CN106676040B - A kind of ash band chain mould and its application and microbial bacterial agent - Google Patents
A kind of ash band chain mould and its application and microbial bacterial agent Download PDFInfo
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Abstract
The present invention relates to microorganisms technical field, in particular to a kind of grey band chain mould and its application and microbial bacterial agent.The deposit number of the ash band chain mould is CGMCC No.13249.The research of the invention finds that the ash band chain mould has the ability of preferable Soluble phosphorus characteristic, stronger synthesis siderophore, it can inhibit a variety of disease fungus activity for causing plant disease.Ash band chain mould of the invention has plant disease-resistant, growth-promoting potential, and the Soluble phosphorus plant growth-promoting unwrapping wire bacteria agent to research and develop environmentally friendly provides good material.
Description
Technical field
The present invention relates to microorganisms technical field, in particular to a kind of grey band chain mould and its application and microbial bacterial agent.
Background technique
The increasingly depleted worldwide problem for having become limitation agricultural sustainable development of phosphate rock resource.Phosphorus is plant growth
Essential elements in order to improve crop yield, during great amount of soluble phosphate fertilizer is manured into soil, but is mostly consolidated by soil in production practice
It is fixed, exist in the form of slightly solubility Phos, can not be absorbed by plants and utilize.Therefore, the activation of soil inavailable phosphorus element becomes
Resource science and fertilizer science major issue urgently to be solved.
Phosphorus-solubilizing bacteria can dissolve insoluble phosphate, activate phosphorus element state.It " can not only mobilize " agricultural land soil phosphorus library
Resource promotes the raising of soil fertility, reduces the usage amount of chemical phosphatic ferfilizer and the consumption of high-grade phosphate rock resource, solves soil
Invalid phosphorus element activation and phosphate rock resource increasingly exhausted problem；Can with effective exploitation using a large amount of low-grade phosphate ore in China and
Tailing improves phosphate fertilizer utilization efficiency, solves the problem of environmental pollution generated in phosphorus ore exploitation and process.Therefore, biologic phosphorus fertilizer
Development and application be the optimal path for solving the problems, such as " phosphorus ".
Currently, there are many research achievement in relation to phosphorus-solubilizing bacteria in research both at home and abroad, but it is true in phosphorus bacteria fertilizer and Soluble phosphorus to focus mostly on
The classification of bacterium and the research field of Soluble phosphorus mechanism, such as disclosure/notification number provide one kind for the patent of invention of CN103834584A
Phosphorus-solubilizing bacteria is named as DDC 95, can be used as phosphorus-solubilizing bacteria auxiliary vegetation.The strain isolation is from soybean rhizosphere, to promotion soybean
It is obvious that effect is absorbed and utilized to the inorganic phosphorus element of indissoluble in soil.For another example disclose/notification number be CN102618449A invention mention
A kind of phosphorus-solubilizing bacteria is supplied, which is smelly aspergillus Aspergillus foetidus, and preservation registration number is
CGMCC No.5857.The phosphorus-solubilizing bacteria is used for the cultivation of tobacco leaf for producing microbial manure, passes through the functions such as Soluble phosphorus, potassium decomposing
The direct effect of microorganism discharges in soil and is not easy absorbed nutrient, improves soil quality, promotes in soil beneficial to micro-
The breeding of biology improves beneficial microorganism quantity in soil, the nutrient balance in soil is adjusted, to improve cigarette strain pair
The Balance Absorption of mineral nutrition improves quality of tobacco and utilization rate of fertilizer, and reduces environmental pollution.
Actinomyces because of its impoverishment tolerant, extreme environment survival ability is strong, produces the multiple function such as various active substance and disease-resistant growth-promoting
It imitates and is concerned, but is very few to the research of Soluble phosphorus actinomycetes population and Soluble phosphorus mechanism both at home and abroad, limit Soluble phosphorus actinomyces
Using.
Summary of the invention
In view of this, the present invention provides a kind of grey band chain mould and its application and microbial bacterial agents.The actinomyces have
Preferable Soluble phosphorus characteristic.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of grey band chain mould, deposit number is CGMCC No.13249.
The present invention is screened from the rhizospheric environment of this specified plant of Rhizosphere of Crops soil has plant disease-resistant, growth-promoting potential
Grey band chain mould, with obtain it is good with plant compatibility, be easy to the Soluble phosphorus plant growth-promoting actinomycetes strain colonized in plant rhizosphere,
Soluble phosphorus plant growth-promoting unwrapping wire bacteria agent to research and develop environmentally friendly provides good material.
In embodiment provided by the invention, deposit number is the screening technique of the grey band chain mould of CGMCC No.13249
Are as follows: the fresh soil sample of the Rhizosphere of Crops of acquisition is adopted and is diluted with water, dilution is coated on NBRIP culture medium, is cultivated, is pure
Change, obtains grey band chain mould.
In the present invention, the formula of NBRIP culture medium are as follows: glucose 10g, Ca3(PO4)2 5g、MgCl2 5g、MgSO4
0.25g、KCl 0.2g、(NH4)2SO40.1g is dissolved in 1L deionized water, adjusts pH7.2, sterilizing.20g is added in the culture medium
Agar is solid NBRIP culture medium.
In embodiment provided by the invention, the temperature that when screening cultivates is 28 DEG C.
In embodiment provided by the invention, the time that when screening cultivates is 48h.
Preferably, the ash band chain mould carries out the culture of fermentation seed using ISP4 culture medium.
In the present invention, ISP4 Liquid Culture based formulas: soluble starch 10g, K2HPO41g, MgSO4·7H2O 1g,
(NH4)2SO42g, CaCO32g is dissolved in 1L water, adjusts pH7.2, sterilizing.
Preferably, the cultural method of grey band chain mold fermentation seed are as follows: by mature bacterial strain and spore inoculating in ISP4 liquid
120r/m in body culture medium, 28 DEG C of 5~7d of culture.
In the present invention, which carries out fermented and cultured using solid fermentation culture medium.
In the present invention, the formula of solid fermentation culture medium are as follows: corn quarrel 550g, rice 400g, millet 50g, CaCO3
10g, it is 60% that the Gause I fluid nutrient medium being concentrated with 2 times, which adjusts moisture content, sterilizing.
Preferably, the solid fermentation cultural method of grey band chain mould are as follows: by grey band chain mould liquid fermentation strain inoculated
In solid fermentation culture medium, 28 DEG C of 5~7d of culture.
Preferably, the storage method of the ash band chain mould are as follows: the bacterium is placed preservation in a cool and dry place or 0~4 DEG C
Under the conditions of save.
The present invention also provides the ash band chain moulds to dissolve the application in invalid phosphorus.
The present invention also provides application of the ash band chain mould in synthesis siderophore.
The present invention also provides application of the ash band chain mould in the disease fungus activity for inhibiting to cause plant disease.
In embodiment provided by the invention, causing the disease fungus of plant disease is Fusarium graminearum, cucumber fusarium axysporum
Bacterium, ginseng hyphal cluster germ, ginseng pine root fungus and Ginseng Blight bacterium.
The present invention also provides a kind of microbial bacterial agents, including grey band chain mould of the invention.
Preferably, further including acceptable auxiliary material in Pesticide Science in microbial bacterial agent.
The present invention provides a kind of grey band chain mould and its application and microbial bacterial agents.The deposit number of the ash band chain mould
For CGMCC No.13249.The present invention at least has one of following advantage:
1, the research of the invention finds that the ash band chain mould has preferable Soluble phosphorus characteristic；
2, the research of the invention finds that the ash band chain mould has the ability of stronger synthesis siderophore；
3, the research of the invention finds that the ash band chain mould can inhibit a variety of disease fungus activity for causing plant disease.
4, the grey band chain mould of the present invention has plant disease-resistant, growth-promoting potential, plants to research and develop environmentally friendly Soluble phosphorus
Object growth-promoting unwrapping wire bacteria agent provides good material.
Biological deposits explanation
Classification naming: grey band chain mould (Streptomyces griseoplanus) was deposited on November 08th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), collection address are Chaoyang District, Beijing City
The institute 3 of North Star West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.13249.
Detailed description of the invention
Fig. 1 shows growth of the bacterial strain PSA1 on NBRIP culture medium；
Fig. 2 shows that bacterial strain PSA1 phosphate solubilization ability measures；
Fig. 3 shows cultural characteristic of the bacterial strain PSA1 in different culture medium；
Fig. 4 shows the building of bacterial strain PSA1 phylogenetic tree；
Fig. 5 shows that grey band chain mould PSA1 produces siderophore situation；
Fig. 6 shows grey band chain mould PSA1 to the inhibiting effect of Fusarium graminearum (Fusarium graminearum)；Wherein
Culture dish shown in 6-1 is added with grey band chain mould PSA1, and 6-2 is control；
Fig. 7 shows grey band chain mould PSA1 to cucumber fusarium axysporum (Fusarium oxysporium
F.sp.cucumerinum inhibiting effect)；Wherein culture dish shown in 7-1 is added with grey band chain mould PSA1, and 7-2 is control；
Fig. 8 shows inhibition of the grey band chain mould PSA1 to ginseng hyphal cluster germ (Sclerotinia libertiana Fuck.)
Effect；Wherein culture dish shown in 8-1 is added with grey band chain mould PSA1, and 8-2 is control；
Fig. 9 shows grey band chain mould PSA1 to the inhibiting effect of ginseng pine root fungus (Fusarium solani)；Wherein 9-1
Shown culture dish is added with grey band chain mould PSA1, and 9-2 is control；
Figure 10 shows grey band chain mould PSA1 to the inhibiting effect of Ginseng Blight bacterium (Fusarium oxysporum)；Wherein
Culture dish shown in 10-1 is added with grey band chain mould PSA1, and 10-2 is control.
Specific embodiment
The invention discloses a kind of grey band chain moulds and its application and microbial bacterial agent, those skilled in the art to use for reference
Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.Method of the invention and application by compared with
Good embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to as described herein
Methods and applications are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Used medium, the city reagent Deng Junkeyou in ash band chain mould provided by the invention and its application and microbial bacterial agent
Field is bought.
In embodiments of the present invention, the formula of NBRIP culture medium are as follows: glucose 10g, Ca3(PO4)2 5g、MgCl2 5g、
MgSO4 0.25g、KCl 0.2g、(NH4)2SO40.1g is dissolved in 1L deionized water, adjusts pH7.2,121.3 DEG C of high steams
Sterilize 20min.It is solid NBRIP culture medium that 20g agar, which is added, in the culture medium.
ISP4 Liquid Culture based formulas: soluble starch 10g, K2HPO41g, MgSO4·7H2O 1g, (NH4)2SO42g,
CaCO32g is dissolved in 1L water, adjusts pH7.2,121.3 DEG C of high pressure steam sterilization 20min.Bacterial strain mature in plate and spore are connect
Kind 120r/m, 28 DEG C of 5~7d of culture in liquid fermentation medium.
Solid fermentation culture medium: rice 400g, millet 50g, CaCO310g is added, the height being concentrated with 2 times in corn quarrel 550g
It is 60% that family name's No.1 fluid nutrient medium, which adjusts moisture content, high pressure steam sterilization 2 hours under the conditions of 110 DEG C.
Below with reference to embodiment, the present invention is further explained:
The screening of 1 Soluble phosphorus actinomyces of embodiment
It weighs 10g to be added in 100mL sterile water from the Rhizosphere of Crops fresh soil sample that Gongzhuling acquires, using plate dilution method
Soil dilution liquid is prepared, is applied on NBRIP culture medium, 28 DEG C of culture 48h, the good bacterium colony of picking growth conditions carries out pure
Change.Bacterial strain after purification is saved backup respectively at 4 DEG C and -80 DEG C.The selection result of Soluble phosphorus actinomyces is as shown in Figure 1.Name
For PSA1 bacterial strain.
The measurement of 2 phosphate solubilization ability of embodiment
The Rhizosphere of Crops actinomyces of the screening of embodiment 1 after purification are inoculated in liquid Gause I culture medium and carry out expansion training
7d is supported, 8000rpm is centrifuged 5min and collects thallus, and 8000rpm is centrifuged 5min after bacterial sediment is resuspended with deionized water, this operation weight
It is 3 times multiple, the Gause I medium component of phage surface is cleaned, sterile working weighs 0.5g thallus (weight in wet base) and is inoculated in
100mL NBRIP fluid nutrient medium (NBRIP culture medium: glucose 10g, calcium phosphate 5g, magnesium chloride 5g, magnesium sulfate 0.25g, sulphur
Sour ammonium 0.1g, potassium chloride 0.2g, distilled water 1000mL, pH 7.0~8.0) in, 28 DEG C of shaking table culture 20d, using the anti-ratio of molybdenum antimony
Color method measures available phosphorus content in different incubation time fermented liquid supernatants, carries out phosphate solubilization ability survey to Rhizosphere of Crops Soluble phosphorus actinomyces
It is fixed.Not connect the culture medium of bacterium as control.
PSA1 is inoculated in liquid NBRIP culture medium, the available phosphorus content in fermented liquid supernatant is measured, as a result such as Fig. 2
It is shown.
The identification of 3 Rhizosphere of Crops Soluble phosphorus actinomyces of embodiment
1, the cultural characteristic of Rhizosphere of Crops Soluble phosphorus actinomyces and physio-biochemical characteristics identification
1 Rhizosphere of Crops Soluble phosphorus actinomyces mycelium morphology of embodiment is observed using inserted sheet method, and referring to the side of Fan Lixia etc.
Soluble phosphorus actinomycetes strain is inoculated into the training of yeast extract malt extract agar using international streptomycete plan culture medium (ISP) by method respectively
Support base (ISP2), oatmeal agar medium (ISP3), inorganic salts starch agar medium (ISP4), glycerol asparagine fine jade
Rouge culture medium (ISP5), peptone yeast extract molysite agar medium (ISP6), tyrosine agar culture medium (ISP7), Cha Shi training
It supports on base and PDA culture medium, 28 DEG C of 5~10d of culture observe and record cultural characteristic of the bacterial strain in 7 kinds of different culture mediums.Ginseng
The physiological and biochemical property identification of Rhizosphere of Crops Soluble phosphorus actinomyces is carried out according to the method for Xu Lihua etc..
Physiology and biochemistry qualification result is as shown in Figure 3:
PSA1 grows vigorous on Gause I culture medium, and bacterium colony is creamy, and luxuriant gas silk is in suede powdery, fibrillae of spores
Directly, flexible, the spacious spiral shape of pine, base silk is good, light yellow to light brown；
PSA1 does not generate soluble pigment on ISP serial culture base and Czapek's medium and PDA culture medium.ISP2 training
Support base: aerial hyphae is luxuriant, and grey to dark gray, substrate mycelium is good, ecru to grey yellowish-brown.ISP3: aerial hyphae is rich
Cyclopentadienyl, grey, substrate mycelium is good, ecru.ISP4: aerial hyphae growth is general, and grey to olive-gray, substrate mycelium is good,
Ecru is to olive-gray.ISP5: aerial hyphae growth is general, grey, substrate mycelium grey.ISP6: aerial hyphae light gray
Yellow, substrate mycelium ecru.ISP7: aerial hyphae olive-gray, substrate mycelium dark gray.
Czapek's medium: aerial hyphae is luxuriant, and for white to light gray, substrate mycelium is good, and ecru is to light brown.PSA1
Bacterial strain gelatin liquefaction, Starch Hydrolysis, nitrate reduction, cellulose hydrolysis, but hydrogen sulfide, melanin are not generated.
PDA culture medium: aerial hyphae is luxuriant, and light grey, substrate mycelium is good, ecru.
By above-mentioned test result as it can be seen that the bacterial strain is preferable using glucose, sucrose, glycerol, sodium citrate effect, to cotton seed
Sugar, inositol, lactose effect are general, and L- rhamnose effect is worst.
2, the Molecular Identification of Rhizosphere of Crops Soluble phosphorus actinomyces
Rhizosphere of Crops actinomyces with Soluble phosphorus characteristic are inoculated in Gause I fluid nutrient medium, 28 DEG C of shaking table cultures
7d vacuumizes freeze-drying 48 hours after thalline were collected by centrifugation.Thallus after freeze-drying extracts genomic DNA.Reference
The method of Hanane Hamdali et (2008) carries out 16S rDNA amplification.Amplified production serves marine growth engineering services
Co., Ltd's sequencing, sequencing result is compared with 16S rRNA sequence relevant in GenBank with Clustal W, passes through
MEGA6.0 software carries out Phylogenetic Analysis to bacterial strain, constructs systematic evolution tree using adjacent method (neighbor-joining),
It is examined with Bootstrap method (1000 repetitions).
16S rDNA Molecular Identification result: using bacterial strain PSA1 total DNA as template, the 16S rDNA of PSA1 bacterial strain is carried out
PCR amplification is simultaneously sequenced.Sequencing result is submitted to NCBI GenBank (KX226414).With Clustal W to sequencing result with
Relevant 16S rRNA sequence is compared in GenBank, carries out Phylogenetic Analysis to bacterial strain by MEGA6.0 software, adopts
Systematic evolution tree is constructed with adjacent method (neighbor-joining), is examined with Bootstrap method (1000 repetitions).As a result such as
Fig. 4 shows that bacterial strain PSA1 and reported grey band chain mould Streptomyces griseoplanus affiliation are nearest, together
Source property is consistent with Blast result up to 99%.Therefore, bacterial strain PSA1 is primarily determined on taxonomy as grey band chain mould
Streptomyces griseoplanus strain PSA1。
Embodiment 4 produces the active qualitative analysis of siderophore
Rhizosphere of Crops actinomyces with Soluble phosphorus function are inoculated in CAS solid medium, and (CAS detects culture medium
(100mL): 20% glucose 1mL, 10% acid hydrolyzed casein 3mL, 1mmol/L CaCl2100 μ L, 1mmol/L MgSO4·
7H2O 2mL, agar powder 1.8g, 115 DEG C of sterilizings are slowly added to 10 × phosphate buffer of filtration sterilization when being cooled to 60 DEG C
It is mixed with each 5mL of CAS detection liquid.CAS detects liquid: 2mL 1mmol/L FeCl3It is added and is dissolved in containing 0.012g chromazurine (CAS)
In 10mL deionized water, it is poured slowly into the 8mL containing 0.015g cetyl trimethylammonium bromide (CTAB) after being sufficiently mixed and goes
In ionized water, filtration sterilization is kept in dark place.10 × phosphate buffer (pH6.8): every 100mL buffer contains Na2HPO4·
2H2O 2.427g, NaH2PO4·2H2O 0.5905g；KH2PO4·3H2O 0.075g；NHCl40.25g；NaCl 0.125g,
Adjust pH6.8.Used time dilutes 10 times.By PSA1 strain inoculated in CAS solid culture primary surface, 28 DEG C of culture 15d observe bacterium colony
Whether surrounding has transparent circle.
As a result as shown in Figure 5: after Rhizosphere of Crops Soluble phosphorus actinomyces PSA1 is inoculated in CAS solid plate culture 15d, at it
There is apparent orange red color ring in periphery of bacterial colonies, illustrates that Rhizosphere of Crops Soluble phosphorus actinomyces PSA1 has the work of synthesis siderophore
Property.
The screening of 5 Rhizosphere of Crops soil Soluble phosphorus actinomyces antagonistic activity of embodiment
Phytopathogen strain and source: ginseng miliary damping-off germ (Rhizoctonia solani Kuhn), ginseng bacterium
Core germ (Sclerotinia libertiana Fuck.), Ginseng Blight bacterium (Fusarium oxysporum), ginseng
Maize ear rot bacterium (Fusarium solani), Fusarium graminearum (Fusarium graminearum), cucumber fusarium axysporum
(Fusarium oxysporium f.sp.cucumerinum), Fusarium moniliforme (Fusarium moniliforme), corn
Curved spore leaf spot fungi (Curvularia lunata), botrytis cinerea (Botrytis cirerea).The above bacterial strain is by Ji
Self-employed tree cultivator sparetime university learn to farm institute's plant pathology laboratory offer.
Cause the disease fungus of plant disease as indicator bacteria using above-mentioned 9 kinds, using opposite culture method, screening has antagonism living
The Soluble phosphorus actinomyces of property.It is inoculated with indicator bacteria in PDA culture medium surface one end, the other end is inoculated with Rhizosphere of Crops Soluble phosphorus actinomyces, often
Group test is repeated 3 times, and culture terminates to cultivate when covering with entire culture dish to pathogen, determines the short of money of actinomyces with antibacterial bandwidth
Resistant activity.It is compareed with not connecing the plate of actinomyces.As a result as shown in Fig. 6~10.
The results show that embodiment 1, which screens obtained Rhizosphere of Crops Soluble phosphorus actinomyces, can obviously inhibit Fusarium graminearum
(Fusarium graminearum), cucumber fusarium axysporum (Fusarium oxysporium f.sp.cucumerinum), people
Join hyphal cluster germ (Sclerotinia libertiana Fuck.), ginseng pine root fungus (Fusarium solani) and ginseng
The growth of wilt (Fusarium oxysporum) shows that the bacterium has the activity of antagonistic phytopathogen.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (4)
1. a kind of application of ash band chain mould (Streptomyces griseoplanus) in the invalid phosphorus of dissolution, feature exist
In deposit number is CGMCC No.13249.
2. a kind of application of ash band chain mould (Streptomyces griseoplanus) in synthesis siderophore, feature exist
In deposit number is CGMCC No.13249.
3. a kind of ash band chain mould (Streptomyces griseoplanus) is inhibiting the disease fungus for causing plant disease living
Property in application, which is characterized in that its deposit number be CGMCC No.13249.
4. application according to claim 3, which is characterized in that the disease fungus for causing plant disease is F.graminearum schw
Bacterium, cucumber fusarium axysporum, ginseng hyphal cluster germ, ginseng pine root fungus and Ginseng Blight bacterium.
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