CN104928221B - One plant of Pseudomonas cepacia bacterial strain QBA 3 for having inhibitory action to ash arrhizus bacteria and its application - Google Patents
One plant of Pseudomonas cepacia bacterial strain QBA 3 for having inhibitory action to ash arrhizus bacteria and its application Download PDFInfo
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- CN104928221B CN104928221B CN201510394929.1A CN201510394929A CN104928221B CN 104928221 B CN104928221 B CN 104928221B CN 201510394929 A CN201510394929 A CN 201510394929A CN 104928221 B CN104928221 B CN 104928221B
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- A—HUMAN NECESSITIES
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- 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 invention discloses one plant of Pseudomonas cepacia bacterial strain QBA 3 for having inhibitory action to ash arrhizus bacteria and its application, by the QBA 3 of screening acquisition, its Classification And Nomenclature is Pseudomonas cepacia to the present inventionPseudomonas cepacia, China typical culture collection center is preserved in, its deposit number is CCTCC M 2015402.The present invention is by ash arrhizus bacteria spore germination, germ tube elongation and the experiment of excised leaf, prove that the bacterial strain QBA 3 that the present invention obtains acts on the high inhibition to ash arrhizus bacteria, the biological prevention and control agent of ash arrhizus bacteria can be prepared into preventing and treating, there is good market application foreground.
Description
Technical field
The invention belongs to microbial technology field, and in particular to one plant of onion vacation unit cell for having inhibitory action to ash arrhizus bacteria
Bacteria strain QBA-3 and its application.
Background technology
Ash arrhizus bacteria is important plant pathogenic fungi widely distributed in a kind of world wide, and the germ host types are numerous
More, stem, leaf, flower and the fruit that can infect 235 kinds of plants such as the vegetables such as tomato, cucumber and grape, strawberry fruit cause grey mold
Disease.The disease can not only occur in the host plant season of growth, can also occur during the storage of agricultural product, therefore endanger
Seriously.Especially facility cultivation mode is widely applied in recent years, causes that the infection process source is wide, spread speed is fast, ash
The generation of mildew is increasingly severe, and it occurs to often result in serious economic loss with popular.It is reported that northern facility tomato is grey
The generation of mildew is generally heavier, and economic loss caused by the general time is up to 20%--30%, and the serious time more than 50% is very
To total crop failure.
Ash arrhizus bacteria reproduction speed is very fast, and it is very more to produce conidial quantity, thus be easy to morph and
Develop immunity to drugs, it is a variety of to benzimidazole class conventional in agricultural production, dicarboximide class, carbamates etc. at present
Bactericide produces resistance, therefore does not only result in the reduction of preventive effect, and causes medicament usage amount to continue to increase.The mistake of chemical agent
Degree uses, and not only causes agricultural product agriculture residual exceeded, endangers the health of consumer, also results in environmental pollution, and it is flat to destroy ecology
Weighing apparatus, influence the sustainable and healthy development of agricultural.
Biological control is one of important measures of control of plant disease, and biocontrol microorganisms are by inducing the production of host plant disease resistance
Purpose raw or that preventing and treating or mitigation disease are reached by the antagonism to ash arrhizus bacteria.Have Trichoderma, withered grass at present
Relevant report of the multiple-microorganisms such as bacillus, Serratieae, streptomycete to ash arrhizus bacteria antagonism.Bacteria agent or system
Agent can reduce or substitute the use of chemical pesticide, so as to can effective controlling disease, the use band of chemical pesticide can be avoided again
The negative effect come.
The content of the invention
Object of the present invention is to provide the Pseudomonas cepacia bacterial strain QBA-3 that one plant has inhibitory action to ash arrhizus bacteria
And its application.The present invention obtains one plant of pseudomonad bacterium for being capable of strong inhibition ash arrhizus bacteria conidia germination by screening
Strain QBA-3, by inoculation experiments, the ferment filtrate of the strain fermentation thing is 92.5% to the prevention effect of graw mold of tomato, real
Testing result proves that the preventing and treating of the bacterial strain or its preparation to gray mold has broad application prospects.
For achieving the above object, the present invention is achieved using following technical scheme：
The invention provides the Pseudomonas cepacia bacterial strain QBA-3 that one plant has inhibitory action to ash arrhizus bacteria, its life of classifying
Entitled Pseudomonas cepacia Pseudomonas cepacia, are preserved in China typical culture collection center, and its deposit number is
CCTCC M 2015402。
The bacterial strain QBA-3 has flagellum.
Present invention also offers described Pseudomonas cepacia bacterial strain QBA-3 to prepare the biological prevention and control agent of preventing and treating ash arrhizus bacteria
In application.
Further improvement to above-mentioned technical proposal：The bacterium solution and ferment filtrate of the Pseudomonas cepacia bacterial strain QBA-3
Inhibitory action is respectively provided with to ash arrhizus bacteria spore and germ tube elongation.
Further improvement to above-mentioned technical proposal：Bacterial content is in the bacterium solution of the Pseudomonas cepacia bacterial strain QBA-3
OD600Conidial to ash arrhizus bacteria sprouting and the elongation of germ tube are respectively provided with during=0.314-0.565 acts on compared with high inhibition.
Further improvement to above-mentioned technical proposal：The preparation of the ferment filtrate of the Pseudomonas cepacia bacterial strain QBA-3
Step is：The bacterial strain QBA-3 is inoculated into liquid YEPD culture mediums the concussion and cultivate 72-96h at 25 DEG C -28 DEG C, then
Centrifugation, filtering, obtains the ferment filtrate containing QBA-3 bacterium.
Further improvement to above-mentioned technical proposal：The bacterial strain QBA-3 is inoculated into culture medium under the conditions of 130rpm
Carry out concussion and cultivate.
Further improvement to above-mentioned technical proposal：Filter through a diameter of 0.22um is filtered.
Compared with prior art, advantages of the present invention and have the technical effect that：The present invention screens has suppression to ash arrhizus bacteria
The bacterial strain QBA-3 of making, categorized discriminating is Pseudomonas cepacia, and by ash arrhizus bacteria spore, germ tube elongation and from
The experiment of body blade, it was demonstrated that the bacterial strain QBA-3 that the present invention obtains, which has, to be acted on the high inhibition of ash arrhizus bacteria, can be prepared
Into the biological prevention and control agent of preventing and treating ash arrhizus bacteria, there is good market application foreground.
Brief description of the drawings
Fig. 1 is the experimental result that the ferment filtrate of QBA-3 bacterial strains of the present invention suppresses to ash arrhizus bacteria spore germination；
Fig. 2 is that the ferment filtrate of QBA-3 bacterial strains of the present invention extends the experimental result suppressed to ash arrhizus bacteria germ tube.
Embodiment
Technical scheme is described in further detail below in conjunction with the drawings and specific embodiments.
Embodiment 1
First, the separation and screening of bacterial strain
On March 19th, 2013 is in 20 parts of Laiyang City greenhouse collection tomato leaf sample, every vanes 75% alcohol-pickled 10
Second, then soaked 30 seconds in 0.1% mercuric chloride, aseptic water washing 3 times, be transferred in the mortar of sterilization treatment and grind, add nothing
Bacterium water is diluted to 10ml, continues to be diluted to 10 with sterilized water-5Times, take 100ul to apply LB flat board (10 g/l of tryptone, ferment respectively
Female 5 g/l of extract, 5 g/l of sodium chloride, pH7.0), cultivate under the conditions of 30 DEG C.
Picking single bacterium colony, and bacterium single bacterium colony is inoculated in YEPD culture mediums (3 g/l of yeast extract, peptone 10
G/l, 20 g/l of glucose) concussion and cultivate 24h under the conditions of 25 DEG C of 120rpm, then centrifuged under the conditions of 25 DEG C of 12000rpm, on
Filter filtering clearly through a diameter of 0.22um, is inoculated into quiescent culture in filtrate, with grey mold by the conidium of ash arrhizus bacteria
The conidium of germ, which is inoculated into YEPD, to be compared, the ash in detection bacterium filtrate when compareing conidia germination rate up to 90%
The conidial germination rate of mildew bacterium, inhibiting rate of the antivirus to ash arrhizus bacteria conidia germination is calculated, filters out one
Strain is conidial to ash arrhizus bacteria to sprout the bacterial strain QBA-3 with very high inhibition power.
2nd, the taxonomic identification of QBA-3 bacterial strains
1st, Molecular Identification：
1), primer：27f/1492r
27f：5′-AGAGTTTGATCCTGGCTCAG-3′(SEQ ID No:1)；
1492r：5′-TACGGCTACCTTGTTACGACTT-3′(SEQ ID No:2)；
2), PCR reaction systems are as follows：2×PCR Master Mix：12.5ul；Primer 2 7f：1ul；Primer 1492r：
1ul；Template：1ul；ddH2O：9.5ul；
PCR response procedures are as follows：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extensions
1.5min；30 circulations, 72 DEG C of renaturation；
Glue reclaim PCR primer, carries out TA clones, and picking single bacterium colony send sequencing company to be sequenced.
3) it is, as follows through sequencing, the 16SDNA fragment sequences of the bacterial strain QBA-3：
16sDNA sequences (SEQ ID No:3)：
AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGAAGCTTGC
TTCTCTTGAGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGAC
GCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGAT
TAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAA
CTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCAT
GCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTTACCTAATACGTGATT
GTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTT
AATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTAGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGA
ACTGCATTCAAAACTGACTGACTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATA
TAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAAC
AGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCA
GCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACA
AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCTAGA
GATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT
AAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGA
CAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTC
GGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACT
CGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACA
CCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTTACCACGGTGTG
ATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTA；
4), conclusion：Compared by ncbi database, bacterium QBA-3 to be detected and pseudomonad YJQ-1216S DNA,
GenBank:Gb | AY569288.1 | sequence identity be 99%.
2nd, Physiology and biochemistry is identified：
Reference《Common bacteria system identification handbook》Described in relevant pseudomonas physiological and biochemical property, select 18
Kind physiological and biochemical test identifies that experimental result is as shown in table 1 to purpose bacterial strain QBA-3, it is determined that the bacterial strain screened
QBA-3 is Pseudomonas cepacia.
The bacterial strain QBA-3 of table 1 physiological and biochemical property parameter
Note：N=does not report ∨=indefinite
The Pseudomonas cepacia QBA-3 that the present invention is screened carries out culture presevation, depositary institution：Chinese Typical Representative culture
Collection (CCTCC)；Address：Luojiashan, Wuchang, Wuhan City, Hubei Province, Wuhan University's collection；Preservation date：2015 6
The moon 25, Pseudomonas cepacia Pseudomonas cepacia deposit number is CCTCC M 2015402.
3rd, biological and ecological methods to prevent plant disease, pests, and erosion experiment of the QBA-3 bacterial strains to ash arrhizus bacteria
1st, inhibitory action of the ferment filtrate of QBA-3 bacterium to spore germination
Experimental method：The single bacterium colony of picking is inoculated into (3 g/l of yeast extract, egg in liquid 3ml YEPD culture mediums
White 10 g/l of peptone, 20 g/l of glucose) concussion and cultivate 12h under the conditions of 25 DEG C of 130rpm.Take the bacterium solution of culture by 2% volume
Concentration is inoculated in 20mlYEPD culture mediums, concussion and cultivate 96h under the conditions of 28 DEG C of 130rpm, then under the conditions of 25 DEG C of 12000rpm
Centrifugation, filter filtering of the supernatant through a diameter of 0.22um, obtains the ferment filtrate (fermentation containing QBA-3 bacterium fermentates
The tunning of QBA-3 bacterium, only QBA-3 bacterium is not contained in filtrate).
To the ferment filtrate carry out various concentrations dilution (specific diluted concentration be 10%, 25%, 50%, 75%,
100%) the filtrate 30ul of various concentrations, wherein gray mold (, are separately added into the concave slide of sterilizing using the detection of concave slide method
The conidial concentration of bacterium is 6 × 105/ ml, 25 DEG C of moisturizing cultures, control conidia germination rate are different dense up to being detected when 90%
Degree ferment filtrate processing in conidial germination rate and calculate inhibiting rate of the filtrate to conidia germination).
Experimental result suppresses ash arrhizus bacteria spore germination as shown in figure 1, the ferment filtrate of 10%-100% concentration all has
Effect, and with the raising of filter liquor concentration, the inhibiting rate of ash arrhizus bacteria conidia germination is significantly improved, i.e., to grey mold
The inhibiting rate of germ conidia germination is in dose dependent.
2nd, the influence that the ferment filtrate of QBA-3 bacterium extends to germ tube：
Experimental method：Detected using concave slide method ibid, after cultivating 6h-12h, micro- sem observation simultaneously measures.It is real
Result is tested as shown in Fig. 2 in the ferment filtrate content is respectively 0-100% processing, at same time point, with filtrate
The raising of concentration, higher to the inhibiting rate of germ tube elongation, i.e., the inhibiting rate to the elongation of ash arrhizus bacteria conidium germ tube is in dosage
Dependence.
3rd, experiment of the ferment filtrate of QBA-3 bacterium to excised leaf：
Experimental method：Respectively with the consistent healthy tomato leaf of the ferment filtrate spraying treatment growing ways of the various concentrations,
The sterile water process of CK is compareed, after filtrate is dried completely on blade, point connects the conidium 5 × 10 of ash arrhizus bacteria5Individual/ml,
23 DEG C of moisturizing processing, investigate incidence, calculate disease index, the result of inoculation experiments is as shown in table 2, QBA-3 hair after 3 days
Ferment filtrate, up to 92.5%, effectively can be effectively prevented and treated graw mold of tomato the prevention effect of graw mold of tomato.
Preventing and treating experimental result of the QBA-3 strain fermentations filtrate of table 2 to ash arrhizus bacteria
4th, the inhibitory action of bacterium solution containing QBA-3 bacterium to ash arrhizus bacteria
The present embodiment also carried out the bacterium solution containing QBA-3 bacterial strains to the Inhibition test of ash arrhizus bacteria spore germination, to bud
The Inhibition test of pipe elongation and excised leaf experiment, test result indicates that when the bacterial content of QBA-3 in bacterium solution is OD600=
Sprouting conidial to ash arrhizus bacteria, the elongation of germ tube are respectively provided with obvious inhibitory action during 0.314-0.565, work as OD600=
When 0.503, inhibition is best, and inhibiting rate is up to 99.5%.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality
Example is applied the present invention is described in detail, for the person of ordinary skill of the art, still can be to foregoing implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replaced
Change, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.
SEQUENCE LISTING
<110>Qingdao Agricultural University
<120>One plant of Pseudomonas cepacia bacterial strain QBA-3 for having inhibitory action to ash arrhizus bacteria and its application
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
tacggctacc ttgttacgac tt 22
<210> 3
<211> 1501
<212> DNA
<213>Pseudomonas cepacia
<400> 3
agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggtagagaga agcttgcttc tcttgagagc ggcggacggg tgagtaatgc ctaggaatct 120
gcctggtagt gggggataac gttcggaaac ggacgctaat accgcatacg tcctacggga 180
gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt 240
ggtgaggtaa tggctcacca aggcgacgat ccgtaactgg tctgagagga tgatcagtca 300
cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga atattggaca 360
atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag 420
cactttaagt tgggaggaag ggcagttacc taatacgtga ttgttttgac gttaccgaca 480
gaataagcac cggctaactc tgtgccagca gccgcggtaa tacagagggt gcaagcgtta 540
atcggaatta ctgggcgtaa agcgcgcgta ggtggttagt taagttggat gtgaaatccc 600
cgggctcaac ctgggaactg cattcaaaac tgactgacta gagtatggta gagggtggtg 660
gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg 720
accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 780
accctggtag tccacgccgt aaacgatgtc aactagccgt tgggagcctt gagctcttag 840
tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg ttaaaactca 900
aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggccttg acatccaatg aactttctag agatagattg gtgccttcgg 1020
gaacattgag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg ggcactctaa 1140
ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1200
acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa gccgcgaggt 1260
ggagctaatc ccataaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1320
agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc ccgggccttg 1380
tacacaccgc ccgtcacacc atgggagtgg gttgcaccag aagtagctag tctaaccttc 1440
ggggggacgg ttaccacggt gtgattcatg actggggtga agtcgtaaca aggtaaccgt 1500
a 1501
Claims (6)
1. one plant of Pseudomonas cepacia bacterial strain QBA-3 for having inhibitory action to ash arrhizus bacteria, it is characterised in that：Its Classification And Nomenclature is
Pseudomonas cepacia Pseudomonas cepacia, are preserved in China typical culture collection center, and its deposit number is
Sprout conidial to ash arrhizus bacteria of CCTCC M 2015402, the bacterial strain QBA-3 has very high inhibition power；The bacterial strain
QBA-3 has flagellum.
2. the answering in the biological prevention and control agent for preparing preventing and treating ash arrhizus bacteria of the Pseudomonas cepacia bacterial strain QBA-3 described in claim 1
With, it is characterised in that：The bacterium solution and ferment filtrate of the Pseudomonas cepacia bacterial strain QBA-3 is stretched to ash arrhizus bacteria spore and germ tube
Length is respectively provided with inhibitory action.
3. Pseudomonas cepacia bacterial strain QBA-3 according to claim 2 is in the biological prevention and control agent for preparing preventing and treating ash arrhizus bacteria
Application, it is characterised in that：Bacterial content is OD in the bacterium solution of the Pseudomonas cepacia bacterial strain QBA-3600During=0.314-0.565
Conidial to ash arrhizus bacteria sprouting and the elongation of germ tube are respectively provided with to be acted on compared with high inhibition.
4. Pseudomonas cepacia bacterial strain QBA-3 according to claim 2 is in the biological prevention and control agent for preparing preventing and treating ash arrhizus bacteria
Application, it is characterised in that：The preparation process of the ferment filtrate of the Pseudomonas cepacia bacterial strain QBA-3 is：By the bacterial strain
QBA-3 is inoculated into liquid YEPD culture mediums the concussion and cultivate 72-96h at 25 DEG C -28 DEG C, is then centrifuged for, and filtering, is contained
The ferment filtrate of QBA-3 bacterium.
5. Pseudomonas cepacia bacterial strain QBA-3 according to claim 4 is in the biological prevention and control agent for preparing preventing and treating ash arrhizus bacteria
Application, it is characterised in that：The bacterial strain QBA-3, which is inoculated into culture medium under the conditions of 130rpm, carries out concussion and cultivate.
6. Pseudomonas cepacia bacterial strain QBA-3 according to claim 4 is in the biological prevention and control agent for preparing preventing and treating ash arrhizus bacteria
Application, it is characterised in that：Filter through a diameter of 0.22um is filtered.
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| CN109055238B (en) * | 2018-08-29 | 2020-04-28 | 青岛农业大学 | Aspergillus flavus strain TR32 with inhibiting effect on plant pathogenic bacteria and application thereof |
| CN110862937B (en) * | 2019-11-05 | 2020-05-26 | 青岛农业大学 | Burkholderia forestensis MP5 strain with bacteriostatic action and application thereof |
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| CN101698827A (en) * | 2009-09-10 | 2010-04-28 | 中国农业大学 | Erythrochromogenes and use thereof in biological control of diseases |
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