CN106062173A - Mixed strains for inhibiting seed germination of plant, and use thereof as herbicide - Google Patents
Mixed strains for inhibiting seed germination of plant, and use thereof as herbicide Download PDFInfo
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- CN106062173A CN106062173A CN201580000156.2A CN201580000156A CN106062173A CN 106062173 A CN106062173 A CN 106062173A CN 201580000156 A CN201580000156 A CN 201580000156A CN 106062173 A CN106062173 A CN 106062173A
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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
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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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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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
- A01N63/32—Yeast
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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
- C12N1/16—Yeasts; Culture media therefor
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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
- C12N1/20—Bacteria; Culture media therefor
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
Abstract
The present invention provides mixed strains for inhibiting seed germination of a weed plant consisting of Lactobacillus strains and yeast strains; a herbicide comprising the mixed strains, a culture of the strains or a concentrate of the culture fluid of the strains and agrochemically acceptable carriers; a preparation method of the herbicide comprising a step of culturing the mixed strains; and a method for removing weeds by treating soil with the mixed strains, the culture of the strains or the concentrate of the culture fluid of the strains.
Description
Technical field
The present invention relates to a kind of for suppressing the hybrid bacterial strain of plant seed germination and as the purposes of herbicide, more in detail
Carefully, relating to a kind of hybrid bacterial strain for suppressing ruderal plant germination, it is made up of lactobacilli strain and yeast strain；
Also providing for a kind of herbicide, it is characterized by, it comprises the culture fluid of described bacterial strain, the culture of described bacterial strain or described bacterial strain
Acceptable carrier on concentrated solution and Pesticide Science；Also providing for the preparation method of a kind of herbicide, it includes cultivating described mixed vaccine
The step of strain；Also providing for the minimizing technology of a kind of weeds, it is by with described bacterial strain, the culture of described bacterial strain or described bacterial strain
The concentrated solution of culture fluid be processed to soil realize.
Background technology
In Agro-ecological System, the kind of ruderal species is the most, and, its harm is huge.Weeds exist with crop contention crop
Necessary moisture, nutrition and sunray in growth, thus reduce quality and the quantity of crop, therefore become reduction crop raw
The important limiting factor of producing property.At present, for controlling weeds, although using various organic synthesis herbicide, but drawn by weeds
The production loss of the crop risen is the biggest, and this is owing to the lasting use of herbicide can cause the ecological change of weeds bacterium colony
Change, and can induce for caused by the patience of herbicide.So far, the organic synthesis weeding used in a large number for controlling weeds
Environmental pollution and the problem of person poultry toxicity caused by agent, i.e. sprout inhibitor or stem and leaf inorganic agent are very serious, therefore, always
Need to develop that toxicity is low, the new microbe herbicide of environmental protection.
This microbial herbicide is as can substitute for the environmental protection prevention and controls method of existing synthetic herbicide and by people
Accept.Recently, for controlling weeds, carrying out a lot of microbe and deriving the research of phytotoxin (phytotoxins).
Although it addition, KR published patent the 2004-0034764th discloses a kind of new strains streptomyces 8E12 and profit
With its herbicide, KR published patent the 2009-0005917th discloses a kind of germination composite inhibiting and production thereof
Method, but do not disclose heretofore described " for suppressing the hybrid bacterial strain of plant seed germination and as herbicide
Purposes ".
Summary of the invention
Solve the technical problem that
The present invention proposes according to above-mentioned requirements, the present inventor with the microbial nutrition agent processed in plant to plant
Seed carries out the result processed, it is thus identified that germinateing after being suppressed, from described microbial nutrition agent, pure lines isolate 4 kinds of lactobacilluss
Belong to bacterial strain and 4 primary yeasts.Further, in these bacterial strains, use as by Lactobacillus brevis LBV-61 bacterial strain, Lactobacillus plantarum subspecies
LPT30 bacterial strain and Dare are furnished with DAG-A culture fluid and the work of the culture fluid of the hybrid bacterial strain of spore torula TDB-34 bacterial strain composition
For by lactobacillus paracasei tough and tensile subspecies LPC63 bacterial strain, Lactobacillus buchneri LBN62 bacterial strain, pichia kudriavzevii
The DAG-B culture fluid of the culture fluid of the hybrid bacterial strain of PKD-51 bacterial strain and Zygosaccharomyces bailii ZBA-22 bacterial strain composition, the most right
The result processed is carried out as the seed of Carex baccans Nees, Herba Digitariae, Amaranthus retroflexus, Herba Erigerontis Annui and the Herba Abutili plant of ruderal plant, it is thus identified that with
The culture fluid mixing other lactobacillus strains and yeast strain and cultivate is used to compare when processing, each ruderal plant seed
Germination the most suppressed.Thereby confirm that the two of the present invention overlaps hybrid bacterial strains and suppresses the germination of ruderal plant seed respectively, from
And effectively can use as herbicide, and complete the present invention.
Technical scheme
In order to solve the problems referred to above, the present invention provides the hybrid bacterial strain of a kind of germination for suppressing ruderal plant,
It is made up of lactobacilli strain and yeast strain.
Further, the present invention provides a kind of herbicide, it is characterized by, described herbicide comprises described hybrid bacterial strain, described bacterium
Acceptable carrier in the culture of strain or the concentrated solution of the culture fluid of described bacterial strain and Pesticide Science.
Further, the present invention provides the preparation method of a kind of herbicide, described preparation method to include cultivating described hybrid bacterial strain
Step.
Further, the present invention provides the minimizing technology of a kind of weeds, and described method is by with described hybrid bacterial strain, described bacterial strain
Culture or the concentrated solution of culture fluid of described bacterial strain be processed to soil realize.
Beneficial effect
In the present invention, it is thus identified that the two set hybrid bacterial strain culture fluid being made up of lactobacilli strain and yeast strain suppress respectively
The germination of ruderal plant seed, thus the herbicidal effect of ruderal plant is excellent.Therefore, the present invention comprise hybrid bacterial strain as having
The herbicide of effect composition the most usefully can use as environment-friendlybiological biological pesticide agent.
Accompanying drawing explanation
Fig. 1 illustrates germination form (the representing without processing of the left side of the Carex baccans Nees ruderal plant seed after processing 1 week with DAG-A
DAG-A is without processing；The process that DAG-A culture fluid is diluted to 1/4 and carries out by 25 expressions on the right).
Fig. 2 illustrates germination form (the representing without processing of the left side of the Herba Digitariae ruderal plant seed after processing 1 week with DAG-B
DAG-B is without processing；The process that DAG-B culture fluid is diluted to 1/8 and carries out by 12.5 expressions on the right).
Fig. 3 illustrates germination form (the nothing process table on the left side of the Amaranthus retroflexus ruderal plant seed after processing 1 week with DAG-A
Show that DAG-A is without processing；The process that DAG-A culture fluid is diluted to 1/8 and carries out by 12.5 expressions on the right).
Fig. 4 illustrates germination form (the nothing process table on the left side of the Herba Erigerontis Annui ruderal plant seed after processing 1 week with DAG-A
Show that DAG-A is without processing；The process that DAG-A culture fluid is diluted to 1/8 and carries out by 12.5 expressions on the right).
Fig. 5 illustrates germination form (the representing without processing of the left side of the Herba Abutili ruderal plant seed after processing 1 week with DAG-A
DAG-A is without processing；The process that DAG-A culture fluid is diluted to 1/32 and carries out by 3.13 expressions on the right).
Fig. 6 illustrates that the hybrid bacterial strain culture fluid after rice transplanting 2 weeks is without field on the left for the treatment of region and hybrid bacterial strain culture fluid treatment region
Right side field appearance (B and C be hybrid bacterial strain culture fluid without field on the left for the treatment of region (B) and hybrid bacterial strain culture fluid treatment region on the right side of
The enlarged drawing in field (C)).
Detailed description of the invention
In order to realize the purpose of the present invention, the present invention provides a kind of mixed vaccine for suppressing ruderal plant germination
Strain, it is made up of lactobacilli strain and yeast strain.
In the hybrid bacterial strain of a specific example of the present invention, described lactobacilli strain can be Lactobacillus
(Lactobacillus sp.) bacterial strain, is preferably selected from Lactobacillus plantarum subspecies LPT30 (Lactobacillus plantarum
Subsp.plantarum LPT30) bacterial strain (KCTC12710BP), Lactobacillus brevis LBV-61 (Lactobacillus brevis
LBV-61) bacterial strain (KCTC 12711BP), Lactobacillus buchneri LBN62 (Lactobacillus buchneri LBN62) bacterial strain
(KCTC 12712BP) and lactobacillus paracasei tough and tensile subspecies LPC63 (Lactobacillus paracasei
Subsp.tolerans LPC63) more than one bacterial strain in bacterial strain (KCTC12713BP), but it is not limited to this.
In the hybrid bacterial strain of a specific example of the present invention, described yeast strain can be red selected from Delhi, storehouse A Ziweibi
Yeast PKD-51 (Pichia kudriavzevii PKD-51) bacterial strain (KCTC 12714BP), Dare are furnished with spore torula TDB-
34 (Torulaspora delbrueckii TDB-34) bacterial strain (KCTC 12715BP), Zygosaccharomyces bailii ZBA-22
(Zygosaccharomyces bailii ZBA-22) bacterial strain (KCTC 12716BP) and double spore zygosaccharomyces ZBP-52
More than one bacterial strain in (Zygosaccharomyces bisporus ZBP-52) bacterial strain (KCTC 12717BP), but not
It is defined in this.
Described four kinds of lactobacillus strains and four primary yeast bacterial strains, on November 13rd, 2014, are deposited in Korea S typical case respectively
Culture collection centerDeposit number is volume shown in each bacterial strain name unquote
Number.
In the hybrid bacterial strain of a specific example of the present invention, described hybrid bacterial strain can be preferably by Lactobacillus brevis LBV-61
(Lactobacillus brevis LBV-61) bacterial strain (KCTC 12711BP), Lactobacillus plantarum subspecies LPT30
(Lactobacillus plantarum subsp.plantarum LPT30) bacterial strain (KCTC 12710BP) and Dare are furnished with spore
Torula TDB-34 (Torulaspora delbrueckii TDB-34) bacterial strain (KCTC 12715BP) forms, but is not limited to
This.
In the hybrid bacterial strain of a specific example of the present invention, described hybrid bacterial strain can be preferably tough and tensile by lactobacillus paracasei
Subspecies LPC63 (Lactobacillus paracasei subsp.tolerans LPC63) bacterial strain (KCTC 12713BP), cloth
Family name lactobacillus LBN62 (Lactobacillus buchneri LBN62) bacterial strain (KCTC 12712BP), Delhi, storehouse A Ziweibi
Red yeast PKD-51 (Pichia kudriavzevii PKD-51) bacterial strain (KCTC 12714BP) and Zygosaccharomyces bailii ZBA-
22 (Zygosaccharomyces bailii ZBA-22) bacterial strain (KCTC 12716BP) forms, or can be by class cheese milk
Tough and tensile subspecies LPC63 of bacillus (Lactobacillus paracasei subsp.tolerans LPC63) bacterial strain (KCTC
12713BP), Lactobacillus buchneri LBN62 (Lactobacillus buchneri LBN62) bacterial strain (KCTC 12712BP), Ku De
Li Aziwei Pichia sp. PKD-51 (Pichia kudriavzevii PKD-51) bacterial strain (KCTC 12714BP) and double spore connect
Close yeast ZBP-52 (Zygosaccharomyces bisporus ZBP-52) bacterial strain (KCTC 12717BP) composition, but do not limit
Due to this.
In the hybrid bacterial strain of a specific example of the present invention, described ruderal plant is as annual ruderal plant, and it can
Think grassy weed or broad leaved weed, it may be preferred to for the one in Carex baccans Nees, Herba Digitariae, Amaranthus retroflexus, Herba Erigerontis Annui and Herba Abutili
Above ruderal plant, but it is not limited to this.
Especially, use in advance when the soil of the farming predetermined area is carried out ploughing and weeding, therefore, do not interfere with the cultivation of desire property on opportunity
The germination of specific crop seed, all the more so when generally using the coating seed of seeds company's supply.
The hybrid bacterial strain of the present invention is in addition to described Carex baccans Nees, Herba Digitariae, Amaranthus retroflexus, Herba Erigerontis Annui and Herba Abutili, to any
Annual ruderal plant seed all can illustrate germination inhibitory activity.Especially in the situation of the specific crop seed to be cultivated
Under, process owing to seeds company the most implements crop protection by operations such as seed coatings, therefore,
Even if using the hybrid bacterial strain of the present invention to process, being still able to that ruderal plant seed is demonstrated, and specificity germinates and suppressing to live
Property.
It addition, the present invention provides a kind of herbicide, it is characterized by, described herbicide comprises described hybrid bacterial strain, described bacterium
Acceptable carrier in the culture of strain or the concentrated solution of the culture fluid of described bacterial strain and Pesticide Science.This carrier is not by particularly
Limiting, it is selected from pharmaceutics or nutritionally acceptable carrier and diluent.
By the described bacterial strain that obtains from the step cultivating bacterial strain of the present invention or the culture of described bacterial strain or described bacterium
When the concentrated solution of the culture fluid of strain uses as additive, can directly add described bacterial strain or the culture of described bacterial strain or institute
State the concentrated solution of the culture fluid of bacterial strain and use, or can also be used together with other additive, and it is possible to according to routine
Method suitably use.The combined amount of effective ingredient suitably can be determined according to its application target.Additive is excellent
Elect the nutrient of the described bacterial strain being made up of molasses and tomato fruit juice as, most preferably, can be by 500g molasses (Brix sugar
Degree (Brix) 79.8 °), 50g tomato fruit juice is dissolved in 1L water the microbial nutrition agent prepared, but is not limited to this.
It addition, the present invention provides the preparation method of a kind of herbicide, described preparation method to include cultivating described hybrid bacterial strain
Step.
The method cultivating bacterial strain of the present invention, can cultivate according to the method that this area generally utilizes.
It addition, the present invention provides the minimizing technology of a kind of weeds, described method is by with described hybrid bacterial strain, described bacterial strain
Culture or the concentrated solution of culture fluid of described bacterial strain be processed to soil realize.Below according to embodiment to the present invention
It is explained in more detail.But, following embodiment is only for illustrating the present invention, and the scope of the present invention is not limited to
This.
Material and method
The separation of bacterial strain and qualification
After the microbial nutrition agent being applied to garden crop arable land being diluted with using normal saline, by 0.1ml's
Its suspension is applied in BCP culture medium (BCP plate count agar plate (the Plate Count Agar of BCP, Eiken company respectively
) and PDA culture medium (peptone potato dextrose agar plate (the Bacto Potato of PDA, Difco company Plate)
Dextrose Agar Plate)) on, BCP cultivates at 37 DEG C, PDA cultivates at 30 DEG C, and observes the bacterium colony bred.?
BCP gathers the bacterium colony making periphery of bacterial colonies yellowing, and in BCP, again carries out Secondary Culture, thus separated lactic acid bacteria,
It addition, the bacterium colony bred in a pda is carried out Secondary Culture, thus separate yeast.
According to 16S rDNA sequence, lactic acid bacteria is carried out systematics classification.From 37 DEG C, MRS culture medium (MRS,
The milk peptonized acidfast bacilli MRS agar plate (Bacto Lactobacilli MRS Agar Plate) of Difco company) middle training
Support in the thalline of 72 hours and be extracted DNA, with PCR (primer: 27F, 5'-AGA GTT TGA TCC TGG CTC AG-3'(SEQ
ID No:9) and 1492R, 5'-GGT TAC CTT GTT ACG ACT T-3'(SEQ ID No:10)/30 cycles: 95 DEG C 15 points
Clock, 95 DEG C 20 seconds, 55 DEG C 40 seconds, 72 DEG C 1 minute 30 seconds, 72 DEG C 5 minutes, afterwards 10 DEG C unlimited) it is expanded and refines,
Then DNA analysis instrument ABI 3730XL (Applied biosystems (Applied Biosystems)) is used to analyze base
After sequence, with sequence alignment (CLUSTAL) X (Thomson et al., 1994) and MEGA program validation Systematic position.
It addition, according to 18S rDNA sequence, yeast is classified.I.e. from cultivating in a pda 30 DEG C 48 hours
Thalline in, be extracted DNA, with PCR (primer: ITS1,5'-TCC GTA GGT GAA CCT GCG G-3'(SEQ ID No:
11) and ITS4,5'-TCC TCC GCT TAT TGA TAT GC-3'(SEQ ID No:12)/30 cycles: 95 DEG C 15 minutes, 95
DEG C 20 seconds, 55 DEG C 40 seconds, 72 DEG C 1 minute 30 seconds, 72 DEG C 5 minutes, afterwards 10 DEG C unlimited) it is expanded and refines, then
After analyzing base sequence by method as above, it is thus identified that Systematic position.
Method for culturing microbes
Lactic acid bacteria class is inoculated in respectively MRS (the milk peptonized acidfast bacilli MRS meat soup (Bacto of MRS, Difco company
Lactobacilli MRS Broth)) in culture medium, and quiescent culture 96～120 hours at 32 DEG C, thus obtain viable bacteria
Number is 108c.f.u.ml-1The complete culture solution of levelYeast class is inoculated in PDB (PDB, Difco respectively
The peptone potato dextrose broth (Bacto Potato Dextrose Broth) of company) in culture medium, and at 30 DEG C
Shaken cultivation 48～72 hours, thus obtaining viable count is 108c.f.u.ml-1The complete culture solution of level.To above obtain
The complete culture solution of each bacterial strain obtained is inoculated in molasses culture medium (Molasses Broth) with 0.5% respectively, and 24 ± 2
Quiescent culture 4～6 weeks at DEG C, thus implement this cultivation.By 5g yeast extract, 50g molasses (Brix Scale 79.8 °) and
10g tomato fruit juice is dissolved in 1L water, thus is prepared for for this molasses culture medium cultivated (pH6.5 ± 0.2,25 DEG C).
The recovery of culture fluid
During cultivation, the viable count of microorganism is controlled as, and rises at Initial stage of culture, reduces, with lactic acid after mid-term
The viable count of mushroom is compared, and the viable count of yeast class is the lowest.In the viable count of late stage of culture, lactic acid bacteria class is 106cfu/ml
Level, yeast class is 104Cfu/ml level.During cultivation, the pH of culture fluid gradually decreases from pH 6.5, until pH in latter stage reaches
3.2～4.2 scopes.
The preparation of mixed-culture medium DAG-A and DAG-B
First, according to aforementioned micro organism cultural method, respectively by Lactobacillus brevis LBV-61 (Lactobacillus brevis
LBV-61) bacterial strain (KCTC 12711BP) and Lactobacillus plantarum subspecies LPT30 (Lactobacillus plantarum
Subsp.plantarum LPT30) bacterial strain (KCTC 12710BP) quiescent culture 120 hours in MRS culture medium, and by Dare
It is furnished with spore torula TDB-34 (Torulaspora delbrueckii TDB-34) bacterial strain (KCTC12715BP) to cultivate at PDB
Shaken cultivation 60 hours in base.Thus obtained culture fluid is inoculated in molasses culture medium with 0.5% respectively, and again stands
Cultivate 6 weeks, and thus reclaimed mixed-culture medium DAG-A.
Respectively by tough and tensile for lactobacillus paracasei subspecies LPC63 (Lactobacillus paracasei
Subsp.tolerans LPC63) bacterial strain (KCTC 12713BP) and Lactobacillus buchneri LBN62 (Lactobacillus
Buchneri LBN62) bacterial strain (KCTC 12712BP) quiescent culture 120 hours in MRS culture medium, and by Delhi, storehouse Ah hereby
Prestige Pichia sp. PKD-51 (Pichia kudriavzevii PKD-51) bacterial strain (KCTC 12714BP), Zygosaccharomyces bailii
ZBA-22 (Zygosaccharomyces bailii ZBA-22) bacterial strain (KCTC 12716BP) or double spore zygosaccharomyces ZBP-52
(Zygosaccharomyces bisporus ZBP-52) bacterial strain (KCTC 12717BP) shaken cultivation 60 in PDB culture medium
Hour.Thus obtained culture fluid is inoculated in molasses culture medium with 0.5% respectively, and quiescent culture 6 weeks again, and thus
Reclaim mixed-culture medium DAG-B.
The mensuration of germination inhibitory activity
Object ruderal plant seed is the barnyard grass of grassy weed, Herba Digitariae seed, the Amaranthus retroflexus of broad leaved weed, Herba Erigerontis Annui, piemarker
The seed of fiber crops.For concentration for the treatment of, using test medicine i.e. culture fluid stock solution 100 as benchmark, be diluted to 1/2 times every time and
Use.That is, carry out successively by 100,50,25,12.5,6.25,3.13,1.56,0.78,0.39,0.20,0.10,0.05 etc. dilute
Release and test.60 × 15mm culture dish spreads two 55mm filter paper, and after 10-20 weed seed is inoculated into above,
5ml is processed respectively according to the concentration of mixed-culture medium, and, replace culture fluid to process with 5ml distilled water, thus carry out
Controlled trial (without treatment region).Put into after culture dish Parafilm is sealed and growth case is taken care of and observes.Now, growth
12 hours (30 DEG C)/dark condition 12 hours (25 DEG C) that case is adjusted to optical condition.The number of individuals germinateed after processing one week i.e. produces
The numeral of young root carries out observing counting, and is expressed as a percentage relative to the treatment region germination percentage without the germination percentage in treatment region.
Comprise the preparation of the hybrid bacterial strain culture fluid of microbial nutrition agent
In order to use mixed-culture medium, for field area 1 section step (Korea S's unit of land area, one section of step is 300
Level ground, i.e. 1.48 mu) (10a) prepare the water of 300L, adds the DAG-B and 1 of 500ml wherein, and 000ml nutrient also mixes
It is sprayed at surface afterwards.Now, nutrient is that 500g molasses (Brix Scale 79.8 °), 50g tomato fruit juice are dissolved in 1L water
In and prepare (pH 6.5 ± 0.2,25 DEG C).Add nutrient be in order to improve the dilution of mixed-culture medium and spray after
The stability that the soil of hybrid bacterial strain is taken root.
Utilize the field test of the culture fluid of hybrid bacterial strain
In order to confirm for makingHerbicidal effect, spring in 2014 be positioned at Zhongqing Namdo, Korea opinion mountain city
The field done weapon practice in city's sun ground is tested.Commercial law has been observed in the management in field.I.e. in mid-April, the soil in field is carried out ploughing and weeding
After, carry out pouring on May about 10 and raked the soil level and rice transplanting (three smooth rice).District has been carried out using the ridge in north-south as center
Point, i.e. left side is without treatment region, and right side is treatment region (seeing Fig. 6), for treatment region, carries out the soil in field in mid-April
After ploughing and weeding, application of mixed-culture medium DAG-B and nutritional solution immediately.The nutrient of DAG-B and 1,000ml of 500ml is diluted
After 300L water, uniformly it is sprayed onto on every section of step (10a) of soil surface.After carrying out rice transplanting, for without treatment region and treatment region
Weeds produce state compare observation.
Embodiment 1: the separation of bacterial strain and qualification result
According to aforesaid method, separate four kinds of lactic acid bacterias, four primary yeasts, and the rDNA base sequence analyzing them (sees
SEQ ID No:1 to 8), and carry out systematics classification.Its result, lactic acid bacteria LBV-61 bacterial strain is accredited as Lactobacillus brevis
(Lactobacillus brevis), LPT30 are accredited as Lactobacillus plantarum subspecies (Lactobacillus plantarum
Subsp.plantarum), LPC63 is accredited as lactobacillus paracasei tough and tensile subspecies (Lactobacillus paracasei
Subsp.tolerans), LBN62 is accredited as Lactobacillus buchneri (Lactobacillus buchneri).Further, yeast TDB-
34 bacterial strains are accredited as that Dare is furnished with spore torula (Torulaspora delbrueckii), PKD-51 is accredited as Delhi, storehouse
A Ziwei Pichia sp. (Pichia kudriavzevii), ZBA-22 are accredited as Zygosaccharomyces bailii
(Zygosaccharomyces bailii), ZBP-52 are accredited as double spore zygosaccharomyces (Zygosaccharomyces
bisporus)。
Embodiment 2: there is the screening of the hybrid bacterial strain of germination inhibitory activity
Confirm by the present invention separate four kinds of lactobacillus strains and four primary yeasts have when mixing and cultivate
Germination inhibitory activity, and be thus respectively combined as 12 shown in table 1 below kind situation and be prepared for hybrid bacterial strain, thus obtain
Mixed-culture medium.
To these mixed-culture mediums, the germination of grassy weed (Carex baccans Nees), broad leaved weed (Amaranthus retroflexus) seed is suppressed to live
Property compares, and shows its result in Table 1.Wherein, with the extension rate of the mixed-culture medium illustrating 0% germination percentage
Represent germination inhibition concentration.I.e. 100 expression culture fluid stock solutions are tested, and culture fluid is diluted to 1/2 by 50 expressions
And test.As shown in table 1, according to the combination of bacterial strain, germination inhibitory activity is variant.(Lactobacillus brevis is combined at No. 3
LBV-61 (Lactobacillus brevis LBV-61)+Lactobacillus plantarum subspecies LPT30 (Lactobacillus
Plantarum subsp.plantarum LPT30)+Dare is furnished with spore torula TDB-34 (Torulaspora
Delbrueckii TDB-34)) bacterial strain and No. 10 combination (lactobacillus paracasei tough and tensile subspecies LPC63 (Lactobacillus
Paracasei subsp.tolerans LPC63)+Lactobacillus buchneri LBN62 (Lactobacillus buchneri LBN62)
+ pichia kudriavzevii PKD-51 (Pichia kudriavzevii PKD-51)+Zygosaccharomyces bailii ZBA-22
(Zygosaccharomyces bailii ZBA-22)) activity in bacterial strain is higher.Further, in combining at No. 10, connect with double spores
Closing yeast ZBP-52 (Zygosaccharomyces bisporus ZBP-52) bacterial strain replaces Zygosaccharomyces bailii ZBA-22 also to show
Go out identical result.
Sum it up, the hybrid bacterial strain that the four kinds of lactobacillus strains separated in the present invention and four primary yeasts carry out mixing exists
Although germination inhibitory activity aspect has certain difference, but all has germination inhibitory activity in all combinations.
Table 1 is mixed combination and the germination inhibition concentration (extension rate) of bacterial strain
* the germination inhibition concentration extension rate of the mixed-culture medium illustrating 0% germination percentage represents.I.e. 100: use
Culture fluid stock solution；50: cultivation is diluted to 1/2 and uses.
Embodiment 3: the germination inhibitory activity of screened hybrid bacterial strain culture fluid
No. 3 and No. 10 combinations to greater activity shown in embodiment 2 have carried out further test.No. 3 are combined
The named DAG-A of culture fluid, by No. 10 named DAG-B of culture fluid combined, and expands object ruderal plant, i.e. to Carex baccans Nees,
Herba Digitariae (grassy weed) and Amaranthus retroflexus, Herba Erigerontis Annui, the seed of Herba Abutili (broad leaved weed) have carried out the test of germination inhibitory activity.
The weed seed germination percentage of the concentration according to DAG-A and DAG-B is represented in table 2 to table 6, after processing 1 week
Germination form represent in Fig. 1 to Fig. 5 with the form of photo.Although DAG-A and DAG-B hybrid bacterial strain is in the kind according to concentration
Sub-germination inhibitory activity aspect has difference, but the germination inhibitory activity for all grassy weeds and broad leaved weed is excellent
Different.
Table 2 is according to the germination percentage of the Carex baccans Nees of the concentration of DAG-A and DAG-B
| DAG concentration | DAG-A | DAG-B |
| 50.00 | 0.0* | 0.0 |
| 25.00 | 0.0 | 0.0 |
| 12.50 | 82.6 | 0.0 |
| 6.25 | 87.0 | 77.8 |
| 3.13 | 95.7 | 105.6 |
| 1.56 | 100.0 | 111.1 |
| 0.78 | 104.3 | 100.0 |
| 0.39 | 104.3 | 94.4 |
| 0.20 | 95.7 | 105.6 |
| 0.10 | 95.7 | 111.1 |
| 0.05 | 91.3 | 100.0 |
* relative to the germination percentage of the treatment region without the germination percentage in treatment region
Table 3 is according to the germination percentage of the Herba Digitariae of the concentration of DAG-A and DAG-B
| DAG concentration | DAG-A | DAG-B |
| 50.00 | 0.0* | 0.0 |
| 25.00 | 0.0 | 0.0 |
| 12.50 | 0.0 | 0.0 |
| 6.25 | 87.5 | 0.0 |
| 3.13 | 81.3 | 75.0 |
| 1.56 | 81.3 | 100.0 |
| 0.78 | 93.8 | 80.1 |
| 0.39 | 106.3 | 90.0 |
| 0.20 | 81.3 | 85.3 |
| 0.10 | 93.8 | 100.0 |
| 0.05 | 100.0 | 95.0 |
* relative to the germination percentage of the treatment region without the germination percentage in treatment region
Table 4 is according to the germination percentage of the Amaranthus retroflexus of the concentration of DAG-A and DAG-B
* relative to the germination percentage of the treatment region without the germination percentage in treatment region
Table 5 is according to the germination percentage of the Herba Erigerontis Annui of the concentration of DAG-A and DAG-B
| DAG concentration | DAG-A | DAG-B |
| 50.00 | 0.0* | 0.0 |
| 25.00 | 0.0 | 0.0 |
| 12.50 | 40.0 | 0.0 |
| 6.25 | 87.5 | 62.5 |
| 3.13 | 95.2 | 62.5 |
| 1.56 | 100.0 | 105.0 |
| 0.78 | 81.0 | 100.0 |
| 0.39 | 100.0 | 87.5 |
| 0.20 | 104.8 | 97.5 |
| 0.10 | 95.2 | 105.0 |
| 0.05 | 104.8 | 110.0 |
* relative to the germination percentage of the treatment region without the germination percentage in treatment region
Table 6 is according to the germination percentage of the Herba Abutili of the concentration of DAG-A and DAG-B
| DAG concentration | DAG-A | DAG-B |
| 50.00 | 0.0* | 0.0 |
| 25.00 | 0.0 | 0.0 |
| 12.50 | 0.0 | 0.0 |
| 6.25 | 83.4 | 0.0 |
| 3.13 | 110.0 | 52.1 |
| 1.56 | 104.0 | 106.0 |
| 0.78 | 96.0 | 94.0 |
| 0.39 | 98.2 | 102.6 |
| 0.20 | 104.0 | 100.0 |
| 0.10 | 94.4 | 98.0 |
| 0.05 | 100.0 | 100.0 |
* relative to the germination percentage of the treatment region without the germination percentage in treatment region
Embodiment 4: utilize the result of the test of being responsible for a task until it is completed of the culture fluid of hybrid bacterial strain
Mid-April test medicine process after to rice transplanting, the rainfall having 20mm due to April 17, have 27-29 day
The rainfall of 60mm, therefore, maintains the moisture state of soil, the rainfall having more than 10mm due to the May 11 after pouring, because of
This, carried out rice transplanting on 13rd.After rice transplanting, the weeds for treatment region with without treatment region produce state and compare sight
Examine.Appearance after shooting rice transplanting 15 days expression are in figure 6.As shown in Figure 6, left side without the weeds rudiment in treatment region,
Making overall field is green, and on the contrary, the weeds in the treatment region of right side produce and substantially reduce, and field also seems that comparison is clean.
Further, no longer hoe up weeds in treatment region and the most do not produce impact, and be continued until heading stage.
[preserving number]
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12710BP
Preservation day: 20141113
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12711BP
Preservation day: 20141113
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12712BP
Preservation day: 20141113
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12713BP
Preservation day: 20141113
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12714BP
Preservation day: 20141113
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12715BP
Preservation day: 20141113
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12716BP
Preservation day: 20141113
Preservation mechanism name: Korea S's Type Tissue Collection
Preserving number: KCTC12717BP
Preservation day: 20141113
Claims (12)
1., for suppressing a hybrid bacterial strain for ruderal plant germination, described hybrid bacterial strain is by lactobacilli strain and yeast
Strain forms.
The most according to claim 1 for suppressing the hybrid bacterial strain of ruderal plant germination, it is characterised in that described breast
Acid bacterium is Lactobacillus (Lactobacillus sp.) bacterial strain.
The most according to claim 2 for suppressing the hybrid bacterial strain of ruderal plant germination, it is characterised in that described breast
Bacillus genus strain is selected from Lactobacillus plantarum subspecies LPT30 (Lactobacillus plantarum subsp.plantarum
LPT30) bacterial strain (KCTC 12710BP), Lactobacillus brevis LBV-61 (Lactobacillus brevis LBV-61) bacterial strain (KCTC
12711BP), Lactobacillus buchneri LBN62 (Lactobacillus buchneri LBN62) bacterial strain (KCTC 12712BP) and class
Tough and tensile subspecies LPC63 of lactobacillus casei (Lactobacillus paracasei subsp.tolerans LPC63) bacterial strain
More than one bacterial strain in (KCTC 12713BP).
The most according to claim 3 for suppressing the hybrid bacterial strain of ruderal plant germination, it is characterised in that described ferment
Mother strains is selected from pichia kudriavzevii PKD-51 (Pichia kudriavzevii PKD-51) bacterial strain (KCTC
12714BP), Dare is furnished with spore torula TDB-34 (Torulaspora delbrueckii TDB-34) bacterial strain
(KCTC12715BP), Zygosaccharomyces bailii ZBA-22 (Zygosaccharomyces bailii ZBA-22) bacterial strain (KCTC
12716BP) and double spore zygosaccharomyces ZBP-52 (Zygosaccharomyces bisporus ZBP-52) bacterial strain (KCTC
More than one bacterial strain in 12717BP).
The most according to claim 1 for suppressing the hybrid bacterial strain of ruderal plant germination, it is characterised in that described mixed
Close bacterial strain by Lactobacillus brevis LBV-61 (Lactobacillus brevis LBV-61) bacterial strain (KCTC 12711BP), plant breast
Bacillus subspecies LPT30 (Lactobacillus plantarum subsp.plantarum LPT30) bacterial strain (KCTC
12710BP) and Dare is furnished with spore torula TDB-34 (Torulaspora delbrueckii TDB-34) bacterial strain (KCTC
12715BP) composition.
The most according to claim 1 for suppressing the hybrid bacterial strain of ruderal plant germination, it is characterised in that described mixed
Close bacterial strain by tough and tensile subspecies LPC63 of lactobacillus paracasei (Lactobacillus paracasei subsp.tolerans
LPC63) bacterial strain (KCTC 12713BP), Lactobacillus buchneri LBN62 (Lactobacillus buchneri LBN62) bacterial strain
(KCTC 12712BP), pichia kudriavzevii PKD-51 (Pichia kudriavzeviiPKD-51) bacterial strain (KCTC
12714BP) and Zygosaccharomyces bailii ZBA-22 (Zygosaccharomyces bailii ZBA-22) bacterial strain (KCTC
12716BP) composition.
The most according to claim 1 for suppressing the hybrid bacterial strain of ruderal plant germination, it is characterised in that described mixed
Close bacterial strain by tough and tensile subspecies LPC63 of lactobacillus paracasei (Lactobacillus paracasei subsp.tolerans
LPC63) bacterial strain (KCTC 12713BP), Lactobacillus buchneri LBN62 (Lactobacillus buchneri LBN62) bacterial strain
(KCTC 12712BP), pichia kudriavzevii PKD-51 (Pichia kudriavzeviiPKD-51) bacterial strain (KCTC
12714BP) and double spore zygosaccharomyces ZBP-52 (Zygosaccharomyces bisporus ZBP-52) bacterial strain (KCTC
12717BP) composition.
The most according to claim 1 for suppressing the hybrid bacterial strain of ruderal plant germination, it is characterised in that described miscellaneous
Grass plant is grassy weed or broad leaved weed.
9. a herbicide, it is characterised in that described herbicide comprise the hybrid bacterial strain according to any one of claim 1 to 8,
Acceptable carrier in the culture of described bacterial strain or the concentrated solution of the culture fluid of described bacterial strain and Pesticide Science.
Herbicide the most according to claim 9, it is characterised in that described herbicide comprises further by molasses and western red
The microbial nutrition agent of Fructus Kaki juice composition.
The preparation method of 11. 1 kinds of herbicides, described preparation method includes cultivating mixing according to any one of claim 1 to 8
Close the step of bacterial strain.
The minimizing technology of 12. 1 kinds of weeds, described method includes with the hybrid bacterial strain according to any one of claim 1 to 8, institute
State the step that soil is processed by the concentrated solution of the culture of bacterial strain or the culture fluid of described bacterial strain.
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| PCT/KR2015/001201 WO2016085039A1 (en) | 2014-11-28 | 2015-02-05 | Mixed strains for inhibiting seed germination of plant, and use thereof as herbicide |
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| JP2003231605A (en) * | 2002-02-06 | 2003-08-19 | Miroku Hado:Kk | Oxygen-containing herbicide |
| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
| CN103497988A (en) * | 2013-09-23 | 2014-01-08 | 广州康代生物科技有限公司 | Fermentation production method of safe efficient lactobacillus product |
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| KR100991298B1 (en) * | 2008-08-01 | 2010-11-01 | 경상대학교산학협력단 | Biotic pesticide using Arthrobacter zoysiae with inhibition activity of germinaton and growth of seeds |
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| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
| CN103497988A (en) * | 2013-09-23 | 2014-01-08 | 广州康代生物科技有限公司 | Fermentation production method of safe efficient lactobacillus product |
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| CN110358699A (en) * | 2019-05-10 | 2019-10-22 | 基因赛奥(大连)生物科技发展有限公司 | A kind of complex microorganism deodorizing microorganism and the preparation method and application thereof |
| CN110358699B (en) * | 2019-05-10 | 2022-12-23 | 基因赛奥(大连)生物科技发展有限公司 | Compound microbial deodorant bacterial agent and preparation method and application thereof |
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