CN103173362A - Endophytic fungus promoting casuarina equisetifolia photosynthesis - Google Patents
Endophytic fungus promoting casuarina equisetifolia photosynthesis Download PDFInfo
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Abstract
The invention relates to an endophytic fungus promoting casuarina equisetifolia photosynthesis. The endophytic fungus is aspergillussp. casuarina equisetifolia rhizospheric fungus 7, which is registered and collected in China General Microbiological Culture Collection Center on June 28th, 2012. The collection number is CGMCC No. 6305. The casuarina equisetifolia rhizospheric fungus 7 is inoculated in casuarina equisetifolia hydroponic seedling. Comprehensive evaluation is carried out according to various indicators of photosynthesis, and it is further proved that the fungus has a promotion effect upon photosynthesis. When a functional endophytic fungus beneficial for promoting photosynthesis is found, the endophytic fungus can be used in production. Through a fungus solution watering method, the endophytic fungus is inoculated on casuarina equisetifolia seedlings. The endophytic fungus has substantial effects in promoting plant photosynthesis and increasing casuarina equisetifolia dry matter accumulation.
Description
Technical field
The present invention relates to a strain and promote the photosynthetic endogenetic fungus of Horsetail Beefwood.
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
[1]But the endophyte that really begins in large quantity research plant originates in the eighties in last century, is 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.At least found endophyte in the gramineae farm crop of more than 80 genus kind more than 290 in worldwide
[2]The endomycorrhiza that separates from plant at present can be divided into according to its unique function that has: the plant growth-promoting bacterias such as vinelandii, solid potassium bacterium, solid phosphorus bacterium
[3 ~ 4], have a biocontrol microorganisms of disease and insect resistance
[5 ~ 8]Promote plant to the resistance bacterium of the repair ability of poor environment with having.Endophyte report aspect promotion photosynthesis is few, only has and find a kind of endophyte with photosynthesis ability on paddy rice, and it also is proved to be beanpod Aeschynomene
[9]Vinelandii Bredyrhizobium strain in the stem knurl.
The Casuarinaceae plant is the symbiotrophy type plant that has frankia bacterium (Frankia), endophyte root fungus and ectotrophic mycorrhiza concurrently, therefore, at present more frankia (Frankia), Ralstonia solanacearum (Solanasearum), the blue or green withered false pseudomonas bacillus (Pseudomonas soIanacearum Smith) etc. of relating to of research direction about Horsetail Beefwood mycology aspect, although the short fruit of coming into force after Horsetail Beefwood artificial inoculation VA Mycorrhizal Fungi is unquestionable, but only stagnate in the research of root nodule bacterium root fungus, the research of Horsetail Beefwood endogenetic fungus aspect there is not yet report
[10-11]Use bacterial strain in forefathers' research, mostly be external fungi, simultaneously also not from promoting photosynthetic basic factor to remove to seek endogenetic fungus, improve its science and reliability
[12]Confirm to promote photosynthetic endophyte aspect to there is not yet report.
Summary of the invention
The object of the present invention is to provide a strain to promote the photosynthetic endogenetic fungus of Horsetail Beefwood.
A strain provided by the invention promotes the photosynthetic endogenetic fungus of Horsetail Beefwood, described endogenetic fungus be aspergillus (
Aspergillus sp.) Horsetail Beefwood Flora of Rhizosphere Fungi 7, on June 28th, 2012 in the registration preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No. 6305.
Separation, the purifying of this bacterium comprise:
A, material: root, branch, the phyllade sprig of the different year that collects are divided into three parts, clean up with tap water, minute 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 for the last time is contained in the small beaker of sterilization, rules in contrast on flat board, thorough with the sterilization of check sample, generates if any bacterium colony after some days, shows that the sample surfaces sterilization is not thorough, need continue to adjust sterilization method
[13-15]
The selection of C, inoculation position: phyllade sprig: divide the phyllade sprig of three parts in upper, middle and lower of getting plant, each phyllade sprig is divided into again a tip, branch middle part and 3 processing of branch base portion; Branch: upper, middle and lower is divided into 3 positions, and wherein the 3rd position is branch stem junction; Root: be divided into 2 parts of the tip of a root and foundation;
D, inoculation: the explant after sterilizing cuts with vaccinating lancet, is laid in media surface, observes the colony growth situation cultivate 5-7 days in 28 ℃ of constant incubators after.The breeding of some bacterial strains can be first carried out separation and purification with the bacterial strain of its generation rapidly; 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, sal epsom 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 the cultivation, and incubation time is 48h;
E, different types of endogenetic fungus access plate culture medium that separation is bred carry out purifying, obtain the above-mentioned function endogenetic fungus of single culture after process is put for 2-3 time and connect purifying, transfers;
It is on plating medium, and bacterium colony is white felted fine hair, is inlaid with blackish green particulate state projection fine hair in bacterium colony, and matrix color is yellow-green colour.Conidiophore is upright, and simple, the top is spherical, and the top living stigma; Conidium (stalk spore) monospore is spherical; With reference to the fungi identification handbook, it is accredited as Aspergillus (Aspergillus sp.), is kept at China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.6305.
Above-mentioned a kind of preparation method who promotes the photosynthetic endogenetic fungus application of Horsetail Beefwood bacterium liquid, with above-mentioned bacterial strain access liquid nutrient medium, the 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, sal epsom 0.5g/L, other is sterilized water, pH value 6.4 ± 0.2.
Above-mentioned a kind of application method that promotes the photosynthetic endogenetic fungus application of Horsetail Beefwood bacterium liquid is the bacterium liquid of using this bacterial strain, and transplanted seedling tree in earlier stage dips in root and is used for the forest land and waters root.
Above-mentioned a kind of application method that promotes the photosynthetic endogenetic fungus application of Horsetail Beefwood bacterium liquid is with 9.0 * 10
5Cfu/ml bacterium liquid waters in the rhizosphere of every strain Horsetail Beefwood in the forest land by every strain 100ml.
Above-mentioned a kind of application method that promotes the photosynthetic endogenetic fungus application of Horsetail Beefwood bacterium liquid is the bacterium liquid 5.0 * 10 of using this bacterial strain
5Cfu/ml dips in seedling root 10min before Rooted Cuttings is planted.
The bacterial strain that the present invention relates to separation and purification from the Horsetail Beefwood plant obtains, aimed strain be aspergillus (
Aspergillus sp.) Horsetail Beefwood Flora of Rhizosphere Fungi 7, on June 28th, 2012 in the registration preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No. 6305.
Horsetail Beefwood Flora of Rhizosphere Fungi 7 is inoculated in the Horsetail Beefwood Rooted Cuttings, comprehensively passes judgment on according to photosynthetic indices, confirm that further it has promoter action to photosynthesis, search out promoting that the useful function endogenetic fungus of photosynthesis can be applicable to produce.
Water by bacterium liquid the method for executing and be inoculated in Casuarina equisetifolia Seedling, inoculate used afterwards in 30 days Handy Fluor Cam fluorescence imaging instrument that Photon Systems Instruments. (PSI) company produces night 19:00-22:00 measure the chlorophyll fluorescence parameters of Horsetail Beefwood nursery stock.The chlorophyll fluorescence parameters Fm that confirmation Horsetail Beefwood Flora of Rhizosphere Fungi 7 is processed and Fv contrast CK and have improved 39.57% and 42.83%.Φ PSII contrasts has increased by 31.52%.The NPQ value that connects vaccine wood contrasts and has reduced 22.1%.Show that its dry-matter accumulation for the photosynthesis that promotes plant and increase Horsetail Beefwood has significant effect.
Description of drawings
Fig. 1 is the different comparisons that meet lower nursery stock Fm, Fv of bacterium processing.
Fig. 2 is the different comparisons that meet bacterium processing plant nursery stock Fv/Fm.
Embodiment
1, the application in the water planting breeding
Get the Horsetail Beefwood Flora of Rhizosphere Fungi 7 for preparing and use bacterium liquid, make 5.0 * 10
5Cfu/ml bacterium liquid dips in root 10min before Rooted Cuttings is planted.Can improve and plant seedlings surviving rate more than 10%.
2, rhizosphere soil waters and executes application
Test materials is No. one, Huian Rooted Cuttings, and nursery stock is potted plant in forest ecology institute of University Of Agriculture and Forestry In Fujian field test ground in July, 2011.
After Rooted Cuttings root system length is neat, it is moved into gold zone loam and the soil of sand with the ratio mixing of 3:1, and be sub-packed in approximately 500 diameter 22cm, in the flowerpot of high 15cm.Every basin is put into the 3.36KG mixed soil of equal mass, connects the accuracy of bacterium in order to guarantee the later stage nursery stock, splashes into 10 ~ 15 formaldehyde (formalin) thimerosal in every basin soil, and with plastics film capping basin mouth, disinfecting time is 24h.Treat that in soil, formaldehyde infiltration sterilization fully, is removed plastics film, with remaining formaldehyde evaporation, in order to avoid affect the transplanting success of seedling.Through the restorative growth of month, applying bacterial strain concentration at the Horsetail Beefwood rhizosphere was 9.0 * 10
5The bacterium liquid 100ml of cfu/ml repeats 3 times, processes as blank with distilled water solution.
The preparation of bacterium liquid: with Horsetail Beefwood Flora of Rhizosphere Fungi 7 access liquid nutrient mediums, substratum is improvement Martin substratum, every L contains peptone 5.0g, yeast extract powder 2.0g, glucose 20.0g, dipotassium hydrogen phosphate 1.0g, sal epsom 0.5g, and other is sterilized water, pH value 6.4 ± 0.2.Through the cultivation of 24h, utilize blood counting chamber to calculate bacterial concentration, bacterium liquid is diluted to 1.0 * 10 with ultrapure water
6Cfu/ml to 9.0 * 10
6Cfu/ml is standby.Inoculate the mensuration of carrying out the indexs such as photosynthesis after rear 30 days, detection method and result are as follows:
2.1 watering, bacterial strain executes the analysis of rear Horsetail Beefwood chlorophyll fluorescence parameters
The Handy Fluor Cam fluorescence imaging instrument that application Photon Systems Instruments. (PSI) company produces is in the chlorophyll fluorescence parameters of 19:00-22:00 mensuration Horsetail Beefwood nursery stock at night, first nursery stock is put into camera bellows before mensuration, abundant dark reaction 20min, subsequently nursery stock is put under camera, focuses till can see leaf image clearly.
The initial parameter of measuring has: Fo (minimum, basis or constant fluorescence intensity etc.) is that all electronics of lightsystemⅡ (PS II) reactive center all are in the fluorescence quantum yield when opening fully, and it is relevant with the chlorophyll concentration of blade; After Fm (maximum fluorescence) was vanes dark adatpation 20min, lightsystemⅡ (PS II) reactive center was in the fluorescence quantum yield when closing fully, can reflect that the electronics of PS II transmits situation; Fo ': (minimum fluorescence under light) is the fluorescence intensity when under the light adaptation state, all PS II center is all opened; Fm ' (maximum fluorescence under light) is the fluorescence intensity when under the light adaptation state, all PS II center is all closed.
Other kinetic parameter:
Original fluorescence parameter (Fm, Fv) and other kinetics chlorophyll fluorescence parameters (Fv/Fm, Φ PSII, Rfd and NPQ) to each nursery stock under different treatment are carried out one-way analysis of variance, to check the significance of its variation, multiple comparisons adopts the Duncan method, and all data processing and chart production all adopt Excel and SPSS software processes.
Rooted Cuttings Fm, the Fv of Different treatments and Fv/Fm value
In the middle of fluorometric analysis, Fm and Fv are all reflecting the photochemical activity of lightsystemⅡ.Maximum fluorescence (being Fm) is that lightsystemⅡ (being the PS II) reactive center is in the fluorescence quantum yield under buttoned-up status, it both can reflect that the electronics of lightsystemⅡ transmits situation, also can reflect only no situation about being suppressed (when Fm reduces); Variable fluorescence (being Fv) is poor for Fm and Fo's, is reflecting electron acceptor(EA)---the reduction situation of elementary quinone acceptor (being QA) of lightsystemⅡ, the research discovery, and the intensity of variation of Fv value is reflecting the ability of restraining oneself that plant is coerced surrounding environment
[16]
Fm from Fig. 1 can find out, Horsetail Beefwood Flora of Rhizosphere Fungi 7 is processed lower nursery stock, its Fm value compared with the control, difference reaches conspicuous level, contrasts and has improved 39.57%.
Fv from Fig. 1 can find out, under 7 effects of Horsetail Beefwood Flora of Rhizosphere Fungi, the Fv value of nursery stock is significantly higher than adjoining tree, has improved respectively 42.83%.
In fluorescence parameter, the actual light chemical efficiency of PS II (being Fv/Fm) is the important parameter of research plant photosynthesis physiological status, the potential photosynthetic capacity that is reflecting plant, it simultaneously is also the important parameter of research plant stress, under non-envrionment conditions of coercing, the species of plant and growth conditions are little on its impact, this parameter value remains between 0.75 and 0.85, in case be subject to extraneous coercion, Fv/Fm presents obvious downtrending, and the reducing of its value means the photochemistry infringement of lightsystemⅡ reactive center and the increase of luminous energy heat dissipation
[17]
Fig. 2 has reflected that intuitively different inoculation methods are on the impact of Horsetail Beefwood nursery stock Fv/Fm.The Fv/Fm value of Horsetail Beefwood Flora of Rhizosphere Fungi 7, CK compares with contrast, has increased by 2.32%.Illustrated that connecing the bacterium processing is conducive to improve the potential photosynthetic capacity of nursery stock to a certain extent, strengthens its adaptive faculty of condition to external world.
The value of Different treatments Rooted Cuttings Φ PSII, Rfd and NPQ
Φ PSII can accurately reflect the actual photosynthesis efficient situation of the current PSII reactive center of plant, and its value is larger, and photosynthetic structures electronics transmission capacity is stronger, participates in photochemically reactive luminous energy share larger, more is conducive to improve the photosynthetic capacity of plant
[18]Respectively connect under the bacterium processing, the Φ PSII of Horsetail Beefwood Flora of Rhizosphere Fungi 7 contrasts and has increased by 31.52%;
The share that the luminous energy that non-photochemical quenching coefficient (NPQ) absorbs for PS II antenna pigment dissipates with the form of heat, its value is larger, represent that plant is used for the luminous energy of heat dissipation more, the corresponding minimizing of actual luminous energy of photosynthesis of plant, thus reduce the photosynthetic efficiency of plant
[17-18]This test-results shows, processes by the tieback of Horsetail Beefwood Flora of Rhizosphere Fungi 7, and the NPQ value that connects vaccine wood contrasts to compare and has a declining tendency, and has reduced 22.1%.
The eigenwert of Horsetail Beefwood Rooted Cuttings chlorophyll fluorescence parameters under table 1 P inoculation method different treatment
* in the same columns value in table, there are different lowercase persons to represent significant difference (P＜0.05)
The present invention finds that a strain can promote the photosynthetic endogenetic fungus of Horsetail Beefwood, this strain Horsetail Beefwood endogenetic fungal bacterial strain is adopted dip in root or bacterium liquid and water the method for executing and be inoculated in Casuarina equisetifolia Seedling.By inoculating the chlorophyll fluorescence test of rear plant, draw the photosynthesis ability of bacterial strain all largely higher than contrast, show that it has very large function for the photosynthesis usefulness that promotes plant, thereby confirm that further obtaining this strain endogenetic fungus can promote Horsetail Beefwood photosynthesis.
Reference
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Claims (3)
1. a strain promotes the photosynthetic endogenetic fungus of Horsetail Beefwood, it is characterized in that: described endogenetic fungus be aspergillus (
Aspergillus sp.) Horsetail Beefwood Flora of Rhizosphere Fungi 7, on June 28th, 2012 in the registration preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No. 6305.
2. the application bacterium liquid of an endogenetic fungus as claimed in claim 1 is characterized in that: the preparation method of described application bacterium liquid, be with described aspergillus (
Aspergillus sp.) Horsetail Beefwood Flora of Rhizosphere Fungi 7 access liquid nutrient mediums, shaking table shaking culture, culture temperature are 28 ℃, incubation time 48～72h utilizes blood counting chamber to calculate bacterial concentration, and bacterium liquid is diluted to 1.0-9.0 * 10 with ultrapure water
6Cfu/ml, 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, sal epsom 0.5g/L, other is sterilized water, pH value 6.4 ± 0.2.
3. the purposes of an application as claimed in claim 2 bacterium liquid, it is characterized in that: described applications bacterium liquid waters root for the forest land or transplanted seedling tree dips in root in earlier stage.
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| CN111004727A (en) * | 2019-12-25 | 2020-04-14 | 福建农林大学 | Endophytic fungus Z1 for increasing biomass of casuarina equisetifolia in high-salt environment |
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| CN101516805A (en) * | 2006-07-25 | 2009-08-26 | 发育研究院 | Novel compositions of fungal inocula, method for the preparation thereof and use thereof for improving the growth of cultures |
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| CN111004727A (en) * | 2019-12-25 | 2020-04-14 | 福建农林大学 | Endophytic fungus Z1 for increasing biomass of casuarina equisetifolia in high-salt environment |
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