CN104004667A - Endophytic fungus capable of accelerating phosphorus absorption of cedar - Google Patents
Endophytic fungus capable of accelerating phosphorus absorption of cedar Download PDFInfo
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- CN104004667A CN104004667A CN201410246284.2A CN201410246284A CN104004667A CN 104004667 A CN104004667 A CN 104004667A CN 201410246284 A CN201410246284 A CN 201410246284A CN 104004667 A CN104004667 A CN 104004667A
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Abstract
The invention provides an endophytic fungus (Paecilomycessp.) capable of accelerating phosphorus absorption of cedar and a separation, purification, inoculation, cultivation and application method thereof. The endophytic fungus is (Paecilomycessp.)AJ6 and is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCCNO.9186. The endophytic fungus bacterial strain related by the invention is separated and purified from a cedar plant, and is inoculated to a cedar tissue culture seedling, according to comprehensive evaluation of indicators of photosynthesis, the endophytic fungus is further proven to have the effect of accelerating phosphorous absorption of the cedar, functional endophytic fungus beneficial to acceleration of phosphorous absorption of the plant is found, and the functional endophytic fungus can be applied to production.
Description
Technical field
This invention relates to the endogenetic fungus (Paecilomyces sp.) that a strain can promote that Phosphorus In Chinese Fir absorbs, and comprises that its separation, purification, inoculation and cultivation connect and application method.
Background technology
The research of endophyte of plant started from for 19 end of the centurys, and Vogl isolates the first strain endophyte from rye grass Lolium temulentum L. seed.But the endophyte really starting in large quantity research plant originates in the eighties in last century, be mainly to conduct a research in the vegetation of Temperate Region in China, subtropical zone and torrid areas.
Forefathers can isolate endophyte from majority of plant, therefore can infer that endophyte is ubiquitous in plant.In worldwide, at least in the gramineae farm crop of more than 80 genus kind more than 290, found endophyte.The unique function that at present separated endomycorrhiza has according to it from plant can be divided into: the plant growth-promoting bacterias such as vinelandii, solid potassium bacterium, solid phosphorus bacterium, have the biocontrol microorganisms of disease and insect resistance and have and promote the resistance bacterium of plant to the repair ability of poor environment.
Xie Qingmei and Huang Jianhe (1985) evaluation to the morphological specificity of China fir endomycorrhiza and mycorrhizal fungi, mainly does anatomy analysis and the mycelia obtaining from Rhizosphere Soil with wet screening-decantation and chlamydospore with the symbiotic structure of Chinese fir root bark layer tissue and endomycorrhiza fungi and observes.Result shows, according to chlamydosporic Morphologic Characteristics in the anatomy analysis of China fir endomycorrhiza and rhizosphere soil, determines that China fir endomycorrhiza is to belong to one clump of branch type endomycorrhiza of vesicle (being called for short VA mycorhiza).Further research China fir endomycorrhiza, to the growing and the effect of resistance aspect of China fir, adequately and reasonably utilizes endomycorrhiza preparation to promote the problem of Growth of Chinese Fir, has great economic worth.Particularly, in the barren red soil region of south China nutrient, research endomycorrhiza strengthens the absorption of Phosphorus in Soil, is major issue urgently to be resolved hurrily in the China fir fast growing of current Red Soil Region of South China.In summary, about the research of the endogenetic fungus aspect of China fir, only from root position, carry out identification research, and Lignum seu Ramulus Cunninghamiae Lanceolatae leaf position and stem position are not all related to the research of endogenetic fungus.In addition, relevant endogenetic fungus also exists blank on the impact research of China fir self growth.
Acid phosphatase (Acid Phosphatase Activity, hereinafter to be referred as APA) is ubiquitous a kind of very important lytic enzyme in soil and plant body, and its active height has close contacting with the phosphorus element Condition of abundance or deficiency in plant materials and soil.Plant can adapt to low-phosphorus stress by improving the absorbed dose of the physiological property increase phosphorus of self, as plant materials internal secretion acid phosphatase enzyme activation insoluble inorganic phosphorus and organophosphorus.
Summary of the invention
The object of the present invention is to provide a strain can promote the endogenetic fungus that Phosphorus In Chinese Fir absorbs, this fungi be Paecilomyces varioti (
paecilomyces sp.) AJ6, in the registration preservation of in May, 2014 20 China Committee for Culture Collection of Microorganisms common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCC NO.9186.
For achieving the above object, the present invention adopts following technical scheme:
Separation, the purifying of this bacterium comprise:
A, material: the root, stem section, the leaf that collect different year are divided into three parts, with tap water, clean up, divide and install in valve bag, in 4 ℃ of Refrigerator stores;
---sterilized water embathes once---10% clorox soaks 7min---, and sterilized water embathes once for B, sterilization: 70% alcohol-pickled 30s.Whether the water after sample embathes is for the last time contained in the small beaker of sterilizing, at flat lining out in contrast, thorough with the sterilization of check sample, after some days, if any bacterium colony, generates, and shows that sample surfaces sterilization is not thorough, needs to continue to adjust sterilization method;
The selection of C, inoculation position: leaf tip, leaf central part and 3 processing of leaf base; Branch: upper, middle and lower is divided into 3 positions, wherein the 3rd Wei Zhijing junction, position; Root: be divided into 2 parts of the tip of a root and foundation;
D, inoculation: the explant after sterilization is cut with vaccinating lancet, be laid in media surface, observe colony growth situation cultivate 5-7 days in 28 ℃ of constant incubators after.Some bacterial strain breedings rapidly, can first be carried out separation and purification by the bacterial strain of its generation; The bacterial strain of poor growth and comparatively small amt can extend observing time, until the stable rear purifying of its growth;
Solid culture: use improvement Martin solid medium, formula for peptone 5.0g/L, yeast extract powder 2.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, agar 14.0g/L, other is sterilized water, pH value 6.4 ± 0.2, culture temperature is 28 ℃, training method is dull and stereotyped cultivation, and incubation time is 48h;
E, different types of endogenetic fungus access plate culture medium that separation is bred carry out purifying, after 2-3 point connects purifying, switching, obtain the above-mentioned of single culture
paecilomyces sp.function stem;
Point inoculation purifying: the transfering loop after sterilization is slightly clicked to thalline surface, then clicked inoculating surfaces, the point that the method is more traditional is planted culture method inoculum size more easy to control, thereby improves the inoculation efficiency of endogenetic fungus and recall rate.
Above-mentioned a kind of preparation method that can promote the endogenetic fungus application bacterium liquid that Phosphorus In Chinese Fir absorbs, by above-mentioned bacterial strain access liquid nutrient medium, shaking table shaking culture, culture temperature is 28 ℃, incubation time 48～72h, utilize blood counting chamber to calculate bacterial concentration, bacterium liquid is diluted to 1.0-9.0 * 10 with ultrapure water
6it is standby that cfu/ml is finished product; Liquid nutrient medium: be improvement Martin substratum, wherein peptone 5.0g/L, yeast extract powder 2.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, other is sterilized water, pH value 6.4 ± 0.2.
Above-mentioned a kind of application method that can promote the endogenetic fungus application bacterium liquid that Phosphorus In Chinese Fir absorbs, is the bacterium liquid of this bacterial strain of application, and transplanted seedling tree in earlier stage dips in root and waters root for forest land.
Above-mentioned a kind of application method that can promote the endogenetic fungus application bacterium liquid that Phosphorus In Chinese Fir absorbs is with 9.0 * 10
5cfu/ml bacterium liquid, waters in the rhizosphere of every strain China fir in forest land by every strain 100ml.
Above-mentioned a kind of application method that can promote the endogenetic fungus application bacterium liquid that Phosphorus In Chinese Fir absorbs is the bacterium liquid 5.0 * 10 of this bacterial strain of application
5cfu/ml dips in seedling root 10min before Rooted Cuttings is planted.
The bacterial strain the present invention relates to separation and purification from China fir plant obtains, through being inoculated in China fir tissue cultured seedling, according to photosynthetic indices, comprehensively pass judgment on, further confirm that it has the effect that promotes that Phosphorus In Chinese Fir absorbs to plant strain growth, the useful function endogenetic fungus searching out promoting plant phosphorus to absorb can be applicable to produce.
Under Different stress gradient, while coercing to 15d, inoculation seedling leaves APA activity is all low than contrast, and all high than contrast after this.Under normal condition, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 7.25% than contrast, 127.20%, 166.81%; Under slight stress conditions, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 82.19% than contrast, 32.58%, 161.47%; Under moderate stress conditions, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 66.42% than contrast, 79.85%, 152.03%; Under severe water stress condition, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 10.82% than contrast, 22.74%, 141.50%.Find out thus, AJ6 bacterial strain has and alleviates the effect that phosphorus is coerced China Fir Seedling.
The root system APA activity of inoculating strain AJ6 seedling is up and down fluctuation, throughout under reason condition respectively higher by 11.39% than contrast, 18.06%, 111.94%, 140.03%.
The China Fir Seedling soil APA of inoculation AJ6 is all high than control group under different treatment condition.Wherein, rhizosphere soil APA, respectively than contrast high by 126.40%, 203.32%, 48.98%, 135.96%; Non-rhizosphere soil APA respectively than contrast high by 92.91%, 54.92%, 69.53%, 34.53%.
The China Fir Seedling phosphorus content of inoculation AJ6 is all high than control group under different treatment condition.Wherein, over-ground part phosphorus content respectively than contrast high by 115.12%, 68.01%, 46.97%, 51.90%; Underground part phosphorus content respectively than contrast high by 59.47%, 32.31%, 30.56%, 46.80%.
Accompanying drawing explanation
fig. 1the impact of AJ6 on China Fir Seedling blade activity of acid phosphatase under low-phosphorus stress.
fig. 2the impact of AJ6 on China Fir Seedling root system activity of acid phosphatase under low-phosphorus stress.
fig. 3the impact of AJ6 on China Fir Seedling soil acidity phosphatase activity under low-phosphorus stress.
fig. 4coerce the impact of lower AJ6 on China Fir Seedling plant phosphorus content.
Embodiment
embodiment 1
1, the application in water planting breeding
Get the above-mentioned endogenetic fungus application bacterium liquid preparing, make 5.0 * 10
5cfu/ml bacterium liquid dips in root 10min before tissue cultured seedling is planted.Can improve and plant seedlings surviving rate more than 10%.
2, rhizosphere soil waters and executes application
This test adopts earth culture pot experiment, tests the China fir tissue cultured seedling that China Fir Seedling used provides for forest-science academy, Fujian Province.Select the consistent nursery stock of growing way to be colonizated in diameter 15cm in May, 2013, in the plastic tub of high 10cm.Soil used is the yellow soil that strictly passes through fumigation.Through weighing, every basin is put into the 3kg soil of equivalent.Through bimestrial restorative growth, continuous three days (July 5, July 6 and July 7) apply the 100mL bacterium liquid of same concentrations respectively at China fir rhizosphere (R) and tree crown (G).Totally 5 dominant bacterias that utilize above-mentioned test to obtain, 4 repetitions of every kind of bacterium liquid, use the aqueous solution as blank.Inoculate and after 60 days, carry out the mensuration of indices.
Bacterium solution preparation: strains tested is accessed to the liquid nutrient medium of 50mL, the cultivation of process 72h in constant-temperature shaking incubator.By stroke-physiological saline solution, press decimal dilution method by the dilution of bacterium liquid, utilize blood counting chamber to calculate bacterial concentration and be mixed with 5.5 * 10
6cfu/mL.
The determination of activity of B11 acid phosphatase
The determination of activity of B11 acid phosphatase is with reference to the method for (1991) such as Lin Qimei.After fresh leaf blots with distilled water is clean, accurately taking 0.05g puts into immediately containing 5mL(pH5.0) acetic acid-sodium-acetate buffer of 0.2mol/L and the tool plug test tube of 5mL 5mmol/L 4-NPP, cover glass stopper, at 30 ℃ of constant incubators, dark place keeps 30min.After taking-up, add 1mL 1mol/L NaOH termination reaction, under 405nm, carry out colorimetric.The unit of blade activity of acid phosphatase is μ gg
-1h
-1.
Root system activity of acid phosphatase is measured
Root system activity of acid phosphatase is measured the method with reference to (2006) such as Chen Yongliangs.After fresh sample is cleaned and blotted, take at random several compared with radicula and take 0.1g, then adding sodium-acetate acetic acid-sodium-acetate buffer (pH5.0) 5mL of 0.2mol/L.Under condition of ice bath, grind to form homogenate.Homogenate is placed in centrifuge tube and places 1h at refrigerator, then the centrifugal 30min of 10000r/min.Getting 1mL supernatant liquor reacts.
Soil acidity phosphatase activity is measured
Soil acidity phosphatase activity is measured the method with reference to (2010) such as Zhang Haiwei.Take 0.3g fresh soil sample (< 2mm) in 10mL centrifuge tube, add acetic acid-sodium-acetate buffer (pH5.0) of 4mL 1mol/L, then add the 4-NPP of 2mL 15mmol/L.Be incubated 1h in 37 ℃ of water-baths after, add immediately 1mL 1mol/L NaOH termination reaction, after filtration, under 405nm, carry out colorimetric.Micromole's number of the p-NP that soil acidity phosphatase activity generates with interior every gram of contained acid phosphatase hydrolysis substrate of soil of unit time represents.
Plant phosphorus assay
With reference to the forestry industry standard < < of People's Republic of China (PRC) forest soil analytical procedure > >, adopt H
2sO-HClO
4disappear and boil, molybdenum antimony resistance colorimetric method is measured plant phosphorus content, and each sample is done three repetitions.
Reagent:
1) 2g/L 2, dinitrophenol indicator: 0.2g 2, and dinitrophenol is dissolved in 100mL water.
2) 2mol/L sodium hydroxide solution: 80g sodium hydroxide (analytical pure) is water-soluble, is diluted with water to 1L.
3) 0.5 mol/L sulphuric acid soln: the 28mL vitriol oil (analytical pure) is diluted with water to 1L.
4) molybdenum antimony stock solution: the 153mL vitriol oil (analytical pure) is slowly poured in 400mL water, stirs, cooling.The ammonium molybdate [(NH that 10g is levigate
4)
6mo
7o
244H
2o, analytical pure] be dissolved in the 300mL water of approximately 60 ℃, cooling.Then sulphuric acid soln is slowly poured in ammonium molybdate solution, then added 100mL 5g/L antimonypotassium tartrate (KSbOC
4h
4o
61/2H
2o, analytical pure) solution, last water is settled to 1L, is placed in brown bottle.In this storage liquid, contain 10g/L ammonium molybdate and 2.8mol/L sulfuric acid.
5) the anti-developer of molybdenum antimony: in 100mL molybdenum antimony stock solution, add 1.5g xitix.Matching while using.
6) 100 μ g/mL phosphorus (P) standardized solution: take 0.4394g in the potassium primary phosphate (KH of 50 ℃ of oven dry
2pO
4, analytical pure) in beaker, add water 100mL and dissolve, add 10mL concentrated hydrochloric acid, water constant volume is to 1L.
7) 5 μ g/mL phosphorus (P) standardized solution: draw 10mL 100 μ g/mL phosphorus standardized solution and be diluted with water to scale in 200mL volumetric flask.
Key instrument: volumetric flask (50mL); Glass stick; Spectrophotometer
Determination step:
1) measure: draw liquid 2 ~ 5mL to be measured in 50mL volumetric flask, add water to 15 ~ 20mL, add 12, dinitrophenol indicator, with 2mol/L sodium hydroxide solution, be transferred to yellow, then with 0.5 mol/L sulphuric acid soln, be transferred to faint yellowly, shake up, with suction pipe, add the anti-developer of 5mL molybdenum antimony, shake up, be diluted with water to scale, shake up, after 30min on spectrophotometer, with 2cm optical path cuvette, select 700nm wavelength, with blank reagent solution demodulating apparatus absorption value, to zero, then measure the absorption value of each nitrite ion to be measured.
2) drafting of working curve: draw respectively 5 μ g/mL phosphorus (P) standardized solution 0,1,2,3,4,5,6mL is in a series of 50mL volumetric flasks, and other working conditions are identical with liquid mensuration to be measured, obtain 0,0.1,0.2,0.3,0.4,0.5,0.6 μ g/mL phosphorus standard serial solution, the 0 μ g/mL phosphorus series solution of take adjusts absorption value to zero as reference, then measures the absorption value of each standard serial solution.
Result is calculated:
In formula: W
pfor phosphorus content, g/kg; C is for checking in the concentration of nitrite ion phosphorus (P), μ g/mL from working curve
V is nitrite ion volume, 50mL; M is for drying sample quality, g.
T
sfor dividing, get multiple,
Results and analysis
The impact of different strain on China Fir Seedling blade activity of acid phosphatase under 3.1 low-phosphorus stress
As shown in Figure 1, under Different stress gradient, while coercing to 15d, inoculation seedling leaves APA activity is all low than contrast, and all high than contrast after this.Under normal condition, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 7.25% than contrast, 127.20%, 166.81%; Under slight stress conditions, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 82.19% than contrast, 32.58%, 161.47%; Under moderate stress conditions, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 66.42% than contrast, 79.85%, 152.03%; Under severe water stress condition, inoculation AJ6 seedling leaves APA activity when coercing to 30d, 45d, 60d respectively higher by 10.82% than contrast, 22.74%, 141.50%.Find out thus, AJ6 bacterial strain has and alleviates the effect that phosphorus is coerced China Fir Seedling.
The impact of different strain on China Fir Seedling root system activity of acid phosphatase under low-phosphorus stress
As shown in Figure 2, the root system APA activity of control group reduces gradually along with coercing heighten degree; The root system APA activity of inoculating strain AJ6 seedling is up and down fluctuation, throughout under reason condition respectively higher by 11.39% than contrast, 18.06%, 111.94%, 140.03%.
The impact of different strain on China Fir Seedling soil acidity phosphatase activity under low-phosphorus stress
As shown in Figure 3, the China Fir Seedling soil APA of inoculation AJ6 is all high than control group under different treatment condition.Wherein, rhizosphere soil APA, respectively than contrast high by 126.40%, 203.32%, 48.98%, 135.96%; Non-rhizosphere soil APA respectively than contrast high by 92.91%, 54.92%, 69.53%, 34.53%.
The impact of different strain on China Fir Seedling plant phosphorus content under low-phosphorus stress
As shown in Figure 4, the China Fir Seedling phosphorus content of inoculation AJ6 is all high than control group under different treatment condition.Wherein, over-ground part phosphorus content respectively than contrast high by 115.12%, 68.01%, 46.97%, 51.90%; Underground part phosphorus content respectively than contrast high by 59.47%, 32.31%, 30.56%, 46.80%.
The present invention finds that a strain can promote the endogenetic fungus that Phosphorus In Chinese Fir absorbs, and adopts the method for watering root to be inoculated in China Fir Seedling this strain China fir endogenetic fungal bacterial strain.By inoculating the phosphorus of rear plant, coerce test, show that the activity of acid phosphatase of plant and soil and plant phosphorus content are all largely higher than contrast, show that it coerces and have very large function for alleviating plant phosphorus, thereby further confirm that obtaining this strain endogenetic fungus can promote the absorption to phosphorus under Phosphorus In Chinese Fir stress conditions.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (3)
1. a strain can promote the endogenetic fungus that Phosphorus In Chinese Fir absorbs, it is characterized in that: this fungi is Paecilomyces varioti (Paecilomyces sp.) AJ6, in the registration preservation of in May, 2014 20 China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC NO.9186.
2. a strain as claimed in claim 1 can promote the application bacterium liquid of the endogenetic fungus that Phosphorus In Chinese Fir absorbs, it is characterized in that: the preparation method of described application bacterium liquid, by described Paecilomyces varioti (Paecilomyces sp.) AJ6 bacterial strain access liquid nutrient medium, shaking table shaking culture, culture temperature is 28 ℃, incubation time 48～72h, utilizes blood counting chamber to calculate bacterial concentration, and bacterium liquid is diluted to 1.0-9.0 * 106cfu/ml with ultrapure water, and to be finished product standby; Liquid nutrient medium: be improvement Martin substratum, wherein peptone 5.0g/L, yeast extract powder 2.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, other is sterilized water, pH value 6.4 ± 0.2.
3. a strain as claimed in claim 2 can promote the application bacterium liquid of the endogenetic fungus that Phosphorus In Chinese Fir absorbs to water the application in executing in water planting breeding and rhizosphere soil.
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| CN104762219A (en) * | 2015-04-14 | 2015-07-08 | 福建农林大学 | Endophytic fungus for promoting biomass growth of aleurites montana in low-phosphorus environment |
| CN104818218A (en) * | 2015-04-14 | 2015-08-05 | 福建农林大学 | Endophytic fungus capable of promoting phosphorus absorption of aleurites montana |
| CN110894474A (en) * | 2019-12-25 | 2020-03-20 | 福建农林大学 | Endophytic fungus capable of promoting phosphorus absorption of casuarina equisetifolia in low-phosphorus environment |
| CN111084011A (en) * | 2019-12-25 | 2020-05-01 | 福建农林大学 | Endophytic fungus capable of enhancing activity of casuarina equisetifolia acid phosphatase in low-phosphorus environment |
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| CN104762219A (en) * | 2015-04-14 | 2015-07-08 | 福建农林大学 | Endophytic fungus for promoting biomass growth of aleurites montana in low-phosphorus environment |
| CN104818218A (en) * | 2015-04-14 | 2015-08-05 | 福建农林大学 | Endophytic fungus capable of promoting phosphorus absorption of aleurites montana |
| CN104818218B (en) * | 2015-04-14 | 2018-03-09 | 福建农林大学 | One plant can promote the endogenetic fungus that aleurite montana phosphorus absorbs |
| CN104762219B (en) * | 2015-04-14 | 2018-05-11 | 福建农林大学 | One plant of endogenetic fungus for promoting aleurite montana biomass to increase under low-phosphorous environment |
| CN110894474A (en) * | 2019-12-25 | 2020-03-20 | 福建农林大学 | Endophytic fungus capable of promoting phosphorus absorption of casuarina equisetifolia in low-phosphorus environment |
| CN111084011A (en) * | 2019-12-25 | 2020-05-01 | 福建农林大学 | Endophytic fungus capable of enhancing activity of casuarina equisetifolia acid phosphatase in low-phosphorus environment |
| CN111084011B (en) * | 2019-12-25 | 2022-02-01 | 福建农林大学 | Endophytic fungus capable of enhancing activity of casuarina equisetifolia acid phosphatase in low-phosphorus environment |
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