CN107841468A - A kind of trichoderma viride strain and its application - Google Patents
A kind of trichoderma viride strain and its application Download PDFInfo
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- CN107841468A CN107841468A CN201711449586.XA CN201711449586A CN107841468A CN 107841468 A CN107841468 A CN 107841468A CN 201711449586 A CN201711449586 A CN 201711449586A CN 107841468 A CN107841468 A CN 107841468A
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- trichoderma viride
- hot pickled
- pickled mustard
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
The invention discloses a kind of trichoderma viride strain and its application, the Strain Designation is trichoderma viride（Trichoderma viride）TV1, China typical culture collection center is preserved in, preservation date is on April 10th, 2017, and deposit number is CCTCC NO.M2017182.The bacterial strain has stronger inhibitory action to the disease fungus of hot pickled mustard tube clubroot.Especially there is significant preventive and therapeutic effect to the clubroot of Fuling Zacai.Immersion treatment is carried out to hot pickled mustard tube seedling using Trichoderma TV1 zymotic fluids, and the incidence of disease of hot pickled mustard tube reduces 15%, output increased 17%, has good economic benefit.And the zymotechnique of the bacterial strain is simply, stably, therefore, agriculturally there is very big application prospect.
Description
Technical field
The invention belongs to field of biological control, and in particular to a kind of trichoderma viride strain and its application.
Background technology
Tumorous stem mustard（Hot pickled mustard tube）(Brassica junceaVar. tumidaTsen et Lee) it is Cruciferae Brassica genus
The mutation of leaf mustard kind leaf mustard subspecies great Ye mustard mutation, one liked as our people as the hot pickled mustard tube that Raw material processing forms using it
Kind salted vegetables, as the important hot pickled mustard tube place of production --- Fuling is also therefore well-known throughout the country.At present in the cultivation of hot pickled mustard tube, knee
Disease and the serious production for constraining hot pickled mustard tube of virosis, wherein clubroot morbidity are the most serious, cause hot pickled mustard tube yield and product
The degradation of matter, serious plot of falling ill is by the cultivation of no longer suitable hot pickled mustard tube, the sustainable development of serious threat hot pickled mustard tube industry.
Clubroot（clubroot）Be by plasmodiophora brassica bacteria (Plasmodiophora brassicae Woron caused by)
A kind of soil-borne disease of obligatory parasitism Brassica Crops, brassicaceous vegetable is seriously endangered, and extremely difficult preventing and treating.Plasmodiophora brassicae master
Infect brassicaceous vegetable root, form tumour not of uniform size, smooth or that cracking is coarse, the later stage infected by soil miscellaneous bacteria and
Rot.And the overground part growth retardation of plant, water shortage is listless withers, later stage radical leaves gradually turn yellow death, and whole strain is withered when serious.
With crop in cruciferae cultivation species increase and the expansion of area, commodity seed and north and south dish exchange allocation and transportation, soil acid
Change, China's Cruciferae clubroot occurring area expands year by year in recent years, and the order of severity aggravates year by year, and clubroot turns into be restricted
One of Major Diseases of China's brassicaceous vegetable production sustainable development.
Clubroot mainly by the resting spore in soil, is carried the modes such as bacterium soil and propagated by dish strain sick seedling, current.The bacterium
Can soil long-term survival, its resting sporangium can in subzero 20 DEG C of long-term survivings, once field fall ill, it is difficult to eliminate, field
If block continues to plant crop in cruciferae, its occurring degree increases year after year.Because hot pickled mustard tube belongs to low-temperature epitaxy plant, its growth period
Typically at 2 months of October to next year, for temperature at 0-20 DEG C or so, contemporaneity lacks the replacement kind beyond Cruciferae, because
This crucifer continuous cropping is also the main reason for causing the disease wildness.Production at present is upper mainly to be prevented using agricultural chemicals
Control, but because the disease acts on hot pickled mustard tube root, Fuling Prefecture's consistency of soil is larger in addition, stone is more, winter, rainfall is plentiful makes
Soil turns over that difficulty is big, and agricultural chemicals is difficult to play a role, while long-term largely to produce clubroot disease using agricultural chemicals anti-
The property of medicine, residues of pesticides welding pollute the problems such as also become increasingly conspicuous.Therefore the research to fungi Biocontrol Effect has great
Research Significance and huge application prospect.
Fungi was received much attention as the biocontrol of plant disease factor in recent years, such as the hair shell being separated in soil
Pseudomonas（Chaetomium）Inhibited to a variety of soil-borne plant pathogens, the antimicrobial mechanism of wherein chaetomium globosum is to produce
Antibiotic and cell wall degrading enzyme, C caused by Endophytic Fungi in Brassica napus chaetomium globosum16Sphingol and phytosphingosine can substantially drop
Low clubroot resting spores of bacteria germination rate, can make the rapeseed clubroot incidence of disease be reduced to 20% from 76.74%；Endophytic Fungi in Brassica napusHeteroconium chaetospiraBC2HB1 can provide nitrogen for rape, moreover it is possible to promote the jasmonic, ethene, life of rape
The expression of long element and PR gene, so as to improve the systemic disease resistance of plant, in addition, fungi autoeciousness is in rape
In root system cortical tissue, rapeseed clubroot can be suppressed and infect and colonize；Pink helix poly spore it is mould (Clonostachys rosea) gene expression of phenylalanine, jasmonic or ethylene synthase approach can be raised, so as to improve rape to plasmodiophora brassicae
Resistance.But current biocontrol fungi does not adapt to low temperature, high humidity environment, and then it is true to play suppression pathogenic well
The effect of bacterium.
The content of the invention
For deficiencies of the prior art, it is an object of the invention to provide a kind of trichoderma viride strain and its answer
With mainly for Fuling Zacai low temperature, high humidity and the more growing environment of stone, solving existing biocontrol fungi and agricultural chemicals to Fuling
The problem of hot pickled mustard tube clubroot prevention effect is bad.
To achieve the above object, the present invention adopts the following technical scheme that：, should the invention provides a kind of trichoderma viride strain
Strain Designation is trichoderma viride strain（Trichoderma viride）TV1, China typical culture collection center is preserved in, protected
Hide unit address：Chinese Wuhan Wuhan Universitys, preservation date are on April 10th, 2017, and deposit number is CCTCC NO.
M2017182。
The trichoderma viride strain of the present invention（Trichoderma viride）TV1, belong to Fungi Imperfecti door, Hyphomycetes, silk spore
Mesh, Moniliaceae, trichoderma.Bacterium colony grows comparatively fast in PDA culture medium, figure it is seen that be white when bacterium colony starts,
Densification, it is circular, extended to surrounding, after from bacterium colony center produce green spores, center becomes green.Periphery of bacterial colonies has white hypha
Growth band, last whole bacterium colony all becomes green, and conidium is circular, green.
Electron microscopic observation can be seen that（Fig. 1）, mycelia is crisscross, network-like；Subiculum is thicker, the intensive such as felt of mycelia,
Fine and close clump pencil, initial stage is white, flat, and the later stage is in green because producing conidium.Conidium wall is smooth, does not pierce
It is prominent.Dirty-green when bacterium colony is aging, sporophore clump Shu Shusong, annular arrangement.Conidiophore is born from the side shoot of mycelia, directly
Vertical, branch, sprig is not often expanded to life, top, upper raw spore ball estranged；Main branch is in tree-shaped, level branch many times thereon.
Above-mentioned trichoderma viride strain TV1 can be used for preventing and treating the pathogen of hot pickled mustard tube clubroot.Specifically used method is：It is first
First culture trichoderma viride strain TV1 prepares zymocyte liquid, then the hot pickled mustard tube seedling for cutting root processing is immersed in the zymocyte liquid,
Soak time is 30 ~ 60min, then by the hot pickled mustard tube seedling replanting after processing to field；Mycelial concentration is 5 in the zymocyte liquid
~ 6mg/mL, no spore.
A kind of hot pickled mustard tube clubroot control agent, its active ingredient are trichoderma viride strain TV1.
Compared with prior art, the present invention has the advantages that：
1st, the present invention experiments prove that the present invention trichoderma viride TV1 there is good prevention effect to hot pickled mustard tube clubroot,
And optimal use method when have studied concrete application, test result indicates that, before hot pickled mustard tube seedling carries out field transplanting, use is green
Color Trichoderma TV1 zymotic fluids carry out immersion treatment to hot pickled mustard tube seedling, the incidence of disease of hot pickled mustard tube reduces 15%, output increased 17%,
With good economic benefit.And the zymotechnique of the bacterial strain is simply, stably, therefore, agriculturally there is very big application
Prospect.
2nd, the present invention is wooden compared with the isolated green of field hot pickled mustard tube root table that is light or not falling ill in hot pickled mustard tube clubroot occurring degree
Mould TV1, the bacterial strain can low temperature resistant, high humidity environment, to the wide adaptation range of the natural environmental conditions such as temperature, pH, to bad
The tolerance of environment is strong, can maintain the ecological balance, adapts to the disease ecosystem, and resistance to mutation, therefore is fully able to adapt to Fu
Mound area winter low temperature, high humidity and consistency of soil is larger, stone is more poor environment, and growth and breeding is good.Green wood
Mould TV1 can suppress hot pickled mustard tube root by producing the antibiotic of small molecule and the antibacterial protein of macromolecular or cell-wall degrading enzyme class
Swell sick pathogen growth, breed and infect, can also be captured by fast-growth and breeding moisture and nutrient, occupy space,
Oxygen etc. is consumed, it is anti-it can in addition contain induce hot pickled mustard tube to produce to weaken and exclude the hot pickled mustard tube clubroot disease in Same Habitat
Imperial reaction, therefore trichoderma viride TV1 can not only directly suppress the growth and breeding of hot pickled mustard tube clubroot disease, and can induce
Hot pickled mustard tube produces IRCM Self-Defence System IRCM and obtains disease resistance, therefore reduces the incidence of disease of hot pickled mustard tube and improve the yield of hot pickled mustard tube.Solve
The problem of existing biocontrol fungi and agricultural chemicals are to the pathogen prevention effect of Fuling Zacai clubroot bad.
Brief description of the drawings
Fig. 1 is trichoderma viride TV1 conidium electron-microscope scanning figure；
Fig. 2 is trichoderma viride TV1 colonial morphology figure；
Fig. 3 is hot pickled mustard tube seedling through influence of the trichoderma viride TV1 fermentation liquor treatments to hot pickled mustard tube clubroot disease index；
Fig. 4 is hot pickled mustard tube seedling through influence of the trichoderma viride TV1 fermentation liquor treatments to hot pickled mustard tube yield.
Embodiment
The present invention is described in further detail with reference to specific embodiments and the drawings.
First, isolate and purify
Soil sample from the field hot pickled mustard tube root table that Chongqing City Fuling District takes hot pickled mustard tube clubroot occurring degree relatively light or does not fall ill, claims
5g soil samples are taken in 100mL sterilized water, gradient dilution is carried out respectively to 10 after being mixed in vortex instrument-2、10-3、10-4With 10-5, then take 1mL dilutions to be coated on Trichoderma selective medium [ampicillin respectively（80mg/L）] on surface, 25 DEG C are fallen
Put culture 5-7 days.The macroscopic fungus colony grown in picking culture medium, move on PDA plate, it is incubated, until
Purifying obtains single bacterium colony, and then obtained single bacterium colony is transferred on the test tube slant of PDA culture medium and preserved.
PDA culture medium preparation method is：Potato 200g, glucose 20g, agar 20g, water 1000mL, pH (are not controlled naturally
PH value processed).Length of side 0.5cm blockage is cut into after fresh potato peeling, adds water 1000mL, is reheated 10 minutes after boiling,
Then, with four layers of filtered through gauze, potato nutrient solution is obtained, adds and claims to have in the triangular flask of agar in advance, 121 DEG C of sterilizing 20min, system
Into PDA inclined-planes or flat board.
Selective medium：Ampicillin is added in PDA culture medium, makes the final concentration of 80mg/ of ampicillin
L。
2nd, strain idenfication
Bacterial strain TV1 of the present invention DNA is extracted with CTAB methods, then enters performing PCR amplification rDNA's using universal primer ITS1/ITS4
ITS regions (ribosomes-DNA ITS), obtained purpose fragment size are about 600bp and carry out sequencing analysis, and particular sequence is such as
Shown in SEQ ID NO.1.Sequencing result carries out BLAST comparisons on NCBI, with trichoderma viride（Trichoderma viride）Affiliation is nearest.Identified by ITS sequence analyses and show that bacterial strain of the present invention is trichoderma viride
(Trichoderma viride), it is named as trichoderma viride TV1.
Embodiment
Above-mentioned trichoderma viride TV1 is inoculated on PDA plate culture medium, after 25 DEG C of culture 7d, added on flat board few
Measure PDA liquid medium scraping mycelia, be inoculated in 200mL liquid PDA culture mediums, 25 DEG C, 150rpm culture 36h after, with nothing
Bacterium water dilutes 10 times, and mycelia quality is 0.51g in average every 100mL, obtains trichoderma viride TV1 zymotic fluids, standby.
In hot pickled mustard tube seedling replanting period（On October 15 ~ 25）The consistent hot pickled mustard tube seedling of 600 growing ways of picking carries out cutting root
Processing, and two groups are randomly divided into, one group is control group（Sterilized water）, one group is embodiment group（Trichoderma viride TV1 zymotic fluids）,
Then the seedling after section root is immersed in sterilized water or trichoderma viride TV1 zymotic fluids respectively and handles 30min, then will processing
Hot pickled mustard tube seedling replanting afterwards carries out randomized grouping experimental, each plot area 6.7m to field, field2, transplant 60 hot pickled mustard tubes.And
Above-mentioned experiment is carried out into 3 repetitions to test.
In hot pickled mustard tube water content in harvest（Mid or late Febuary next year）, count all into the hot pickled mustard tube yield of dish, and calculate average value.
Statistical method is as follows：
Hot pickled mustard tube clubroot Severity gradation is set to 5 grades, its standard is：0 grade：Healthy tree.1 grade：Lateral root is fallen ill, and tumour is small and lacks,
It is white, to plant strain growth without or minimal effect, production loss be 0 ~ 5%.2 grades：Tumour is mainly in the middle part of main root, upper and lower lateral root
It is more, or tumour is big and more in lateral root, white or ecru, has certain influence on plant strain growth, but aerial part is without obvious disease
Shape, production loss 6 ~ 20%.3 grades：Main root tumour is big and blackening, rough surface cracking, there is a small amount of lateral root, aerial growth by
Resistance, center vane are changed into green, occur slight wilting, production loss 21 ~ 35% in the sun.4 grades：Main root tumour is cracked or corruption
Rotten blackening, almost without lateral root, aerial growth is seriously obstructed, and plant type is short and small, and leaf color is dark green, and turnip is small and skin depth muscle is more,
Under sunlight, whole strain is seriously wilted, and is easily pulled up, production loss（Do not include the diseased plant without actual cut value）More than 36%.
Disease index=∑（Diseased plant numbers at different levels × sick level the value）/（Investigate total strain number × superlative degree value）×100
As shown in figure 3, after trichoderma viride TV1 fermentation liquor treatments, the disease index of hot pickled mustard tube clubroot is 0.76, and without green
Color Trichoderma TV1 fermentation liquor treatments, the disease index of hot pickled mustard tube clubroot is 0.9；As shown in figure 4, fermented through trichoderma viride TV1
After liquid processing, hot pickled mustard tube yield is 13kg, and without trichoderma viride TV1 fermentation liquor treatments, hot pickled mustard tube yield is 11.1kg.Thus may be used
To see the incidence of disease of the hot pickled mustard tube through trichoderma viride TV1 fermentation liquor treatments reduce 15%, output increased 17%, has good warp
Ji benefit.Confirmed by the field experiments of continuous 2 years, repeatability of the trichoderma viride TV1 to the Biocontrol Effect of hot pickled mustard tube clubroot
Well.
Above it is demonstrated experimentally that trichoderma viride TV1 of the present invention can be grown in the environment of the low temperature, high humidity of Fuling Prefecture it is good
It is good.And it can efficiently suppress the generation of hot pickled mustard tube clubroot, there is preferable biological control effect, and environment will not be caused dirty
Dye, solves the problems, such as that existing biocontrol fungi and agricultural chemicals are bad to the pathogen prevention effect of Fuling Zacai clubroot.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to the skill of the present invention
Art scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover at this
Among the right of invention.
Sequence table
<110>Yangtze Normal University；
<120>A kind of trichoderma viride strain and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 590
<212> DNA
<213>Trichoderma viride (Trichoderma viride)
<400> 1
gatcggaggg atcattaccg agtttacaac tcccaaaccc aatgtgaacc ataccaaact 60
gttgcctcgg cggggtcacg ccccgggtgc gtcgcagccc cggaaccagg cgcccgccgg 120
agggaccaac caaactcttt tctgtagtcc cctcgcggac gttatttctt acagctctga 180
gcaaaaattc aaaatgaatc aaaactttca acaacggatc tcttggttct ggcatcgatg 240
aagaacgcag cgaaatgcga taagtaatgt gaattgcaga attcagtgaa tcatcgaatc 300
tttgaacgca cattgcgccc gccagtattc tggcgggcat gcctgtccga gcgtcatttc 360
aaccctcgaa cccctccggg gggtcggcgt tggggacctc gggagcccct aagacgggat 420
cccggccccg aaatacagtg gcggtctcgc cgcagcctct cctgcgcagt agtttgcaca 480
actcgcaccg ggagcgcggc gcgtccacgt ccgtaaaaca cccaacttct gaaatgttga 540
cctcggatca ggtaggaata cccgctgaac ttaagcatat caaatcccga 590
Claims (6)
1. a kind of trichoderma viride strain, it is characterised in that the bacterium is named as trichoderma viride（Trichoderma viride）
TV1, the deposit number of the strain is CCTCC NO. M2017182.
2. trichoderma viride strain according to claim 1, it is characterised in that the ribosomes-DNA ITS sequences of the bacterial strain are such as
SEQ ID NO：Shown in 1.
3. the application of trichoderma viride strain, it is characterised in that trichoderma viride strain as claimed in claim 1 is used to prevent and treat hot pickled mustard tube root
Swollen disease.
4. the application of trichoderma viride strain according to claim 3, it is characterised in that specific method is：Cultivate Trichoderma viride
Bacterial strain TV1 prepares zymocyte liquid, then the hot pickled mustard tube seedling for cutting root processing is immersed in the zymocyte liquid, and soak time is 30 ~
60min, then by the hot pickled mustard tube seedling replanting after processing to field.
5. the application of trichoderma viride strain according to claim 4, mycelial concentration is 5 ~ 6mg/mL in the zymocyte liquid, nothing
Spore.
6. a kind of hot pickled mustard tube clubroot control agent, it is characterised in that its active ingredient is trichoderma viride strain described in claim 1
TV1。
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ID=61684171
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113897260A (en) * | 2021-11-22 | 2022-01-07 | 江西赛阳酒业有限公司 | Folium artemisiae argyi wine and preparation method thereof |
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Application publication date: 20180327 |