CN103314943A - 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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- CN103314943A CN103314943A CN2013102407253A CN201310240725A CN103314943A CN 103314943 A CN103314943 A CN 103314943A CN 2013102407253 A CN2013102407253 A CN 2013102407253A CN 201310240725 A CN201310240725 A CN 201310240725A CN 103314943 A CN103314943 A CN 103314943A
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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 that expands numerous mycorrhizal fungi for open-air scale.
Background technology
Mycorrhizal fungi (mycorrhizal fungi) refers to form with the plant nutrition root fungi of symbiont, 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 of land more than 90% can both form mycorrhizas homobium with it.Arbuscular mycorrhiza can enlarge root absorbing area, accelerates Plant To Nutrient element N, P, K and organic absorption, improves disease resistance of plant and resistance, the improvement soil structure, strengthen soil fertility, promote revegetation, mycorhiza has obvious ecological effect to the restoration of the ecosystem of coal field.
Arbuscular mycorrhizal fungi is obligate biotroph microorganism, and vegetative propagation has abundant genetic diversity, still can not fully in the in vitro pure culture, can only rely on live plant it is bred up to now.Biological technology of mycorrhiza is as a kind of method that is widely used at present the coal field ecological management, and the expansion of mycorhiza is numerous to become a part indispensable in this technology.
The expanding propagation method of mycorrhizal fungi is numerous at present, such as live body earth culture method, nutrient solution cultivation, stripped dual cultivation etc.Wherein the live body land for growing field crops earth culture method after the improvement is suitable for the application of coal field.The selected culture matrix of the method mostly is local Farmland Soil, must carry out soil disinfection, seed and the Inoculant of host plant 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 use 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 that expands numerous mycorrhizal fungi for open-air scale.
Soil disinfection method be used to expanding numerous mycorrhizal fungi provided by the present invention can comprise the steps:
(1) alcoholic strength is sprayed on soil surface to be sterilized at the liquor of 56 degree above (spending to 68 such as 56 degree), the consumption of described liquor is every cubic metre of described soil to be sterilized with described liquor 0.8-1.5 kilogram (such as 0.8 kilogram or 1.47 kilograms);
(2) will spray the soil described to be sterilized sealing of described liquor, the time of described sealing is 2-7 days;
(3) remove sealing, described soil to be sterilized is dried, namely finish soil disinfection.
In the step (1) of said method, described liquor is sprayed on described soil surface to be sterilized before, as required, also comprise the step that described soil to be sterilized is dried; After described liquor is sprayed on described soil surface to be sterilized, also comprise the step that both are stirred.In an embodiment of the present invention, described liquor is 56 degree liquor, 65 degree liquor or 68 degree liquor.More concrete, described 56 degree liquor are " 56 ° special herd ox is vexed to fall donkey "; Described 65 degree liquor are " 65 ° of vexed donkeys of Mongolian spirits "; Described 68 degree liquor are " put horsewhip for 68 ° and burn cutter grassland spirits ".
In the step (2) of said method, sealing is for fear of the alcohol volatilization, makes it to the soil layer inner penetration.The material that carries 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, the purpose that described soil to be sterilized is dried is for alcohol is thoroughly volatilized, thereby avoids its plant or bacterial strain to plantation to produce harmful effects such as suppressing growth.The time of in the present invention, described soil to be sterilized being dried is 7-10 days.
Further, described soil to be sterilized is dried, soil is frequently stirred up and down in the soil tiling described to be sterilized that specifically will seal 7-10 days, and soil moisture and alcohol can thoroughly be volatilized as early as possible.
In the present invention, described liquor is specially as lower at least a:
1) 56 degree liquor: 56 ° special herds that ox is vexed to fall donkey, and ox Wine Co., Ltd, delicate fragrance type are herded in Ningcheng, the Inner Mongol;
2) 65 degree liquor: 65 ° of vexed donkeys of Mongolian spirits, ox Wine Co., Ltd, delicate fragrance type are herded in Ningcheng, the Inner Mongol;
3) 68 degree liquor: put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine in Ningcheng County, Inner Mongol factory, delicate fragrance type.
A further object of the present invention provides a kind of method that expands numerous mycorrhizal fungi.
The method of the numerous mycorrhizal fungi of expansion provided by the present invention can comprise the steps:
(a) according to the method described above to disinfecting soil;
(b) can with numerous mycorrhizal fungi mix after be seeded in step (a) disinfection soil with described wait expanding wait the plant seed that expands numerous mycorrhizal fungi symbiosis, thereby realize that the described expansion of waiting to expand numerous mycorrhizal fungi is numerous.
In said method, 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) be documented in for Glomus mosseae (Glomus mosseae) BGC XJ01(" Cui Weidong; dragon a surname's 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 " literary composition); Described Glomus intraradices (Glomus intraradices) be documented in for Glomus intraradices (Glomus intraradices) BGC AH01(" 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 the zinc pollution. Acta Ecologica Sinica; 2012,32(7): 2127-2134 " literary composition).
In expanding the method for numerous mycorrhizal fungi, 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 the said method step (b), described " can wait to expand numerous mycorrhizal fungi and mix with described with the plant seed of waiting to expand numerous mycorrhizal fungi symbiosis ", be with described plant seed with contain the described microbial inoculum that expands numerous mycorrhizal fungi of waiting and mix, 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: contain the microbial inoculum of described Glomus mosseae (Glomus mosseae) BGC XJ01, Chinese arbuscular mycorrhiza germplasm resource bank be numbered BGC XJ01, its spore density is 126/gram microbial inoculum.
Microbial inoculum B: contain the microbial inoculum of described Glomus intraradices (Glomus intraradices) BGC AH01, Chinese arbuscular mycorrhiza germplasm resource bank be numbered BGC AH01, its spore density is 158/gram microbial inoculum.
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.The phosphorus of described available phosphorus for can directly being absorbed by plant.
In the present invention, described soil is the soil of the sinking land in coalmining areas, is specially the Yulin City Da Liu of Shaanxi Province tower---the soil that live chickens rabbit mining collapse is located in, and available phosphorus in the soil (phosphorus that can directly be absorbed by plant) content is lower than 10 mg/kg.
Experimental results show that, adopt method provided by the present invention to carry out soil disinfection, expand numerous mycorrhizal fungi, not only can effectively reduce disease fungus in the soil, bacterium and actinomycetic quantity, can also improve the quality of mycorrhizal fungi, as increasing Mycorrhizal Infection Incidence, mycelial density and spore density.As disinfectant, its main component of liquor is alcohol and water (accounting for the 98%-99% of total amount) with liquor in the present invention, and is dissolved in the numerous micro-organic compounds (only accounting for the 1%-2% of total amount) of kind such as wherein acid, ester, alcohol, aldehyde.Can easily buy liquor at coal field, its Disinfection Effect that reaches is better than formaldehyde equally, has solved the defective that alcohol is difficult to buy, and has also satisfied the requirement of the ecological agriculture, and is nontoxic.Method of the present invention is easy and simple to handle, and is with low cost, remarkable in economical benefits.The method wide adaptability makes mycorhiza become a kind of feasible new method in coal field ecological reconstruction, soil remediation.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Related liquor has following several among the following embodiment:
56 degree liquor: 56 ° special herds that ox is vexed to fall donkey, and ox Wine Co., Ltd, delicate fragrance type are herded in Ningcheng, the Inner Mongol;
65 degree liquor: 65 ° of vexed donkeys of Mongolian spirits, ox Wine Co., Ltd, delicate fragrance type are herded in Ningcheng, the Inner Mongol;
68 degree liquor: put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine in Ningcheng County, Inner Mongol factory, delicate fragrance type.
Related arbuscular mycorrhizal fungi and microbial inoculum thereof among the following embodiment:
Glomus mosseae (Glomus mosseae) BGC XJ01 and microbial inoculum thereof: be numbered BGC XJ01 at Chinese arbuscular mycorrhiza germplasm resource bank, contain in the microbial inoculum of Glomus mosseae (Glomus mosseae) BGC XJ01, the spore density of Glomus mosseae (Glomus mosseae) BGC XJ01 is 126/gram microbial inoculum." Cui Weidong; dragon a surname's 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 the 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, contain in the microbial inoculum of Glomus intraradices (Glomus intraradices) BGC AH01, the spore density of Glomus intraradices (Glomus intraradices) BGC AH01 is 158/gram microbial inoculum." 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 the zinc pollution. Acta Ecologica Sinica, 2012,32(7): 2127-2134 " record Glomus intraradices (Glomus intraradices) BGC AH01 in the literary composition.The public can buy from Chinese arbuscular mycorrhiza germplasm resource bank.
Related and the plant arbuscular mycorrhizal fungi symbiosis among the following embodiment:
Alfalfa: kind is Algonquin, buys and plant grass cultivation Co., Ltd (middle kind of grass cultivation) in Beijing.
Embodiment 1, laboratory soil disinfection
Used soil sample is taken from lawn in China Mining Univ. (Beijing) campus in the present embodiment, available phosphorus in the soil (phosphorus that can directly be absorbed by plant) content is lower than 10 mg/kg (adopt method---the sodium bicarbonate lixiviate of available phosphorus content in the existing conventional determining soil, molybdenum antimony resistance colorimetric method is measured) after measured.
One, fungi, bacterium and actinomycetic detection analysis in the soil before and after the inventive method sterilization
As follows soil sample is carried out disinfection, simultaneously the soil sample before and after the sterilization is carried out fungi, bacterium and actinomycetic detection, thus the Disinfection Effect of Analysis and Identification the method.
1, soil disinfection
(1) get high 11 centimetres, 13 centimetres of basin mouth diameters, the red little red basin of 9 centimetres of diameters fills 0.0034 cubic metre of soil (taking from lawn in China Mining Univ. (Beijing) campus) at the bottom of the basin, and soil is tiled on the newspaper dries.
(2) 65 degree liquor (65 ° of vexed donkeys of Mongolian spirits, ox Wine Co., Ltd, delicate fragrance type are herded in Ningcheng, the Inner Mongol) are sprayed on soil surface, the liquor consumption is 0.8 kilogram every cubic metre, and namely every basin 2.72 restrains, and mixes.
(3) with plastic film sealing, continue to open plastic film after 3 days, the soil tiling of spraying behind the liquor was dried (alcohol thoroughly volatilizees) in 7 days, namely finish sterilization, can be used for connecing bacterium and sowing.
2, fungi, bacterium and actinomycetic detection in the soil sample
Adopt dilution-plate method that fungi, bacterium and actinomycetes in the soil sample (soil and the rear soil of liquor sterilization before the liquor sterilization) are detected, operating procedure is as follows:
(1) preparation of medium
Bacteria culture media (beef extract-peptone agar is used 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).Annotate: the easy moisture absorption of peptone, weighing is wanted rapidly.
Actinomycetes medium (No. 1 medium of improvement Gao Shi is used for measuring the actinomycetes total plate count) is used 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).Facing the time spent adds potassium bichromate solution in No. 1 medium of the Gao Shi that has melted, with the growth of anti-bacteria and mould.Add 3%(3g/100ml in every 300ml medium) potassium bichromate 1ml.
Fungi culture medium (brave red agar is used for measuring the fungus colony sum): 1000ml distilled water adds the red agar of 35g tiger.
(2) sterilization
Culture dish: two is 1 cover, and 6 cover culture dishes are wrapped with newspaper, are fastened with rubber band.Each sample needs a bag culture dish.
Medium: the medium for preparing is packed in the 500ml triangular flask, after the stopper sealing, wrap with newspaper.
Test tube: in test tube, inject 9ml distilled water, with the stopper sealing, put into large beaker fixing, wrap with newspaper.
The 100ml triangular flask: the 50ml distilled water of packing in each triangular flask, good with the tampon plug, and wrap with newspaper.Each sample needs a triangular flask.
1ml pipette: wrap every pipette with newspaper.
Annotate: for preventing accident, need container and the medium of sterilization all to need certain surplus.
With above thing subject to sterilization pack into pressure cooker carry out high temperature and high pressure steam sterilization (121 ℃, 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, probably each culture dish 20ml spends the night, and treats the medium cooled and solidified, and is for subsequent use.Annotate: culture dish needs pour medium into behind the finish-drying again, otherwise medium is difficult for solidifying, can't scraper plate.
(4) detect
1. getting 5g soil sample adding to be measured fills in the 100ml triangular flask of 50ml sterile water.
2. this triangular flask is placed on the 10min that vibrates on the oscillator, forms soil suspension.
3. draw soil supension 1ml in the test tube that the 9ml sterile water is housed, by 10 times of method dilutions, be diluted to required gradient successively.Used pipette is each when drawing suspension, and pressure-vaccum 3-5 time repeatedly in dilution is to reduce error.
4. according to each quasi-microorganism in soil quantity how much select suitable extension rate.The dilution factor that general fungi is adopted 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:
The soil supension of drawing the required gradient of 0.1ml drips in the culture dish, then with glass slicker suspension evenly is applied in media surface immediately.Annotate: inoculate the different dilution suspensions of same sample when the culture dish with same pipette, should be from high dilution, then dilution suspension is hanged down in inoculation successively.
6. inoculate the culture dish of soil supension, be inverted in and cultivate the rear taking-up of certain hour (fungi 2 days, bacterium 3-4 days, actinomycetes 4-5 days) in the 28-30 ℃ of insulating box, found clump count.
7. the result calculates
Clump count (CFU/ml) in every ml soil bacteria suspension=bacterium colony mean (CFU/0.1ml) * extension rate * 10 experiment triplicates, the detection of at every turn testing every kind of bacterium of each soil sample all arrange 2 parallel, the result gets the mean value that repeats three times.
3, result
Before and after according to the method for step 1 soil sample being carried out disinfection, fungi, bacterium and actinomycetic number change are as shown in table 1 in the soil.As can be seen from the table, adopt the described method of step 1 can significantly reduce fungi, bacterium and Population of Actinomycetes in the soil after to disinfecting soil with liquor.
Table 1 alcohol disinfecting soil is on the impact of edaphon
Annotate: the different lowercase alphabet differentials of same column different significantly (p＜0.05).
Two, compare distinct methods to the difference on effect of soil disinfection
Get three parts of soil (taking from lawn in China Mining Univ. (Beijing) campus), 0.0034 cubic metre every part, be tiled on the newspaper and dry.Afterwards three parts of soil samples are handled as follows respectively:
Process one (56 degree liquor sterilization): (56 ° special herds that ox is vexed to fall donkey with 56 degree liquor with sprayer, ox Wine Co., Ltd is herded in Ningcheng, the Inner Mongol, delicate fragrance type) spray is soil surface (the liquor consumption is 5 grams), stir with glass bar, immediately soil is put into No. 8 plastics valve bags (240mm*170mm) and seal sterilization.After 2 days, soil is taken out tiling, dry for subsequent use.
Process two (sterilizations of 0.1% formaldehyde): be 0.1% formalin spray with mass fraction with sprayer at soil surface (0.1% formaldehyde consumption be 5 restrain), stir with glass bar, immediately soil is put into No. 8 plastics valve bags (240mm*170mm) and seal sterilization.After 2 days, soil is taken out tiling, dry for subsequent use.
Process three (blanks): with sprayer 5 gram 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, dry for subsequent use.
After sterilization is finished, detect fungi, bacterium and actinomycetic quantity (detection method and result data are analyzed same step 1) in the soil of three processing, and result data is compared analysis.
The result is as shown in table 2, and as can be seen from the table, 56 degree liquor are best to the Disinfection Effect of soil, compares all significantly reductions of fungi, bacterium and Population of Actinomycetes in the soil after the sterilization with blank.
The different sterilization methods of table 2 are on the impact of edaphon
Annotate: the different lowercase alphabet differentials of same column different significantly (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 the Yulin City Da Liu of Shaanxi Province tower---the soil that live chickens rabbit mining collapse is located in.Available phosphorus in the soil (phosphorus that can directly be absorbed by plant) content is lower than 10 mg/kg (adopt method---the sodium bicarbonate lixiviate of available phosphorus content in the existing conventional determining soil, molybdenum antimony resistance colorimetric method is measured) after measured.
One, soil disinfection and expand numerous Glomus mosseae (Glomus mosseae)
1, experiment grouping and processing
Experimental group (liquor sterilization): the Yulin City Da Liu of Shaanxi Province tower---live chickens rabbit mining collapse is located in, long 3m is set up in the vacant lot of selecting a place not to be interfered, wide 2m, the culture pond of high 0.5m, with sandy soil dig out be deposited near the culture pond stand-by.Cover bottom and the wall of culture pond with the plastic film of a 6 * 8m.At the thick sandy soil of culture pond bottom tiling one deck 0.1m, with sprayer 68 degree liquor (put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine in Ningcheng County, Inner Mongol factory, delicate fragrance type) are ejected on the sandy soil first, stir immediately; Repave one deck sandy soil after stirring, repeating the front soil volume that the thick 0.5m(of sandy soil namely sterilizes to the culture pond of working is 0.5 * 3 * 2=3m
3), the total consumption of liquor is 2.5 kilograms, immediately transparent plastic film is covered tightly sealing, liquor is fully contacted, to reach sterilisation purpose with sandy soil in the pond.Open plastic film after 2 days, disinfection soil is paved airing, stirs to accelerate the volatilization of alcohol in the liquor, treats after 7 days that soil dries, and alcohol all volatilizees, and alcohol-free smell is filled out back culture pond with sandy soil.Alfalfa (the Shen Dong mining area pionner of bud will be urged, alfalfa cultivars is Algonquin) seed and the microbial inoculum that contains Glomus mosseae (Glomus mosseae) BGC XJ01, ratio according to volume ratio 1:1 mixes, be sprinkled upon on the soil that dries of sterilizing, the above covers the thin layer disinfection soil, waters an amount of.Alfalfa is sprouted after a week, and alfalfa rhizosphere soil (from the soil at 1-10 millimeter place, root axle surface) is collected in growth after 16 weeks, be used for following mensuration.
Control group (high pressure steam sterilization): cultivate used matrix and be in the experimental group soil in the culture pond.(121 ℃ 1h), are dried for subsequent use culture matrix through high temperature, high pressure steam sterilization.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 sterilize for subsequent use with 75% alcohol.Test is carried out in China Mining Univ. (Beijing) greenhouse.Alfalfa (alfalfa cultivars the is Algonquin) seed and the microbial inoculum that contains Glomus mosseae (Glomus mosseae) BGC XJ01 of bud will be urged, ratio according to volume ratio 1:1 mixes, on the soil that is sprinkled upon high pressure steam sterilization and dries, the above covers the thin layer sterile soil, waters an amount of.Alfalfa is sprouted after a week, and alfalfa rhizosphere soil (from the soil at 1-10 millimeter place, root axle surface) is collected in growth after 16 weeks, be used for following mensuration.
2, the quality testing of Glomus mosseae (Glomus mosseae) the BGC XJ01 of different sterilization methods productions
(1) mensuration of Mycorrhizal Infection Incidence
The alfalfa fibrous root that cleans up is put in 10%KOH(100gKOH to be dissolved in the 1L water) solution immersion 24h, with the clear water rinsing for several times, no longer be yellow to water.(the bent sharp benzene indigo plant of 0.5g is dissolved in the lactic acid that the 1L volume ratio is 1:1:1: glycerine: in the distilled water mixed liquor with 0.05% bent sharp benzene indigo plant, must use glass bar to be stirred to whole dissolvings, putting into brown wide-mouth bottle deposits) dyeing liquor immersion 12h, the flush away dyeing liquor, root is put in (lactic acid and glycerine volume ratio 1:1:1) in the lactic acid glycerol liquor, then (every contains 15 in film-making, every 1cm-1.5cm, each sample repeats twice), examine under a microscope that to infect the root segment number be n, infection rate (%)=n/15 * 100%.
(2) mensuration of the outer mycelial density of root
The Rhizosphere Soil of getting the alfalfa of 2 gram steps 1 collections places the 500ml conical flask, adding the 250ml running water makes soil form suspension, the 50ml suspension is sub by 300 mesh sieves, wash sieve in agitator with the 100ml running water, stir 30s, place 1min, draw at twice 10ml(and draw 5ml at every turn) to miillpore filter (diameter 50mm, aperture 0.45 μ m) upper vacuum filtration, filter membrane is evenly cut off two halves to be placed respectively on the slide, adding 2-3 drips 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 the 1L volume ratio is 1:1:1: glycerine: in the distilled water mixed liquor again, must use glass bar to be stirred to whole dissolvings, putting into brown wide-mouth bottle deposits), then film-making, examine under a microscope, randomly draw record crosspoint, 30 visuals field (in the microscopy on mycelia and the microscope ocular-net crosspoint of grid).
Computing formula:
(3) mensuration of spore density
Adopt improved wet screening decantation-Determination Staining spore density, specific as follows:
1. take by weighing the Rhizosphere Soil of the alfalfa of constant weight (gram) step 1 collection, be placed on the 20-30min that is soaked in water in the container, make soil loosening.If soil stickiness is very large, also can add various soil dispersing agents.
2. select the soil sieve that the aperture is 0.5-0.034mm that has of a cover cleaning, be piled up successively.The bottom cushions (such as things such as culture dish, wooden units) with an object, and compass screen surface is tilted a little.
3. the aqueous solution that stir to soak with glass bar after parked several seconds, precipitates down large chad and foreign material, and the soil solution that is about to suspend is poured on the soil sieve of last layer aperture maximum at leisure.When toppling over, preferably concentrate to be poured on the point of compass screen surface, do not make whole compass screen surface all speckle with the soil solution.
4. wash gently successively the outsifting that rests on the compass screen surface with clear water, in order to avoid in the residue of upper strata scalping face, mailed mycorrhizal fungal spore;
5. with wash bottle will rest on outsifting on the compass screen surface be flushed to gently one in vitro clean, (the bent sharp benzene indigo plant of 0.5g is dissolved in the lactic acid that the 1L volume ratio is 1:1:1: glycerine: in the distilled water mixed liquor to drip the blue dyeing liquor of 0.05% bent sharp benzene, must use glass bar to be stirred to whole dissolvings, putting into brown wide-mouth bottle deposits), be placed in 90 ℃ of water-baths or the baking oven and heat half an hour, dye;
6. the filtrate after will dyeing is by dusting cover, and the screening on the washing screen, the flush away coloring agent; Outsifting is placed in the clean culture dish, and in the outsifting under flushing, except many thin grit and impurity were arranged, all the other were the mycorrhizal fungal spore of different-diameter.
7. will contain undersize culture dish and be placed under the binocular entity disecting microscope and observe, namely can observe the spore of purple.
Computing formula:
Above mensuration about Mycorrhizal Infection Incidence, mycelial density and spore density all is provided with three experiments and repeats, each each soil sample of repeated experiments each detect index all arrange 2 parallel, the result gets the mean value that repeats three times.
3, result
Respectively to the soil sample disinfection, the Glomus mosseae that final production goes out (Glomus mosseae) BGC XJ01 quality is different according to the method for the described experimental group of step 1 and control group.Compare with the control group of high pressure steam sterilization, Glomus mosseae (Glomus mosseae) the BGC XJ01 quality that experimental group adopts the soil of high spirit sterilization to produce all is improved significantly aspect mycelial density, the spore density outside infection rate, root.Concrete outcome is as shown in table 3.
The different sterilization methods of table 3 are on the impact of Glomus mosseae (Glomus mosseae) BGC XJ01 quality
Annotate: the different lowercase alphabet differentials of same column different significantly (p＜0.05).
Two, soil disinfection and expand numerous Glomus intraradices (Glomus intraradices)
1, experiment grouping and processing
Experimental group: experimental group (liquor sterilization): the Yulin City Da Liu of Shaanxi Province tower---live chickens rabbit mining collapse is located in, long 3m is set up in the vacant lot of selecting a place not to be interfered, wide 2m, the culture pond of high 0.5m, with sandy soil dig out be deposited near the culture pond stand-by.Cover bottom and the wall of culture pond with the plastic film of a 6 * 8m.At the thick sandy soil of culture pond bottom tiling one deck 0.1m, with sprayer 68 degree liquor (put horsewhip for 68 ° and burn cutter grassland spirits, the national red wine in Ningcheng County, Inner Mongol factory, delicate fragrance type) are ejected on the sandy soil first, stir immediately; Repave one deck sandy soil after stirring, repeating the front soil volume that the thick 0.4m(of sandy soil namely sterilizes to the culture pond of working is 0.4 * 3 * 2=2.4m
3, the total consumption of liquor is 2 kilograms), immediately transparent plastic film is covered tightly sealing, liquor is fully contacted, to reach sterilisation purpose with sandy soil in the pond.Open plastic film after 7 days, disinfection soil is paved airing, stirs the volatilization that can accelerate alcohol in the liquor, treats after 7 days that soil dries, and it is complete that alcohol all volatilizees, and alcohol-free smell is filled out back culture pond with soil.Alfalfa (the Shen Dong mining area pionner of bud will be urged, alfalfa cultivars is Algonquin) seed and the microbial inoculum that contains Glomus intraradices (Glomus intraradices) BGC AH01, ratio according to volume ratio 1:1 mixes, be sprinkled upon on the soil that dries of sterilizing, the above covers the thin layer disinfection soil, waters an amount of.Alfalfa is sprouted after a week, and results alfalfa and rhizosphere soil thereof (from the soil at 1-10 millimeter place, root axle surface) are used for following mensuration after 16 weeks of growth.
Control group (high pressure steam sterilization): cultivate the soil that used matrix is culture pond place in the experimental group.Culture matrix through high temperature, high pressure steam sterilization (121 ℃, 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 sterilize for subsequent use with 75% alcohol.Test is carried out in China Mining Univ. (Beijing) greenhouse.Alfalfa (alfalfa cultivars the is Algonquin) seed and the microbial inoculum that contains Glomus intraradices (Glomus intraradices) BGC AH01 of bud will be urged, ratio according to volume ratio 1:1 mixes, on the soil that is sprinkled upon high pressure steam sterilization and dries, the above covers the thin layer sterile soil, waters an amount of.Alfalfa is sprouted after a week, and alfalfa rhizosphere soil (from the soil at 1-10 millimeter place, root axle surface) is collected in growth after 16 weeks, be used for following mensuration.
2, Glomus intraradices (Glomus intraradices) the BGC AH01 quality testing of different sterilization methods productions
The Mycorrhizal Infection Incidence of two processing in the detecting step 1, the outer mycelial density of root and spore density (detection method and result data analysis are with embodiment 2 step 1), and result data compared analysis.
3, result
Respectively to the soil sample disinfection, the Glomus intraradices that final production goes out (Glomus intraradices) BGC AH01 quality is different according to the method for the described experimental group of step 1 and control group.Compare 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 aspect the mycelial density outside Mycorrhizal Infection Incidence and root.Concrete outcome is as shown in table 4.
The different sterilization methods of table 4 are on the impact of Glomus intraradices (Glomus intraradices) BGC AH01 quality
Annotate: the different lowercase alphabet differentials of same column different significantly (p＜0.05).
Claims (7)
1. a soil disinfection method that is used for expanding numerous mycorrhizal fungi comprises the steps:
(1) liquor of alcoholic strength more than 56 degree is sprayed on soil surface to be sterilized, the consumption of described liquor is every cubic metre of described soil to be sterilized with described liquor 0.8-1.5 kilogram;
(2) will spray the soil described to be sterilized sealing of described liquor, the time of described sealing is 2-7 days;
(3) remove sealing, described soil to be sterilized is dried, namely finish soil disinfection.
2. a method that expands numerous mycorrhizal fungi comprises the steps:
(a) according to the described method of claim 1 to disinfecting soil;
(b) can with wait the plant seed that expands numerous mycorrhizal fungi symbiosis and described soil wait expanding after being seeded in the step (a) sterilization after numerous mycorrhizal fungi mixes, thereby realize that the described expansion of waiting to expand numerous mycorrhizal fungi is numerous.
3. method according to claim 1 and 2, it is characterized in that: described mycorrhizal fungi is arbuscular mycorrhizal fungi.
4. method according to claim 3, it is characterized in that: described arbuscular mycorrhizal fungi is Glomus mosseae (Glomus mosseae) or Glomus intraradices (Glomus intraradices).
5. arbitrary described method according to claim 2-4 is characterized in that: described plant seed with waiting to expand numerous mycorrhizal fungi symbiosis is the seed of alfalfa.
6. arbitrary described method according to claim 2-5, it is characterized in that: describedly can wait to expand numerous mycorrhizal fungi and mix with described with the plant seed of waiting to expand numerous mycorrhizal fungi symbiosis, be with described plant seed with contain the described microbial inoculum that expands numerous mycorrhizal fungi of waiting and mix;
Described plant seed is specially 1:1 with the mixed volume ratio of described microbial inoculum.
7. arbitrary described method according to claim 1-6 is characterized in that: described soil is the soil that available phosphorus content is lower than 10 mg/kg.
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