug out be deposited near culture pond stand-by.With bottom and the wall of the covered rearing with plastic film culture pond of one piece of 6 × 8m.68 degree of white wine (put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine factory in Ningcheng County, the Inner Mongol, delicate fragrance type) are ejected on sandy soil with sprayer, stir immediately by the sandy soil that one deck 0.1m that first tiles bottom culture pond is thick; Repave one deck sandy soil after stirring, repeating the soil volume that in previous work to culture pond, namely the thick 0.5m(of sandy soil sterilizes is 0.5 × 3 × 2=3m
3), the total consumption of white wine is 2.5 kilograms, immediately transparent plastic film is covered tightly sealing, white wine is fully contacted, to reach sterilisation purpose with sandy soil in pond.Open plastic film after 2 days, disinfection soil is paved airing, and stir the volatilization accelerating alcohol in white wine, treat that soil dries after 7 days, alcohol all volatilizees, and sandy soil are filled out back culture pond by alcohol-free smell.Alfalfa (the infrared ray light shine pionner of bud will be urged, alfalfa cultivars is Algonquin) seed with containing the microbial inoculum of Glomus mosseae (Glomus mosseae) BGC XJ01, mix according to the ratio of volume ratio 1:1, be sprinkled upon and sterilize on the soil that dries, cover thin layer disinfection soil above, water appropriate.After one week, alfalfa is sprouted, and grows after 16 weeks and collects alfalfa rhizosphere soil (soil from 1-10 millimeter place, root axle surface), for following mensuration.
Control group (high pressure steam sterilization): cultivating matrix used is soil in culture pond in experimental group.Culture matrix, through high temperature, high pressure steam sterilization (121 DEG C, 1h), dries for subsequent use.For trial assembly be set to 20cm × 20cm × 15cm(high × basin mouth diameter × basin at the bottom of diameter) the white plastic basin of specification, clean and use 75% alcohol sterilizing for subsequent use.Test is carried out in China Mining Univ. (Beijing) greenhouse.The alfalfa of bud (alfalfa cultivars is Algonquin) seed and the microbial inoculum containing Glomus mosseae (Glomus mosseae) BGC XJ01 will be urged, mix according to the ratio of volume ratio 1:1, be sprinkled upon high pressure steam sterilization and on the soil dried, cover thin layer sterile soil above, water appropriate.After one week, alfalfa is sprouted, and grows after 16 weeks and collects alfalfa rhizosphere soil (soil from 1-10 millimeter place, root axle surface), for following mensuration.
2, the quality testing of Glomus mosseae (Glomus mosseae) the BGC XJ01 of different sterilization method production
(1) mensuration of Mycorrhizal Infection Incidence
The alfalfa fibrous root cleaned up being put in 10%KOH(100gKOH be dissolved in 1L water) solution soaks 24h, with clear water rinsing several, is no longer yellow to water.By 0.05% bent sharp benzene indigo plant, (the bent sharp benzene indigo plant of 0.5g is dissolved in the lactic acid that 1L volume ratio is 1:1:1: glycerine: in distilled water mixed liquor, glass bar must be used to be stirred to whole dissolving, put into brown, wide-mouth bottle to deposit) dyeing liquor immersion 12h, wash away dyeing liquor, root is put in (lactic acid and glycerine volume ratio 1:1:1) in lactic acid glycerol liquor, then (every sheet is containing 15 in film-making, every root 1cm-1.5cm, each sample repeats twice), examine under a microscope that to infect root segment number be n, infection rate (%)=n/15 × 100%.
(2) mensuration of the outer mycelial density of root
The Rhizosphere Soil getting the alfalfa that 2 grams of steps 1 are collected is placed in 500ml conical flask, add 250ml running water and make Zinc fractions suspension, by 50ml suspension by 300 mesh sieve, with 100ml tap water sieve in agitator, stir 30s, place 1min, draw 10ml(at twice and draw 5ml at every turn) to miillpore filter (diameter 50mm, 0.45 μm, aperture) upper vacuum filtration, filter membrane is evenly cut off two halves to be placed in respectively on slide, add 2-3 again to drip the blue dyeing liquor of 0.05% bent sharp benzene (the bent sharp benzene indigo plant of 0.5g is dissolved in the lactic acid that 1L volume ratio is 1:1:1: glycerine: in distilled water mixed liquor, glass bar must be used to be stirred to whole dissolving, put into brown, wide-mouth bottle to deposit), then film-making, examine under a microscope, randomly draw record crosspoint, 30 visuals field crosspoint of grid (in the microscopy on mycelia and microscopy grids eyepiece).
Computing formula:
(3) mensuration of spore density
Adopt the wet screening decantation-Determination Staining spore density improved, specific as follows:
1. take the Rhizosphere Soil of the alfalfa that constant weight (gram) step 1 is collected, be placed in container the 20-30min that is soaked in water, make soil loosening.If soil stickiness is very large, also various soil dispersing agent can be added.
That 2. selects a set of cleaning has the soil sieve that aperture is 0.5-0.034mm, is piled up successively.The bottom one object cushions (as the thing such as culture dish, wooden unit), and compass screen surface is tilted a little.
3. stir the aqueous solution soaked with glass bar, after parked a few second, large chad and foreign material precipitation are gone down, and the soil solution being about to suspend is poured on the maximum soil sieve in most last layer aperture at leisure.When toppling over, preferably concentrate and be poured on a point of compass screen surface, do not make whole compass screen surface all speckle with the soil solution.
4. the outsifting rested on compass screen surface is rinsed successively gently with clear water, in order to avoid mailed mycorrhizal fungal spore in the residue in scalping face, upper strata;
5. with wash bottle the outsifting rested on compass screen surface is flushed to gently one in vitro clean, (the bent sharp benzene indigo plant of 0.5g is dissolved in the lactic acid that 1L volume ratio is 1:1:1: glycerine: in distilled water mixed liquor to drip the blue dyeing liquor of 0.05% bent sharp benzene, glass bar must be used to be stirred to whole dissolving, put into brown, wide-mouth bottle to deposit), be placed in 90 DEG C of water-baths or baking oven and heat half an hour, dye;
6. the filtrate after dyeing is passed through dusting cover, and the screening on washing screen, wash away coloring agent; Be placed on by outsifting in clean culture dish, in the outsifting rinsed, apart from outside many thin grit and impurity, all the other are the mycorrhizal fungal spore of different-diameter.
7. observe under being placed on binocular entity disecting microscope containing undersize culture dish, namely can observe the spore of purple.
Computing formula:
Above about the mensuration of Mycorrhizal Infection Incidence, mycelial density and spore density, be all provided with three experiments and repeat, repeat to test at every turn each Testing index of each soil sample all arrange 2 parallel, result gets three mean values repeated.
3, result
According to the method for the experimental group described in step 1 and control group respectively to soil sample disinfection, Glomus mosseae (Glomus mosseae) the BGC XJ01 quality that final production goes out is different.Compared with the control group of high pressure steam sterilization, Glomus mosseae (Glomus mosseae) the BGCXJ01 quality that experimental group adopts the soil of high spirit sterilization to produce all is improved significantly in infection rate, the outer mycelial density of root, spore density.Concrete outcome is as shown in table 3.
The different sterilization method of table 3 is on the impact of Glomus mosseae (Glomus mosseae) BGC XJ01 quality
Note: the different lowercase alphabet of same column shows significant difference (p<0.05).
Two, soil disinfection and expand numerous Glomus intraradices (Glomus intraradices)
1, experiment grouping and process
Experimental group: experimental group (white wine sterilization): Shaanxi Province Yulin City great Liu tower---live chickens rabbit mining collapse is located in, long 3m is set up in the vacant lot selecting a place not to be interfered, wide 2m, the culture pond of high 0.5m, sandy soil are dug out be deposited near culture pond stand-by.With bottom and the wall of the covered rearing with plastic film culture pond of one piece of 6 × 8m.68 degree of white wine (put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine factory in Ningcheng County, the Inner Mongol, delicate fragrance type) are ejected on sandy soil with sprayer, stir immediately by the sandy soil that one deck 0.1m that first tiles bottom culture pond is thick; Repave one deck sandy soil after stirring, repeating the soil volume that in previous work to culture pond, namely the thick 0.4m(of sandy soil sterilizes is 0.4 × 3 × 2=2.4m
3, the total consumption of white wine is 2 kilograms), immediately transparent plastic film is covered tightly sealing, white wine is fully contacted with sandy soil in pond, to reach sterilisation purpose.Open plastic film after 7 days, disinfection soil is paved airing, and stir the volatilization can accelerating alcohol in white wine, treat that soil dries after 7 days, alcohol all volatilizees complete, and alcohol-free smell, fills out back culture pond by soil.Alfalfa (the infrared ray light shine pionner of bud will be urged, alfalfa cultivars is Algonquin) seed with containing the microbial inoculum of Glomus intraradices (Glomus intraradices) BGC AH01, mix according to the ratio of volume ratio 1:1, be sprinkled upon and sterilize on the soil that dries, cover thin layer disinfection soil above, water appropriate.After one week, alfalfa is sprouted, and grows results alfalfa and rhizosphere soil (soil from 1-10 millimeter place, root axle surface) thereof after 16 weeks, for following mensuration.
Control group (high pressure steam sterilization): cultivate the soil that matrix used is culture pond place in experimental group.Culture matrix is through high temperature, high pressure steam sterilization (121 DEG C, 1h), air-dry for subsequent use.For trial assembly be set to 20cm × 20cm × 15cm(high × basin mouth diameter × basin at the bottom of diameter) the white plastic basin of specification, clean and use 75% alcohol sterilizing for subsequent use.Test is carried out in China Mining Univ. (Beijing) greenhouse.The alfalfa of bud (alfalfa cultivars is Algonquin) seed and the microbial inoculum containing Glomus intraradices (Glomus intraradices) BGC AH01 will be urged, mix according to the ratio of volume ratio 1:1, be sprinkled upon high pressure steam sterilization and on the soil dried, cover thin layer sterile soil above, water appropriate.After one week, alfalfa is sprouted, and grows after 16 weeks and collects alfalfa rhizosphere soil (soil from 1-10 millimeter place, root axle surface), for following mensuration.
2, Glomus intraradices (Glomus intraradices) the BGC AH01 quality testing of different sterilization method production
The outer mycelial density of the Mycorrhizal Infection Incidence of two process in detecting step 1, root and spore density (detection method and result data analysis are with embodiment 2 step one), and analysis is compared to result data.
3, result
According to the method for the experimental group described in step 1 and control group respectively to soil sample disinfection, Glomus intraradices (Glomus intraradices) the BGC AH01 quality that final production goes out is different.Compared with the control group of high pressure steam sterilization, Glomus intraradices (Glomus intraradices) the BGC AH01 quality that experimental group adopts the soil of high spirit sterilization to produce all is improved significantly in Mycorrhizal Infection Incidence and the outer mycelial density of root.Concrete outcome is as shown in table 4.
The different sterilization method of table 4 is on the impact of Glomus intraradices (Glomus intraradices) BGC AH01 quality
Note: the different lowercase alphabet of same column shows significant difference (p<0.05).
Claims (8)
1., for expanding a soil disinfection method for numerous mycorrhizal fungi, comprise the steps:
(1) alcoholic strength is sprayed on soil surface to be sterilized at the white wine of 56 degree to 68 degree, the consumption of described white wine is every cubic metre of described white wine 0.8-1.5 kilogram of described soil to be sterilized;
(2) will spray the soil sealing described to be sterilized of described white wine, the time of described sealing is 2-7 days;
(3) remove sealing, described soil to be sterilized is dried, namely completes soil disinfection;
The white wine of described 56 degree to 68 degree be following in any one:
1) 56 degree of white wine: 56 ° special herd ox vexed fall donkey, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type;
2) 65 degree of white wine: 65 ° of vexed donkeys of Mongolian spirits, Ningcheng, Inner Mongol Mu Niu Wine Co., Ltd, delicate fragrance type;
3) 68 degree of white wine: put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine factory in Ningcheng County, the Inner Mongol, delicate fragrance type.
2. expand a method for numerous mycorrhizal fungi, comprise the steps:
(a) according to method described in claim 1 to disinfecting soil;
B () can be seeded in the soil after sterilizing in step (a) with wait the plant seed expanding numerous mycorrhizal fungi symbiosis with described after expanding numerous mycorrhizal fungi and mixing, thus wait that the expansion of expanding numerous mycorrhizal fungi is numerous described in realizing.
3. method according to claim 1 and 2, is characterized in that: described mycorrhizal fungi is arbuscular mycorrhizal fungi.
4. method according to claim 3, is characterized in that: described arbuscular mycorrhizal fungi is Glomus mosseae (Glomus mosseae) or Glomus intraradices (Glomus intraradices).
5. method according to claim 2, is characterized in that: described with wait that the plant seed expanding numerous mycorrhizal fungi symbiosis is the seed of alfalfa.
6. method according to claim 2, is characterized in that: described can with wait with described, the plant seed expanding numerous mycorrhizal fungi symbiosis waits that expanding numerous mycorrhizal fungi mixes, be by described plant seed with contain described in wait that the microbial inoculum expanding numerous mycorrhizal fungi mixes.
7. method according to claim 6, is characterized in that: described plant seed is 1:1 with the mixed volume ratio of described microbial inoculum.
8. method according to claim 1 and 2, is characterized in that: described soil is the soil of available phosphorus content lower than 10 mg/kg.
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