CN104357369B - Biological control streptomycete and its application - Google Patents
Biological control streptomycete and its application Download PDFInfo
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- CN104357369B CN104357369B CN201410691633.1A CN201410691633A CN104357369B CN 104357369 B CN104357369 B CN 104357369B CN 201410691633 A CN201410691633 A CN 201410691633A CN 104357369 B CN104357369 B CN 104357369B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/465—Streptomyces
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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
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Abstract
The present invention relates to biological control streptomycete and its application.The biological control streptomycete of the present invention, is named as streptomycete Lnu 12 (Streptomyces sp.lnu 12), deposit number is CCTCCNO：M2014554.The present invention is using flat board face-off method, detection Lnu 12 has antagonism to various plants pathogen, it is wherein most strong to Fusarium oxysporum inhibitory action, antibacterial band mean breadth reaches 19~21mm, there is very strong inhibitory action to Fulvia fulva, tomato early blight bacterium, antibacterial bandwidth is respectively 15mm and 17mm, there is certain inhibitory action to Fusarium Solani, antibacterial bandwidth 13mm, streptomycete Lnu 12 have good application prospect in the biological control of booth vegetable soil-borne disease.
Description
Technical field
The present invention relates to microbial technology field, more particularly to a kind of anti-plurality of plant diseases with efficient, wide spectrum is sick
The streptomycete bacterial strain of opportunistic pathogen and its application.
Biological control streptomycete alleged by the present invention, is named as streptomycete Lnu-12 (Streptomyces sp.lnu-
12), on November 6th, 2014, in China typical culture collection center preservation, deposit number is CCTCC NO：
M2014554。
Background technology
It is the most popular method of current crops pest control using the method for Agro-chemicals control insect, however it is chemical
The be excessively used caused environmental pollution and person poultry safety's problem of agricultural chemicals have turned into the whole world and have paid high attention to focus, are that the mankind can hold
Supervention opens up implementation urgent problem to be solved.In recent years, meet environmental protection, the healthy, biological pesticide of sustainable development theory to open
Hair turns into the Main Means for solving above-mentioned problem, and biological control has caused as an important measures of control of plant disease
The great attention of people.
The content of the invention
It is an object of the invention to provide a kind of environmental protection, the efficient, life of the anti-plurality of plant diseases pathogen of wide spectrum
Thing prevents and treats streptomycete.
It is a further object of the present invention to provide applications of the streptomycete Lnu-12 in phytopathogen is suppressed.
The biological control streptomycete of the present invention, is named as streptomycete Lnu-12 (Streptomyces sp.lnu-12),
On November 6th, 2014, in Wuhan China typical culture collection center preservation, deposit number was CCTCC NO：M2014554.
The present invention streptomycete Lnu-12, be from the green house of vegetables soil of Shenyang City Dongling District separation screening and obtain.By soil
Earth sample dilution 105, using Gause I culture medium, it is separately cultured using conventional plate streaking partition method, picking is put
Line bacterium single bacterium colony, numbering is standby, screens and obtains.
1st, streptomycete Lnu-12 morphological features：
Lnu-12 well-growns on Gause I culture medium, Fig. 1 is spores of the Lnu-12 under light microscope 100X
The gentle raw hypha form of silk, aerial hyphae is flourishing, multi-branched；Fibrillae of spores is generally straight or curve shape, no closing twist；Spore is in circle
Shape, oval.Fig. 2 is Lnu-12 cultural characteristic (front) on Gause I culture medium, and Fig. 3 is that Lnu-12 is trained in Gause I
Cultural characteristic (reverse side) on base is supported, Fig. 2 and 3 is shown in colony edge without water suction spot, and no soluble pigment is produced.Meet strepto-
The feature of Pseudomonas.
2nd, Lnu-12 physio-biochemical characteristics：
Gelatin liquefaction, hydrogen peroxide experiment, cellulose hydrolysis, lecithinase experiment are positive；Milk solidify with peptonize,
Starch Hydrolysis, MR experiments, hydrogen sulfide experiment, the experiment of tryptophan decomposition experiment, nitrate reduction are feminine gender；Using grape
Sugar, fructose, xylose, lactose, starch, sucrose, galactolipin, cellobiose are as carbon source for growth, and biochemical reactions characteristic is with forming sediment
Powder streptomycete is consistent.
3rd, streptomycete Lnu-12 16S rDNA sequence analysis methods：
(1) streptomycete Lnu-12 DNA, which is extracted, uses multigelation method, and performing PCR amplification is entered as template.
(2) PCR primer：Forward primer BSF8/27：5′-AGAGTTTGATCCTGGCTCAG-3′；
Reverse primer BSR1541/1522：5′-AAGGAGGTGATCCAGCCGCA-3′.
(3) PCR primer is synthesized by Shanghai bioengineering Co., Ltd.
(5) PCR amplification conditions：94℃5min；94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 1.5min；30 circulations；72℃5min.
Streptomycete Lnu-12 bacterial strains of the present invention, PCR primer sequencing is completed by Shanghai bioengineering Co., Ltd, the sequence measured
Row recall measured 16S rDNA sequences and it with blast search instrument as shown in SCQ ID No.1 from GeneBank
Related 16S rDNA sequences, then carry out Multiple Sequence Alignment with CLASTAL X softwares, and using the neighbour in MEGA4.1 softwares
Position phase connection (Neighbor-Joining) carries out the structure and tetraploid rice of systematic evolution tree, constructed systematic evolution tree
See Fig. 4.
Streptomycete Lnu-12 bacterial strains are analyzed through round pcr, DNA sequencer, and its 16S rDNA sequence 1424bp will
The 16S rDNA sequences of itself and related strain in GeneBank databases are compared, as a result the starch streptomycete watt with having reported
Grey subspecies (S.diastaticus subsp.Ardesiacus) homology reaches 99%.
Biocontrol bacterial strain Lnu-12 isolated from the green house of vegetables soil of Shenyang Dongling District of the invention is anti-by Physiology and biochemistry
Should, and the 16S rDNA for expanding the bacterial strain are reacted using PCR, by sequencing identification, multisequencing is carried out using CLASTAL X softwares
Compare, the structure of systematic evolution tree is then carried out with the ortho position phase connection (Neighbor-Joining) in MEGA4.1 softwares, reflect
Streptomycete Lnu-12 (Streptomyces sp.lnu-12) bacterial strain is determined for starch streptomycete dark grey subspecies (S.diastaticus
subsp.Ardesiacus)。
The beneficial effects of the invention are as follows：Streptomycete Lnu-12 provided by the present invention has antagonism to various plants pathogen
Effect, wherein most strong to Fusarium oxysporum inhibitory action, antibacterial band mean breadth reaches 19~21mm.It is provided by the present invention
Streptomycete Lnu-12 has the antimicrobial spectrum of high-efficiency broad spectrum, and anti-plant disease ability is stronger.By taking leaf muld of tomato as an example, with Lnu-12
Zymotic fluid has carried out field efficacy experiment, as a result shows Lnu-12 zymotic fluids to dilute after 50 times and has significantly to leaf muld of tomato
Prevention effect, prevention effect can reach 70%, hence it is evident that higher than comparison medicament.
Brief description of the drawings
Streptomycete Lnu-12 (Streptomyces sp.lnu-12), depositary institution：China typical culture collection center,
Referred to as：CCTCC, depositary institution address：China, Wuhan University, postcode：430072.Preservation date is November 6 in 2014
Day, deposit number is CCTCC NO：M2014554.
Fig. 1 is the Lnu-12 gentle raw hypha form (light microscope 100X) of fibrillae of spores.
Fig. 2 is Lnu-12 cultural characteristic (front) on Gause I culture medium.
Fig. 3 is Lnu-12 cultural characteristic (reverse side) on Gause I culture medium.
Fig. 4 is the phylogenetic evolution tree of Lnu-12 bacterial strains.
Embodiment
The streptomycete Lnu-12 of embodiment 1 (Streptomyces sp.lnu-12) bacteriostasis
1st, activate：By streptomycete Lnu-12 (Streptomyces sp.lnu-12) (CCTCC NO：M 2014554),
Operated on aseptic operating platform, picked and be evenly coated in after the bacterium solution of preservation on No. 1 inclined-plane solid plate of Gao Shi with aseptic inoculation ring, 28
DEG C culture 3~5 days.
2nd, using flat board face-off method, Lnu-12 spore of the scraping through overactivation prepares suspension, is evenly coated in No. 1 training of Gao Shi
Support on base solid plate, 28 DEG C are cultivated 3~5 days.
3rd, bacteria cake is made with diameter 7mm aseptic card punch, is positioned over PDA plate center, after 28 DEG C are cultivated 2 days, then at
Six kinds of various pathogenic bacteria bacteria cakes that surrounding is made close to the equidistant inoculation in edge with same method, 28 DEG C are cultivated 4~6 days, observation
Record antibacterial situation.Often processing is repeated 3 times.
Six kinds of different pathogens are：Fusarium Solani (Pepper Root Rot), Fulvia fulva
(Cladosporium fulvum), tomato early blight bacterium (Alternaria solani), Melon fusarium Wilt (Fusariwn
Oxysporum), withered germ of water-melon (Fusarium oxysporum f.sp.niveum), cucumber fusarium axysporum (Fusarium
oxysporum f.sp.cucumerinum)。
4th, result such as table 1
Table 1
| Pathogen | Antibacterial band mean breadth |
| Fusarium Solani (Pepper Root Rot) | 13mm |
| Fulvia fulva (Cladosporium fulvum) | 15mm |
| Tomato early blight bacterium (Alternaria solani) | 17mm |
| Melon fusarium Wilt (Fusariwn oxysporum) | 19mm |
| Withered germ of water-melon (Fusarium oxysporum f.sp.niveum) | 20mm |
| Cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum) | 21mm |
From table 1, streptomycete Lnu-12 of the invention has certain inhibitory action to all strains testeds.It is wherein right
Fusarium oxysporum inhibitory action is most strong, reaches 19~21mm, there is very strong suppression to Fulvia fulva, tomato early blight bacterium
Effect, antibacterial bandwidth is respectively 15mm and 17mm, there is certain inhibitory action, antibacterial bandwidth 13mm to Fusarium Solani.
Prevention effect of the biological control agentses Lnu-12 zymotic fluids of embodiment 2 to leaf muld of tomato
The bacterial strain Lnu-12 that the present invention is obtained 28 DEG C of cultures on Gause I slant medium, produce enough spores
After spore suspension is made, with 3% amount be inoculated in 250ml triangular flasks fermentation medium (corn flour 4.0%,
KNO30.2%th, FeSO40.02%th, NaCl 0.02%, CaCO30.2%, surplus is water) in, 28 DEG C, 180r/min shaken cultivations
5 days.Zymotic fluid 7000r/min centrifuges 20min, and supernatant is put standby in 4 DEG C of refrigerators.
In glasshouse by the way of potted plant inoculation, by Fulvia fulva, 26 DEG C of constant temperature are trained in PDA culture medium
4-5d is supported, takes 7-8 leaf phases tomato seedling to pierce 4 pins in middle part blade with No. 5 syringe needles, then pastes Fulvia fulva bacteria cake
At acupuncture, sterilized water is dipped in sterile absorbent cotton and covers moisturizing thereon, often 15 plants of processing, 3 repetitions, 28-30 DEG C of high-temperature cultivation,
Bacteria cake is removed after moisturizing 48h.By above-mentioned 50 times of dilutions of fermented supernatant fluid, 50% 500 times of carbendazim WP liquid and clear water control are equal
The even face anyway for being sprayed on blade, is dripped for degree with blade face.Disease index is investigated in inoculation after 7 days, calculate prevention effect.
It is positive control present invention employs the antifungal medicine carbendazim commonly used in production, potted plant experiment result is shown,
Using the fermentation condition of above-mentioned optimization, bacterial strain Lnu-12 zymotic fluid has good drug effect to leaf muld of tomato, sick after processing
Feelings index is 20.5, hence it is evident that less than control, prevention effect is up to 69.9%.Better than the carbendazim WP of comparison medicament 50%.
Prevention effect of the Lnu-12 zymotic fluids of table 2 to leaf muld of tomato
Claims (1)
1. the method that streptomycete Lnu-12 suppresses phytopathogen, it is characterised in that method is as follows：By streptomycete Lnu-12 in hair
Fermented in ferment culture medium, fermented supernatant fluid is diluted after 50 times, the plant for suffering from phytopathogen is applied in the way of sprinkling
On thing blade, described phytopathogen is：Melon fusarium Wilt (Fusarium oxysporum), withered germ of water-melon
(Fusarium oxysporum f.sp.niveum) and cucumber fusarium axysporum (Fusarium oxysporum
f.sp.cucumerinum)；Streptomycete (Streptomyces sp.) Lnu-12, in China typical culture collection center
Preservation, deposit number is CCTCC NO：M 2014554；Its 16S rDNA sequence is starch strepto- as shown in SEQ ID No.1
Bacterium dark grey subspecies (Streptomyces.diastaticus subsp.Ardesiacus).
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| CN104357369B true CN104357369B (en) | 2017-10-13 |
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| CN110699304B (en) * | 2019-11-27 | 2021-04-27 | 吉林省林业科学研究院 | Biocontrol streptomyces |
| CN114410518A (en) * | 2022-01-07 | 2022-04-29 | 春华秋实科技集团有限公司 | Compound microbial agent containing streptomyces and application thereof |
| CN116083266B (en) * | 2022-07-28 | 2024-05-03 | 西北农林科技大学 | Streptomyces flagelliformis, biocontrol microbial inoculum and application |
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