CN106497838A - A kind of composite bacteria agent capable of Herba Dendrobii - Google Patents
A kind of composite bacteria agent capable of Herba Dendrobii Download PDFInfo
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- CN106497838A CN106497838A CN201610962563.8A CN201610962563A CN106497838A CN 106497838 A CN106497838 A CN 106497838A CN 201610962563 A CN201610962563 A CN 201610962563A CN 106497838 A CN106497838 A CN 106497838A
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Abstract
The invention belongs to bio-bacterial manure technical field, specifically related to a kind of composite bacteria agent capable of Herba Dendrobii, isolate and purify including actinomycetic in root, the screening of Antagonistic Actinomycetes, the actinomycetes screening of growth-promoting mycorrhizal fungi, growth-promoting actinomycetes kind status determines, optimizes mycorrhizal fungi and cooperate with actinomycetes to promote the composite bacteria agent capable of Herba Dendrobii Cloned seedling growth.The present invention cooperates with the composite bacteria agent capable that actinomycetes obtain in its root using Herba Dendrobii mycorrhizal fungi; Herba Dendrobii cell can be stimulated to produce efficient defence capability; strengthen Herba Dendrobii itself reparation, disease resistance, opposing extreme environment and improve its asexual shoot survival percent ability; promote the growth of Herba Dendrobii Cloned seedling, technical support is provided for large-scale production high-quality seedlings of Dendrobium officinale.
Description
Technical field
The invention belongs to bio-bacterial manure technical field, and in particular to a kind of composite bacteria agent capable of Herba Dendrobii.
Background technology
Herba Dendrobii is the perennial Epiphyte of orchid family Dendrobium, applies wide in terms of the research and development of field of medicaments
General.Modern medicine study shows that Herba Dendrobii has grows lung heat clearing away, tonifying-Yin and nourishing-stomach, liver heat removing and eyesight improving, opposing cancer, reduction blood glucose
Deng medicinal efficacy.Used as the superfine product of health care, Herba Dendrobii is praised highly by successive dynasties physician.
At present as the natural propagation rate of Herba Dendrobii is low, relatively strictly producing region medicinal herb grower excavate for a long time is required to environmental condition
Cause natural resourcess endangered etc. factor.And Herba Dendrobii is small because of seed, without endosperm, therefore under natural conditions
It is unfavorable for obtaining high-volume Herba Dendrobii seedling.Currently, Herba Dendrobii tissue culturing system is successfully established, can amount reproduction Herba Dendrobii without
Property Seedling, but Cloned seedling transplanting survival rate is low, poor growth, long growth cycle the problems such as.
Content of the invention
Above-mentioned technical problem is solved, the present invention proposes a kind of composite bacteria agent capable of Herba Dendrobii, using Herba Dendrobii mycorhiza
Funguses promote its Cloned seedling growth, i.e. actinomycetes Lance base streptomycete with actinomycetes synergism in root（Streptomyces krainskii）Belong to mycorrhizal fungi Epulorhiza（Epulorhizasp.）And Tulasnella（Tulasnellasp.）Collaboration
Promote the growth of Herba Dendrobii Cloned seedling.
Its technical scheme is：
A kind of composite bacteria agent capable of Herba Dendrobii,
（1）Actinomycetic in root isolate and purify：
A. healthy fresh Herba Dendrobii nutrition root system is chosen, root sample is bound up with gauze and is rinsed well through the tap water that flows；
B. sterilization 20s is placed in 75% ethanol, aseptic water washing 2-3 time, then with 0.1% mercuric chloride solution sterilization 20s, aseptic water washing
4-5 time, root sample surface moisture is blotted using sterilizing filter paper；
C. blank control test is set, i.e., is placed in Gause I culture medium with the root sample that has sterilized and is rolled for several times；
D. surface has been sterilized root sample, has been placed in superclean bench and grinds to form homogenate, microwave treatment 30S has been diluted after mixing respectively
Into 10-1、10-2、10-3Root sample diluent, is inoculated in Gause I, tri- kinds of culture medium flat plates of TWYE, YIM38# respectively, is placed in
Culture 7-15 days is inverted in 28 DEG C of constant incubators；
If E. control medium is aseptic being born into, show that root sample surface sterilization is qualified, the actinomycetes bacterium colony that isolates chosen,
Repeatedly purification, obtains actinomycetes strain, is placed in 4 DEG C of refrigerators and saves backup；
（2）The screening of Antagonistic Actinomycetes：
A. using flat board face-off method, fusarium will be gone out for trying Herba Dendrobii pathogenic fungi Alternaria tenuissima, Fusarium lateritiumNees bacterium, layer
Bacterium, the pathogen of Botrytis cinerea, Sclerotium rolfsii are inoculated in PDA culture medium, 28 DEG C of constant temperature culture 5 days, beat the pathogen for taking diameter 5mm
Truffle is inoculated in Gause I culture dish central authorities, and in the same horizontal line, with pathogen with equidistant position both sides, inoculation is same
Etc. the actinomycetes truffle of size, each process 3 repeated experiments；
B. pathogenic fungi is only inoculated with culture dish central authorities, both sides are not inoculated with actinomycetes for control, and each process is repeated 3 times, and it is right to treat
Culture dish is covered with according to pathogenic fungi, suppression ratio of the actinomycetes to 5 kinds of pathogenic fungi is determined, is screened to pathogenic fungi with substantially short of money
The actinomycetes strain of anti-effect；
（3）The actinomycetes screening of growth-promoting mycorrhizal fungi：
A. by the above-mentioned growth-promoting actinomycetes for filtering out respectively with two kinds for examination Herba Dendrobii growth-promoting mycorrhizal fungi Epulorhizas category and
Tulasnella, carries out flat board dual test in Gause I culture medium, each process 3 repeated experiments；
B. with a culture dish central authorities Arbuscular Mycorrhizal Fungi fungus as control, each process is equally repeated 3 times, and mycorrhizal fungi to be compareed is covered with
Culture dish, determines actinomycetes to mycorrhizal fungi suppression ratio, filters out, to Herba Dendrobii mycorrhizal fungi, there are obvious growth-promoting functions
Actinomycetes；
（4）Growth-promoting actinomycetes kind status determines：
The above-mentioned actinomycetic form micro-structural feature for filtering out is observed using streaking inoculation, inserted sheet method, using 16S
RDNA PCR Amplification Analysis, identify that growth-promoting actinomycetes are this base streptomycete of Crane；
（5）Optimize the composite bacteria agent capable that mycorrhizal fungi collaboration actinomycetes promote the growth of Herba Dendrobii Cloned seedling：
A. the Herba Dendrobii Cloned seedling of 4 months seedling ages is chosen, is inoculated in Harvaris culture medium, cultivated 15 days；
B. by growth-promoting actinomycetes Crane, this base streptomycete belongs to and glued membrane bacterium with examination Herba Dendrobii growth-promoting mycorrhizal fungi Epulorhiza is supplied
Belong to according to 1:1:1 ratio is seeded in free of contamination healthy Cloned seedling carries out symbiosis culture, cultivates 60 days.
The present invention cooperates with the composite bacteria agent capable that actinomycetes obtain in its root using Herba Dendrobii mycorrhizal fungi, can stimulate iron sheet
Herba Dendrobii cell produces efficient defence capability, strengthens Herba Dendrobii itself reparation, disease resistance, opposing extreme environment and improves its nothing
Property shoot survival percent ability, promote Herba Dendrobii Cloned seedling growth, provide technology for large-scale production high-quality seedlings of Dendrobium officinale
Support.
Specific embodiment
1st, actinomycetic in root isolate and purify
（1）Healthy fresh Herba Dendrobii nutrition root system is chosen, root sample is bound up with gauze and is rinsed well through the tap water that flows.
（2）It is placed in 75% ethanol sterilization 20s, aseptic water washing 2-3 time, then with 0.1% mercuric chloride solution sterilization 20s, aseptic
Water is rinsed 4-5 time, is blotted root sample surface moisture using sterilizing filter paper.
（3）Blank control test is set, i.e., is placed in Gause I culture medium with the root sample that has sterilized and is rolled for several times.
（4）Surface is sterilized root sample, be placed in superclean bench and grind to form homogenate, microwave treatment 30S.Because detached
It is endogeny rayungus, and the more difficult separation of root system actinomycetes, so must be fully ground, to increase the separation kind of endogeny rayungus
Class；Appropriate microwave treatment may promote the sprouting of actinospore, make the spore of part hardly possible separating payingoff bacteria receive one
Sprout after quantitative microwave radiation, be converted into educable actinomycetes, increase can cultivate actinomycetic type and quantity.
（5）10 are diluted to after mixing respectively-1、10-2、10-3Root sample diluent, be inoculated in respectively Gause I, TWYE,
In tri- kinds of culture medium flat plates of YIM38#.Gradient dilution exactly in order to reduce concentration, reduces separating difficulty.Three kinds of culture medium are general all
It is separating payingoff bacteria, and the actinomycetes flora that every kind of culture medium is directed to is slightly different, therefore the combination of three kinds of culture medium
Increase the species of separating payingoff bacteria using being exactly.
（6）It is placed in 28 DEG C of constant incubators and is inverted culture 7-15 days, if control medium is aseptic being born into, shows root
Sample surface sterilization is qualified.
（7）The actinomycetes bacterium colony that isolates is chosen, through repeatedly after purification, isolating and obtaining 12 plants of actinomycetes strains,
It is placed in 4 DEG C of refrigerators and saves backup.
2nd, the screening of Antagonistic Actinomycetes
（1）Using flat board face-off method, the toVerticilliumdahliaActivitie Activitie S of Relative of examination Herba Dendrobii pathogenic fungi melasma will be suppliedAlternaria tenuissima, stem rot Fusarium lateritiumNees bacteriumFusarium incarnatumAnd layer goes out FusariumspFusarium proliferatum, gray mold the pathogen of Botrytis cinereaBotrytis cinereaAnd the Sclerotium rolfsii of southern blightsclerotium rolfsiiIt is inoculated in PDA culture medium, 28 DEG C of constant temperature culture 5d, beats and take the pathogen truffle of diameter 5mm and be inoculated in Gause I
Culture dish central authorities, in the same horizontal line, are inoculated with the actinomycetes of equal size apart from pathogen with equidistant position both sides
Truffle, each process are tested in triplicate.
（2）Pathogenic fungi is only inoculated with culture dish central authorities, and both sides are not inoculated with actinomycetes for control, and each process is repeated 3 times,
Pathogenic fungi to be compareed covers with culture dish, determines suppression ratio of 12 plants of actinomycetes to 5 kinds of pathogenic fungi, true to cause of disease so as to screen
Bacterium has 4 plants of actinomycetes strains of obvious antagonism.
These pathogenic fungi that chooses, are Herba Dendrobii encountered pathogenic funguses entirely.ToVerticilliumdahliaActivitie Activitie S of RelativeAlternaria tenuissimaThe pathogenic fungi of Herba Dendrobii melasma, Fusarium lateritiumNees bacteriumFusarium incarnatumAnd layer goes out FusariumspFusarium proliferatumIt is the pathogenic fungi of Herba Dendrobii stem rot, the pathogen of Botrytis cinereaBotrytis cinereaIt is
The pathogenic fungi of Herba Dendrobii gray mold, Sclerotium rolfsiisclerotium rolfsiiBe Herba Dendrobii southern blight cause of disease true
Bacterium.Choose these Herba Dendrobii encountered pathogenic eubacteriales is the actinomycetes in order to screen opposing Herba Dendrobii pathogenic fungi.
3rd, the actinomycetes screening of growth-promoting mycorrhizal fungi
The above-mentioned 4 plants of growth-promoting actinomycetes for filtering out are named as the true for examination Herba Dendrobii growth-promoting mycorhiza of S1, S2 respectively with two kinds
Mycocecidium mycorhiza Pseudomonas（Epulorhizasp.）And Tulasnella（Tulasnellasp.）, carry out in Gause I culture medium
Flat board dual test, each process 3 repeated experiments.With a culture dish central authorities Arbuscular Mycorrhizal Fungi fungus as control, each process is same
Sample is repeated 3 times, and mycorrhizal fungi to be compareed covers with culture dish, determines actinomycetes to mycorrhizal fungi suppression ratio.So as to filter out to ferrum
Skin Herba Dendrobii mycorrhizal fungi has 1 plant of the actinomycetes of obvious growth-promoting functions, is named as X.
4th, growth-promoting actinomycetes kind status determines
（1）The identification of kind status is carried out to the above-mentioned 1 plant of actinomycetes to mycorrhizal fungi with growth-promoting functions for filtering out.
（2）Actinomycetic form micro-structural feature is observed using streaking inoculation, inserted sheet method.16S rDNA is equally adopted
PCR Amplification Analysis, identify that growth-promoting actinomycetes X is this base streptomycete of Crane（Streptomyces krainskii）.
5th, optimize the composite bacteria agent capable that mycorrhizal fungi collaboration actinomycetes promote the growth of Herba Dendrobii Cloned seedling
（1）The Herba Dendrobii Cloned seedling of 4 months seedling ages is chosen, is inoculated in Harvaris culture medium, cultivated 15 days.
（2）By this base streptomycete of growth-promoting actinomycetes Crane（Streptomyces krainskii）Examination growth-promoting mycorhiza is true with supplying
Bacterium S1, S2 are seeded in free of contamination healthy Cloned seedling according to following recipe and carry out symbiosis culture, and control accesses aseptic equal big
Little agar block.After co-culturing 60 days, referred to by determining thick Cloned seedling fresh weight, dry weight, plant height, stem, tiller, the growth such as number of taking root
Mark, the optimal inoculating proportion of optimization.
（3）Outcome research goes out actinomycetes Crane this base streptomycete（Streptomyces krainskii）With mycorrhizal fungi tumor
Mycorhiza Pseudomonas（Epulorhizasp.）, Tulasnella（Tulasnellasp.）Co-inoculation and is compareed in Cloned seedling root system
Test is compared, and the growth-promoting functions to Herba Dendrobii Cloned seedling are most notable.
Claims (1)
1. a kind of composite bacteria agent capable of Herba Dendrobii, it is characterised in that its processing technology is followed the steps below：
（1）Actinomycetic in root isolate and purify：
A. healthy fresh Herba Dendrobii nutrition root system is chosen, root sample is bound up with gauze and is rinsed well through the tap water that flows；
B. sterilization 20s is placed in 75% ethanol, aseptic water washing 2-3 time, then with 0.1% mercuric chloride solution sterilization 20s, aseptic water washing
4-5 time, root sample surface moisture is blotted using sterilizing filter paper；
C. blank control test is set, i.e., is placed in Gause I culture medium with the root sample that has sterilized and is rolled for several times；
D. surface has been sterilized root sample, has been placed in superclean bench and grinds to form homogenate, microwave treatment 30S has been diluted after mixing respectively
Into 10-1、10-2、10-3Root sample diluent, is inoculated in Gause I, tri- kinds of culture medium flat plates of TWYE, YIM38# respectively, is placed in
Culture 7-15 days is inverted in 28 DEG C of constant incubators；
If E. compareing, blank cultures are aseptic to be born into, show that root sample surface sterilization is qualified, by the actinomycetes bacterium colony that isolates
Choose, multiple purification obtains actinomycetes strain, be placed in 4 DEG C of refrigerators and save backup；
（2）The screening of Antagonistic Actinomycetes：
A. using flat board face-off method, fusarium will be gone out for trying Herba Dendrobii pathogenic fungi Alternaria tenuissima, Fusarium lateritiumNees bacterium, layer
Bacterium, the pathogen of Botrytis cinerea, Sclerotium rolfsii are inoculated in PDA culture medium, 28 DEG C of constant temperature culture 5 days, beat the pathogen for taking diameter 5mm
Truffle is inoculated in Gause I culture dish central authorities, and in the same horizontal line, with pathogen with equidistant position both sides, inoculation is same
Etc. the actinomycetes truffle of size, each process 3 repeated experiments；
B. pathogenic fungi is only inoculated with culture dish central authorities, both sides are not inoculated with actinomycetes for control, and each process is repeated 3 times, and it is right to treat
Culture dish is covered with according to pathogenic fungi, suppression ratio of the actinomycetes to 5 kinds of pathogenic fungi is determined, is screened to pathogenic fungi with substantially short of money
The actinomycetes strain of anti-effect；
（3）The actinomycetes screening of growth-promoting mycorrhizal fungi：
A. by the above-mentioned growth-promoting actinomycetes for filtering out respectively with two kinds for examination Herba Dendrobii growth-promoting mycorrhizal fungi Epulorhizas category and
Tulasnella, carries out flat board dual test in Gause I culture medium, each process 3 repeated experiments；
B. with a culture dish central authorities Arbuscular Mycorrhizal Fungi fungus as control, each process is equally repeated 3 times, and mycorrhizal fungi to be compareed is covered with
Culture dish, determines actinomycetes to mycorrhizal fungi suppression ratio, filters out, to Herba Dendrobii mycorrhizal fungi, there are obvious growth-promoting functions
Actinomycetes；
（4）Growth-promoting actinomycetes kind status determines：
The above-mentioned actinomycetic form micro-structural feature for filtering out is observed using streaking inoculation, inserted sheet method, using 16S
RDNA PCR Amplification Analysis, identify that growth-promoting actinomycetes are this base streptomycete of Crane；
（5）Optimize the composite bacteria agent capable that mycorrhizal fungi collaboration actinomycetes promote the growth of Herba Dendrobii Cloned seedling：
A. the Herba Dendrobii Cloned seedling of 4 months seedling ages is chosen, is inoculated in Harvaris culture medium, cultivated 15 days；
B. by growth-promoting actinomycetes Crane, this base streptomycete belongs to and glued membrane bacterium with examination Herba Dendrobii growth-promoting mycorrhizal fungi Epulorhiza is supplied
Belong to according to 1:1:1 ratio is seeded in free of contamination healthy Cloned seedling carries out symbiosis culture, cultivates 60 days.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109819868A (en) * | 2019-04-09 | 2019-05-31 | 中国热带农业科学院环境与植物保护研究所 | A kind of dendrobium officinale culture medium and preparation method thereof |
| CN111763627A (en) * | 2020-07-09 | 2020-10-13 | 云南大学 | Fungus for promoting growth of medicinal dendrobium officinale seedlings |
| CN112980701A (en) * | 2021-04-17 | 2021-06-18 | 浙江理工大学 | Composite microbial inoculum for promoting germination and growth of dendrobium officinale seeds |
| CN114381379A (en) * | 2022-01-11 | 2022-04-22 | 云南大学 | Mucuna strain TP-8 capable of improving sprouting capacity of dendrobium seedlings and application thereof |
| CN114395486A (en) * | 2022-01-11 | 2022-04-26 | 云南大学 | Murraya koenigii strain TP-3 with capacity of promoting high growth of dendrobium and application thereof |
| CN114395485A (en) * | 2022-01-11 | 2022-04-26 | 云南大学 | Mucuna strain TP-2 capable of promoting stem growth of dendrobium and application thereof |
| CN114507618A (en) * | 2022-01-11 | 2022-05-17 | 云南大学 | Turkey mycorrhiza strain TP-11 with capacity of promoting growth of new leaves of dendrobium and application thereof |
-
2016
- 2016-10-28 CN CN201610962563.8A patent/CN106497838A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109819868A (en) * | 2019-04-09 | 2019-05-31 | 中国热带农业科学院环境与植物保护研究所 | A kind of dendrobium officinale culture medium and preparation method thereof |
| CN111763627A (en) * | 2020-07-09 | 2020-10-13 | 云南大学 | Fungus for promoting growth of medicinal dendrobium officinale seedlings |
| CN112980701A (en) * | 2021-04-17 | 2021-06-18 | 浙江理工大学 | Composite microbial inoculum for promoting germination and growth of dendrobium officinale seeds |
| CN114381379A (en) * | 2022-01-11 | 2022-04-22 | 云南大学 | Mucuna strain TP-8 capable of improving sprouting capacity of dendrobium seedlings and application thereof |
| CN114395486A (en) * | 2022-01-11 | 2022-04-26 | 云南大学 | Murraya koenigii strain TP-3 with capacity of promoting high growth of dendrobium and application thereof |
| CN114395485A (en) * | 2022-01-11 | 2022-04-26 | 云南大学 | Mucuna strain TP-2 capable of promoting stem growth of dendrobium and application thereof |
| CN114507618A (en) * | 2022-01-11 | 2022-05-17 | 云南大学 | Turkey mycorrhiza strain TP-11 with capacity of promoting growth of new leaves of dendrobium and application thereof |
| CN114507618B (en) * | 2022-01-11 | 2023-06-09 | 云南大学 | Oncomelania mycorrhizal fungus strain TP-11 with dendrobium new leaf growth promoting capability and application thereof |
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Application publication date: 20170315 |