CN105779303B - Dendrobium officinale mycorrhizal fungus Arthrinium sp. strain ZJ11C12 and application of dendrobium officinale mycorrhizal fungus Arthrinium sp. strain ZJ11C12 - Google Patents
Dendrobium officinale mycorrhizal fungus Arthrinium sp. strain ZJ11C12 and application of dendrobium officinale mycorrhizal fungus Arthrinium sp. strain ZJ11C12 Download PDFInfo
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- CN105779303B CN105779303B CN201610172897.5A CN201610172897A CN105779303B CN 105779303 B CN105779303 B CN 105779303B CN 201610172897 A CN201610172897 A CN 201610172897A CN 105779303 B CN105779303 B CN 105779303B
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Abstract
The invention belongs to the field of agricultural microorganisms and relates to a dendrobium officinale mycorrhizal fungus Arthrinium sp. strain ZJ11C12 and application of the dendrobium officinale mycorrhizal fungus Arthrinium sp. strain ZJ11C12. An endophytic fungus is named as Arthrinium sp. ZJ11C12 by classes and is preserved in China General Microbiological Culture Collection Center on December 6, 2013, with the preservation number of CGMCC No.8602. The strain provided by the invention and a dendrobium officinale aseptic seedling symbiose to form a typical fungus root structure; a greenhouse pot culture test proves that the survival rate of seedlings with the fungus root structure is obviously higher than that of common aseptic seedlings after transplanting, and the growth vigor of the dendrobium officinale seedlings is obviously improved. Therefore, the Arthrinium sp. ZJ11C12 can provide good strain resources for developing a plant growth promoting bacterium fertilizer of rare medical plant dendrobium officinale, and has a better application and development prospect.
Description
Technical field
The invention belongs to field of agricultural microbial technology, it is related to a kind of dendrobium candidum mycorrhizal fungi Nodule defects bacterial strain
(Arthrinium sp.) ZJ11C12 and its application.
Background technology
As Chinese Famous traditional medicine, dendrobium candidum (Dendrobium officinale) early in《Compendium of Materia Medica》Iron
The skin stem of noble dendrobium be just listed in it is medicinal,《Sheng Nong's herbal classic》In be listed in top grade,《Taoist Scriptures》In be listed in " Chinese nine big celestial grass "
First of.Dendrobium candidum not only have functions that tonifying-Yin and nourishing-stomach, moisten the lung and relieve the cough, heat-clearing improving eyesight, also strengthen body immunity, eliminate
Tumour, suppresses the effect such as cancer.Because dendrobium candidum is very rare, in recent years, dendrobium candidum artificial cultivation industry is in national model
Enclose interior development rapider.But, the stem of noble dendrobium in growing distinctive complexity increase the difficulty of stem of noble dendrobium industry development.Wherein
One main cause is that the stem of noble dendrobium is mycorrhizal plants, under natural growth conditions, it is necessary to partially or completely relies on and is set up altogether with fungi
Raw relation forms mycorhiza and could realize normal activities.And when carrying out stem of noble dendrobium vast propagation under artificial condition, often because lacking
The participation of few suitable symbiotic effects, seed germination rate is extremely low, and mycorhiza cannot be formed, and causes the stem of noble dendrobium to grow slowly, influence
The development of domestic and international stem of noble dendrobium industry.
Meanwhile, as a kind of new microbial resources, pass extensively is caused including the endogenetic fungus including plant VA Mycorrhizal Fungi
Note.The endogenetic fungus of plant refer to the various tissues that health plant is moved in the certain phase of plant life history or whole stages
With the fungi inside organ.At present, various bioactivators are isolated from endogenetic fungus, including it is antibacterial, antitumor, anti-
The native compounds such as oxidation, wherein be much novel substance, with special bioactivity, in medicine, health products, plant bacterial manure microbial inoculum
Good application prospect is all shown etc. every field, and plant endogenesis epiphyte is the resource of a class sustainable use, to certainly
The right destruction Of resources are few, can solve the problem that the not enough problem of natural resources.
Used as a kind of plant endogenesis epiphyte, its metabolite has antioxidation activity to Nodule defects class VA Mycorrhizal Fungi, to plant
Grow up healthy and sound, disease control it is significant.The present invention provides a kind of new Nodule defects class VA Mycorrhizal Fungi, the bacterial strain energy and iron
Skin stem of noble dendrobium aseptic seedling symbiosis forms typical mycorhiza structure, promotes growth of seedling, improves it and transplants survival rate.
The content of the invention
First purpose of the invention is to provide one plant of Nodule defects ZJ11C12 bacterial strain, and it was in the preservation of December 06 in 2013
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address：BeiChen West Road, Chaoyang District, BeiJing City
No. 1 institute 3, preserving number CGMCC No.8602；Classification And Nomenclature is Nodule defects Arthrinium sp.；
Nodule defects ZJ11C12 of the present invention is separated from Wild Medicinal dendrobium candidum root system.Its separation side
Method is：Fresh dendrobium candidum 4~5cm of feeding root is taken, surface silt is rinsed, the slow stream of clear water washes 30min.Root is put into aseptic
In cup, 75% ethanol postincubation 30s outwells alcohol, adds 0.1% mercuric chloride, sterilizing 5min or so.Outwell mercuric chloride, sterilized water punching
Wash 4~5 times.The crosscutting thin slice into 3~5mm of material is placed in PDA culture medium with scalpel then, tangent plane is towards culture medium.
4~5 pieces of tissues of each culture dish, 25 DEG C of constant temperature lucifuge cultures.Mycelium grows from tissue block after cultivating 3~7 days, and is formed
A certain size bacterium colony, to diffusion growth around centered on tissue block.Another PDA is moved on to from its edge picking mycelium to put down
Plate, repeats to pure culture is obtained, and is transferred to the preservation of PDA inclined-planes.
Culture observation, colony diameter after dark culturing 7d are carried out on ZJ11C12 inoculations to the PDA plate that will be separated to
7.5cm.Aerial hyphae is flourishing, quality villiform, and near cotton-shaped, bacterium colony is white, and edge is irregular.Micro- sem observation mycelia without
Color, tool barrier film, branch, 2.5-5 μm of diameter, conidium has no.
The genomic DNA of ZJ11C12 bacterial strains is extracted using conventional meanses, and utilizes the universal primer in the ITS areas of fungi to expand
Increase its ITS interval, its base sequence is as shown in SEQ ID NO.1.By carrying out blast retrievals in GenBank, find its with
Arthrinium (Arthrinium sp.) bacterial strain ML-2004 (GeneBank accession number AY425967) homology highest, up to 99%,
Identify that the bacterial strain is Arthrinium (Arthrinium sp.) fungi.
Second object of the present invention is that described Nodule defects bacterial strain ZJ11C12 is setting up symbiosis with dendrobium candidum axenic seedlings
System, forms the application in typical mycorhiza structure, or above-mentioned Nodule defects fungi promotes stem of noble dendrobium growth and bioactivator
After the application of accumulation, Nodule defects fungi and stem of noble dendrobium seedling symbiosis, promote seedling replanting survival rate, improve seedling growth.
Bacterial strain of the invention can form typical mycorhiza structure with dendrobium candidum axenic seedlings symbiosis, and be tried by greenhouse pot culture
Checking is real, forms the seedling replanting survival rate after mycorhiza structure and is significantly improved apparently higher than common aseptic seedlings, and growing way.
Arthrinium (Arthrinium sp.) ZJ11C12, Chinese microorganism strain is preserved on December 06th, 2013
Preservation administration committee common micro-organisms center (CGMCC), preserving number CGMCC No.8602.
Brief description of the drawings
Fig. 1 is colony characteristicses figure of the Nodule defects ZJ11C12 bacterial strains of the invention in PDA culture medium；
Fig. 2 is the micro- sem observation picture of mycelia of Nodule defects ZJ11C12 bacterial strains of the invention；
Fig. 3 is the mycorhiza cross section of formation after Nodule defects ZJ11C12 bacterial strains of the invention and dendrobium candidum axenic seedlings symbiosis
Micro-structure diagram；Wherein A, Mycorrhizal Symbiosiss 7d, mycelia start to infect root by (× 100)；B, Mycorrhizal Symbiosiss 14d, mycelia is by passage
Cell enters exodermis (× 400)；C, Mycorrhizal Symbiosiss 21d, mycelia enter cortical cell (× 200)；D, Mycorrhizal Symbiosiss 28d, skin
There are a large amount of hypha bodies (× 200) decayed in confluent monolayer cells；E, Mycorrhizal Symbiosiss 35d, hypha body enter endothelium confluent monolayer cells (× 200)；
F, control root cross section (× 100).
Specific embodiment
Below by way of specific embodiment, the present invention is further described, but not limits the present invention.
Embodiment 1：Nodule defects ZJ11C12's is separately cultured：
Nodule defects ZJ11C12 is, its separation side isolated from the medicinal plant dendrobium candidum for picking up from Yunnan Province Kunming
Method is：Fresh dendrobium candidum 4~5cm of feeding root is taken, surface silt is rinsed, the slow stream of clear water washes 30min.Root is put into aseptic
In cup, 75% ethanol postincubation 30s outwells alcohol, adds 0.1% mercuric chloride, sterilizing 5min or so.Outwell mercuric chloride, sterilized water punching
Wash 4~5 times.The crosscutting thin slice into 3~5mm of material is placed in PDA culture medium with scalpel then, tangent plane is towards culture medium.
4~5 pieces of tissues of each culture dish, 25 DEG C of constant temperature lucifuge cultures.Mycelium grows from tissue block after cultivating 3~7 days, and is formed
A certain size bacterium colony, to diffusion growth around centered on tissue block.Another PDA is moved on to from its edge picking mycelium to put down
Plate, repeats to pure culture is obtained, and is transferred to the preservation of PDA inclined-planes.
Nodule defects ZJ11C12 morphological features of the invention are as follows：
As depicted in figs. 1 and 2, growth temperature of the Nodule defects ZJ11C12 bacterial strains in PDA culture medium is 25 DEG C, the raw bacterium of gas
Silk is flourishing, quality villiform, and near cotton-shaped, bacterium colony is white, and edge is irregular.Micro- sem observation hyphae colorless, has barrier film, point
Branch, 2.5-5 μm of diameter, conidium has no.
The genomic DNA of ZJ11C12 bacterial strains is extracted using conventional meanses, and utilizes the universal primer in the ITS areas of fungi to expand
Increase its ITS interval, its base sequence is as shown in SEQ ID NO.1.By carrying out blast retrievals in GenBank, find its with
Arthrinium (Arthrinium sp.) bacterial strain ML-2004 (GeneBank accession number AY425967) homology highest, up to 99%,
Identify that the bacterial strain is Arthrinium (Arthrinium sp.) fungi.
Arthrinium (Arthrinium sp.) ZJ11C12, Chinese microorganism strain is preserved on December 06th, 2013
Preservation administration committee common micro-organisms center (CGMCC), preserving number CGMCC No.8602.
Embodiment 2：The foundation of dendrobium candidum-Nodule defects bacterial strain ZJ11C12 syntaxial systems
6 monthly ages were grown into more consistent dendrobium candidum axenic tissue culture plant inoculation in the blake bottle containing 50ml symbiotic culture mediums
In, every bottle inoculation 3 young plants, altogether be inoculated with 30 bottles, be placed in 25 DEG C, intensity of illumination 2000Lx, the constant temperature illumination of daily 12~14h of illumination
Incubator preculture one week or so.
Meanwhile, by ZJ11C12 inoculation PDA solid mediums, after cultivating 5d at 25 DEG C, colony edge is taken with card punch
Bacterium block, is inoculated into the 500mL triangular flasks for filling 100mL PDB fluid nutrient mediums, puts 25 DEG C, on the shaking table of rotating speed 200r/min
Shaken cultivation 3d, obtains Nodule defects ZJ11C12 seed liquors.The thalline ball (a diameter of 0.3cm) of the equivalent size in seed liquor is taken,
Bacterium ball is inoculated in stem of noble dendrobium seedling on 2cm culture mediums with transfer needle, culture observation syntaxial system formational situation.
Embodiment 3：Nodule defects bacterial strain ZJ11C12 forms typical mycorhiza structure with dendrobium candidum axenic seedlings symbiosis
By the observation of the dendrobium candidum mycorhiza paraffin section to different symbiosis periods, ZJ11C12 bacterial strains and iron sheet are found
Stem of noble dendrobium seedling can form complete mycorhiza structure.The structure of dendrobium candidum root be followed successively by from outside to inside root quilt, exodermis, cortex,
Endodermis and center pillar.Root is generally made up of 3~4 confluent monolayer cells, and arrangement more rule, cell membrane is thicker, mostly dead cell；Crust
Layer is cell monolayer, is generally dead cell in addition to passage cell, and cell volume is larger, queueing discipline；Cortex is by 5~6 layers of living cells
Composition, 1~2 thin, adjacent with exodermis, endodermis confluent monolayer cells small volume of wall, center section volume is larger, in irregular
It is polygon-shaped；Endodermis cell monolayer, small volume；Cortical cell proportion in dendrobium candidum root is maximum, and cell membrane is thin,
The mycelia of intrusion can be digested, for the growth of orchid provides nutriment[83].Displaing microstructure observing discovery, vaccine symbiosis 7 days
When, the root that mycelia touches dendrobium candidum is organized and in root by outer accumulation (Fig. 3-A)；At 14 days, mycelia is entered by passage cell
Enter exodermis (Fig. 3-B)；At 21 days, mycelia continues to invade the parenchyma cell of cortex, generally poly- with the bending of one or more mycelia
Collection, shows typical mycelia junction structure and hyphae length increases, and some mycelia are then dissolved into the jelly (Fig. 3-C) of dark color；
At 28 days, there are a large amount of hypha bodies (Fig. 3-D) decayed in cortical cell；There is mycelia to occur at 35 days, near endodermis, exodermis
There is the nucleus (Fig. 3-E) that the new hypha body for being formed winds passage cell.In the control group for not connecing bacterium, then it is observed that just
Normal root architecture, and have not seen above-mentioned microbiological contamination structure (Fig. 3-F).
Embodiment 4：Nodule defects bacterial strain ZJ11C12 promotes dendrobium candidum seedling replanting survival rate
According to the above results, by dendrobium candidum seedling replanting to the hole tray after symbiosis 14 days, seedling survival situation is observed.
Hot-house culture, conventional sunshine counts survival rate of seedling after 3 months.(control and treatment group are each for 4000 plants of coprocessing stem of noble dendrobium seedling
2000 plants), the survival rate through counting transplanted seedling is respectively 93.99% and 99.95%, that is, survival rate rises after processing
5.96%, and treatment group stem of noble dendrobium seedling growth is apparently higher than control group.
SEQUENCE LISTING
<110>Hangzhou Pedagogic University
<120>A kind of dendrobium candidum mycorrhizal fungi Nodule defects bacterial strain ZJ11C12 and its application
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 580
<212> DNA
<213>It is artificial synthesized
<400> 1
tccgtaggtg aacctgcgga gggatcatta cagagttata caactcccat accatctgtt 60
aacctaccca gttatgcctc ggcgtaagct cggttggagg cacctgcagc taccctgtag 120
ttgcggactg ccaactccag ccgcggcccg ccggcggtac actaaactct gttttatttt 180
atattctgag cgtcttattt taataagtta aaactttcaa caacggatct cttggttctg 240
gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat 300
catcgaatct ttgaacgcac attgcgccca tcagtattct ggtgggcatg cctgttcgag 360
cgtcatttca acccttaagc ctagcttagt gttgggaatc tgctgtactg cagttcctta 420
aagacagtgg cggagcggcg gtagtcctct gagcgtagta atttattcct cgcttttgtc 480
aggctctgtc ctcccgccat aaaaccccca attttttagt ggttgacctc ggatcaggta 540
ggaatacccg ctgaacttaa gcatatcaat aagcggagga 580
Claims (3)
1. a kind of dendrobium candidum mycorrhizal fungi Nodule defects fungi ZJ11C12, is preserved in Chinese microorganism strain preservation conservator
Can common micro-organisms center, deposit number：CGMCC No.8602, preservation date：On December 06th, 2013；Classification And Nomenclature is section
Water chestnut spore Arthrinium sp.；The address of China Committee for Culture Collection of Microorganisms's common micro-organisms center is：Beijing
The institute 3 of Chaoyang District North Star West Road 1.
2. Nodule defects fungi as claimed in claim 1 is setting up syntaxial system with stem of noble dendrobium aseptic seedling, in formation mycorhiza structure
Using.
3. Nodule defects fungi as claimed in claim 1 promotes the application of stem of noble dendrobium growth and bioactivator accumulation, its feature
It is after Nodule defects fungi and stem of noble dendrobium seedling symbiosis, to promote seedling replanting survival rate, improve seedling growth.
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| CN108148769B (en) * | 2018-03-19 | 2020-11-27 | 临沂大学 | Culture medium for normal-temperature preservation of arthrobotrys fungus strains and preparation method and application thereof |
| CN108719336B (en) * | 2018-05-18 | 2020-12-08 | 中国林业科学研究院林业研究所 | Method for promoting growth of orchids by using basidiomycetes fungi |
| CN111440729B (en) * | 2020-02-19 | 2021-10-22 | 杭州师范大学 | Application and method of dendrobium officinale endophytic fungi in seed seedling culture |
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| 铁皮石斛内生真菌分布;胡克兴等;《微生物学通报》;20100120;第37卷(第1期);第37-42页，主要涉及第38页1.2－1.4，第39页左栏表2 * |
| 铁皮石斛内生真菌研究进展;徐焰平;《微生物学杂志》;20151031;第35卷(第5期);第108-112页，主要涉及第111页左栏第2段 * |
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