CN105176831B - A kind of penicillium oxalicum and its application - Google Patents
A kind of penicillium oxalicum and its application Download PDFInfo
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- CN105176831B CN105176831B CN201510410093.XA CN201510410093A CN105176831B CN 105176831 B CN105176831 B CN 105176831B CN 201510410093 A CN201510410093 A CN 201510410093A CN 105176831 B CN105176831 B CN 105176831B
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Abstract
The invention discloses a kind of penicillium oxalicum and its applications.The penicillium oxalicum preservation is entitledPenicillum oxalicum SG4；Depositary institution: China typical culture collection center；Preservation address is China, Wuhan, Wuhan University；Preservation date: 2015-4-30；Deposit number are as follows: CCTCC M 2015270.The present invention the experiment proved that, penicillium oxalicum thallus has very strong inhibiting effect to citrus Penicillium citrinum bacterium, it can be used for preparing citrus bio-preservative, provide new direction to develop the biological preservation of new bio-preservative and citrus, there is good economic and social benefit.
Description
Technical field
The present invention relates to one plant of penicillium oxalicum and its applications, and in terms of being mainly used in citrus biological preservation, the oxalic acid is green
Mould can be used for antagonism citrus Penicillium citrinum bacterium.
Background technique
Penicillium italicum is mainly caused by Penicillium citrinum germ (Penicillium italicum), and early stage pericarp is soft
Change, water stain shape fades, and hereafter goes out aerial hyphae in scab director, forms the white mustiness object of a thickness, and is extended to rapidly white
Color subcircular mildew.Main harm growth period ripening fruits and storage phase fruit, have seriously affected Aspects In The Development of Citrus Industry.
Traditionally common citrus is fresh-keeping refrigeration, controlled atmosphere and a chemical fresh-keeping method, however refrigerate and controlled atmosphere it is at high cost,
Energy consumption is big and unstable quality is gradually eliminated, and chemical fresh-keeping method is there are potential food safety and problem of environmental pollution,
And long-time service chemicals is fresh-keeping, and citrus pathogenic bacteria can generate drug resistance to it.However biological way of keeping fresh overcome it is above
The unfavorable factor and drawback of fresh-keeping method have many advantages, such as that processing target is clear, at low cost, green and pollution-free.Therefore biology
Preservation technology is increasingly valued by people, if: Cai Wentao in capsicum from isolating and having fungistatic effect to capsicum pathogenic bacteria
Endophyte strain-L and strain-39, and study its biological control effect.The result shows that: strain-39 fermentation liquid acetic acid second
When ester extract quality concentration is 100mg/ml, capsicum and tomato decayed fruit rate decline 51.879% and 58.47% respectively；75% second
When alcohol sediment is 100mg/mL, capsicum rotting rate has dropped 47.50%, and tomato rotting rate has dropped 50.47%.Li Meicong mandarin orange
Isolated G-3, G-4 for having antagonism to Penicillium italicum and citrus common green mold in orangery soil.And use silica gel column chromatography
Isolation technics isolates three kinds of active constituents from its fermentation liquid: active constituent 1 is respectively to the inhibiting rate of Citrus, green mould
48.32%, 60.83%；Active constituent 2 is respectively 48.01%, 57.35% to the inhibiting rate of Citrus, green mould；Activity at
Divide 3 pairs of Citrus, the inhibiting rate of green mould is respectively 35.28%, 55.79%.
Penicillium is a kind of omnivorousness fungi, can be grown in any matrix containing organic matter.The spore of mould is heat-resisting
Property it is stronger, thallus the bread worm is lower, and common acid is its favorite carbon source in beverage, Penicillium production and clinically
Have and is widely applied very much.Such as: the antibiotic clinically applied --- penicillin is exactly extracted from mould, another important
Antibiotic --- griseofulvin is generated by Penicillium griseofulvum, is the best antibiotic for inhibiting fungal dermatopathy.
The present invention isolated one plant of penicillium oxalicum from M. laxiflora leaf, it has well citrus Penicillium citrinum bacterium
Inhibiting effect, the citrus bio-preservative to develop new provide technical support.
Summary of the invention
An object of the present invention is to provide a kind of penicillium oxalicum (Penicillum oxalicum) SG4 bacterial strain, the grass
Sour Penicillium notatum thallus has very strong inhibiting effect to citrus Penicillium citrinum bacterium, can be used for preparing citrus bio-preservative, new to develop
Bio-preservative and citrus biological preservation provide new direction.
Penicillium oxalicum (Penicillum oxalicum) SG4 is from M. laxiflora
(MyricariaLaxiflora) endogenetic fungus obtained in root is preserved in Chinese Typical Representative culture on April 30th, 2015
Collection, deposit number are as follows: CCTCC M2015270.Preservation address is China, Wuhan, Wuhan University.
Penicillium oxalicum (Penicillum oxalicum) SG4 of the present invention has following microbiology in PDA culture medium
Characteristic: white mycelium filiform is shorter, a large amount of celadon spores.
It is an object of the present invention to provide a kind of application of penicillium oxalicum on the rotten microbial inoculum of preparation biological control citrus.Institute
The citrus stated rots for disease caused by citrus Penicillium notatum.
The present invention also provides a kind of penicillium oxalicums to prepare the application on antistaling agent.The penicillium oxalicum may be used also
To replace with the fermentation liquid of penicillium oxalicum bacteria strain or the extract of fermentation liquid.
The present invention is using the DNA of penicillium oxalicum (Penicillum oxalicum) SG4 as template, upstream primer ITS1:
5 '-TCCGTAGGTGAACCTGCG-3 ', downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' carry out bacterium colony PCR
Amplification, obtains the 18S rDNA partial sequence (SEQ ID NO:1) of 560bp.The sequence included by Blast and GenBank into
Row compares, it is found that the homology of SG4 and penicillium oxalicum is 100%.It is built chadogram (Fig. 4), is identified bacterial strain (SG4) with MEGA5
It is nearest with the affiliation of one plant of P.oxalicum (HQ680452).
Detailed description of the invention
Fig. 1 is the colonial morphology of penicillium oxalicum (Penicillum oxalicum) SG4 of the invention.
Fig. 2 is the microscopic morphology of penicillium oxalicum (Penicillum oxalicum) SG4 of the invention.
Fig. 3 is penicillium oxalicum (Penicillum oxalicum) SG4 thallus of the invention to citrus Penicillium citrinum bacterium antagonism
Function and effect.
Fig. 4 is the 18S rDNA systematic growth of penicillium oxalicum (Penicillum oxalicum) SG4 bacterial strain of the invention
Tree.
Specific embodiment
Embodiment one: the separation and preservation of penicillium oxalicum (Penicillum oxalicum) SG4
M. laxiflora root is acquired on the domestic kermes dam in Three Gorges basin Yichang within 2012, be inoculated in and add after surface sterilization
Working concentration is that the PDA solid medium of 25 μ g/ml penicillin separates fungi.
Surface sterilization: taking M. laxiflora root tap water to rinse 5 times, and after aseptic filter paper is dried, 75% alcohol impregnates
1min, aseptic water washing 3 times, then 60S is impregnated with 3% sodium hypochlorite, it then uses aseptic water washing 5 times, aseptic filter paper washes dry root
Surface moisture.
Culture: being cut into 0.5*0.5cm size for the root after surface sterilization with sterile scissors, is inoculated in plus working concentration is 25
μ g/ml penicillin P DA solid medium (potato 200g/L, glucose 20g/L, agar 20g/L, Ph6.0-6.5), is placed in 28
It is cultivated at DEG C.
Bacterial strain purifying: after growing bacterium colony on plate, new PDA solid medium is inoculated according to form picking single colonie
In, it is placed at 28 DEG C and cultivates.
Bacterial strain preservation: after purification to bacterial strain, the pure strain inoculated of picking is in PDA slant medium, 28 DEG C of culture 4d, with sterilizing
Atoleine envelope be hidden in 4 DEG C of preservations.
The bacterial strain penicillium oxalicum (Penicillum oxalicum) is subjected to preservation, the entitled SG4 of preservation；Preservation list
Position: China typical culture collection center；Preservation address is China, Wuhan, Wuhan University；Preservation date: 2015-4-30；It protects
Hiding number are as follows: CCTCC M 2015270.
Embodiment two: penicillium oxalicum (Penicillum oxalicum) SG4 identification
The analysis of 18S rDNA sequence: colony PCR amplification 18S rDNA sequence is carried out using universal primer.Upstream primer
ITS1:5 '-TCCGTAGGTGAACCTGCG-3 ', downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '.Reactant
It is as follows:
Reaction condition: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 1min, 56 DEG C of annealing 30sec, 72 DEG C of extension 1.min, 33
Circulation, last 72 DEG C of extensions 10min.PCR product is detected with 1% agarose gel electrophoresis, and the band of picking 750bp or so carries out
The raw work sequencing in sea is served in gel recycling.
The sequence measured is committed to GenBank, and obtains accession number KR024319.By this sequence in GenBank into
Row Blast is compared, and chooses the sequence MEGA5 software building 18S rDNA phylogenetic tree of the higher bacterial strain of homology.
Apply example three: the preparation of penicillium oxalicum (Penicillum oxalicum) SG4 thallus
Actication of culture: going bail for the penicillium oxalicum strain streak inoculation for depositing 4 DEG C of refrigerators in PDA plate, at 28 DEG C, culture
4d obtains thallus.
Example IV: penicillium oxalicum (Penicillum oxalicum) SG4 thallus makees citrus Penicillium citrinum bacterium antagonism
With.
The preparation of agar block: M. laxiflora endogenetic fungal bacterial strain is inoculated in respectively by PDA culture using plate streak
On base, 4d is cultivated at 28 DEG C, is punched on being covered with the agar plate for examination bacterium with punch (diameter 10mm), to prepare bacterium
Cake.Tablet face-off method detects thallus to citrus Penicillium citrinum bacterium bacteriostatic activity: Penicillium citrinum bacterium is inoculated in PDA slant medium, 28
DEG C culture 4d scrapes all spores with oese in test tube plus 9ml sterile water, and spore suspension is made, takes 500 μ l citruses
Penicillium citrinum bacterium spore suspension, coating uniformly, M. laxiflora endogenetic fungus bacteria cake are placed on plate, the bacterium in an area of plate
Cake forward direction places (bacterium is face-up), and another area is oppositely positioned (bacterium is face-down), cultivates 4d respectively at 28 DEG C, observes result.
Antibacterial result:
SEQUENCE LISTING
<110>applicant SanXia University
<120>a kind of penicillium oxalicum and its application
<210> SG4
<211> 546
<212> DNA
SEQ ID NO:1
GGCGTCGGAA GGATCATTAC CGAGTGAGGG CCCTCTGGGT CCAACCTCCC ACCCGTGTTTA
TCGTACCTTG TTGCTTCGGC GGGCCCGCCT CACGGCCGCC GGGGGGCATC CGCCCCCGGGC
CCGCGCCCGC CGAAGACACA CAAACGAACT CTTGTCTGAA GATTGCAGTC TGAGTACTTG
ACTAAATCAG TTAAAACTTT CAACAACGGA TCTCTTGGTT CCGGCATCGA TGAAGAACGCA
GCGAAATGCG ATAAGTAATG TGAATTGCAG AATTCAGTGA ATCATCGAGT CTTTGAACGCA
CATTGCGCCC CCTGGTATTC CGGGGGGCAT GCCTGTCCGA GCGTCATTGC TGCCCTCAAGC
ACGGCTTGTG TGTTGGGCTC TCGCCCCCCG CTTCCGGGGG GCGGGCCCGA AAGGCAGCGG
CGGCACCGCG TCCGGTCCTC GAGCGTATGG GGCTTCGTCA CCCGCTCTGT AGGCCCGGCCG
GCGCCCGCCG GCGAACACCA TCAATCTTAA CCAGGTGACC TCGGATCAGG TAGGACCCGA
Claims (2)
1. penicillium oxalicum (Penicillum oxalicum) SG4 preparing disease caused by biological control citrus Penicillium notatum
Application on microbial inoculum, the penicillium oxalicum (Penicillum oxalicum) SG4 is preserved on April 30th, 2015
China typical culture collection center, deposit number are as follows: CCTCC M 2015270, preservation address are Hubei, Wuhan, and Wuhan is big
It learns.
2. application described in claim 1, which is characterized in that penicillium oxalicum (Penicillum oxalicum) SG4 be from
The endogenetic fungus obtained in the root of M. laxiflora.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929430A (en) * | 2017-02-10 | 2017-07-07 | 三峡大学 | One plant of penicillium oxalicum SY 15 with strong anti-oxidation and its application |
| CN107691479A (en) * | 2017-09-22 | 2018-02-16 | 北京联合大学 | A kind of penicillium oxalicum microbial inoculum and its preparation method and application |
| CN108823110B (en) * | 2018-07-26 | 2021-07-16 | 福州工微生物科技有限公司 | Strain for producing griseofulvin and application thereof |
| CN108841733B (en) * | 2018-07-26 | 2021-09-28 | 福州工微生物科技有限公司 | Strain and method for producing griseofulvin serving as major component of tranexamycin |
| CN108841889B (en) * | 2018-07-26 | 2021-09-28 | 福州工微生物科技有限公司 | Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation |
| CN109608517A (en) * | 2019-01-18 | 2019-04-12 | 三峡大学 | A kind of novel antimicrobial peptide and the application in citrus is fresh-keeping |
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| WO2006032530A1 (en) * | 2004-09-24 | 2006-03-30 | Bionext | Compositions for use against one or more pathogens |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006032530A1 (en) * | 2004-09-24 | 2006-03-30 | Bionext | Compositions for use against one or more pathogens |
| CN101031203A (en) * | 2004-09-24 | 2007-09-05 | 生物耐克斯特公司 | Compositions for use against one or more pathogens |
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| 拮抗菌的分离、筛选及对柑橘霉变的抑制效果;黄丽婵;《中国优秀硕士学位论文全文数据库 农业科技辑》;20120315(第03期);D046-64 * |
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