CN107691479A - A kind of penicillium oxalicum microbial inoculum and its preparation method and application - Google Patents
A kind of penicillium oxalicum microbial inoculum and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of penicillium oxalicum bacteria agent, including cultivating seeds, expansion culture, liquid film extraction, prepares microbial inoculum.The penicillium oxalicum bacteria agent can prevent and treat a variety of diseases of garden stuff by single bacterial strain, to the bacteriostasis rates of a variety of diseases of garden stuff such as Prospect on Kiwifruit Bacterial Canker, Bacterial septicaemia syndrome, ring rot of apple, dry rot of potato and peach brown rot up to more than 90%, has a broad antifungal spectrum, it can effectively reduce cost accounting.
Description
Technical field
The present invention relates to microorganism field, more particularly to a kind of penicillium oxalicum microbial inoculum and preparation method thereof and in preventing and treating fruits and vegetables
Application in Major Diseases.
Background technology
The plant pathogenic fungis such as peach brown rot, Bacterial septicaemia syndrome, Prospect on Kiwifruit Bacterial Canker, ring rot of apple are endangered to crops
Evil is serious, and difficulty of prevention and cure is big.Chemical pesticide is the effective means for preventing and treating plant disease at present, is ensureing higher yield of crops
Huge effect has been played with terms of stable yields.But residual, resistance caused by long-term a large amount of unreasonable uses of traditional chemical agricultural chemicals
It is rampant again, cause the common concern of society.Seek and develop increasingly to compel the novel agrochemical of human health and ecological safety
Cut.
Emulsion liquid membrane extraction has solvent extraction concurrently and film permeates two technical characterstics, can be divided into emulsion liquid membrane by its structure
(emulsion liquid membrane) and supported liquid membrane (supported liquid membrane) two major classes.Emulsion
Film is the sphere that liquid surfactant is formed, and solution is divided into interior phase and foreign minister, liquid film only has several molecule thicks, unit volume
Surface area up to 1000-3000m2/m3, have that rate of extraction is fast, separation is completed with one step of concentration, can be from low concentration solution
The advantages that effectively reclaiming solute, but the stability problem of liquid film is relatively difficult to resolve certainly.Supported liquid membrane is to borrow the liquid phase for playing centrifugation
Capillarity is helped to be fixed in porous polymeric membranes.Supported liquid membrane can make extraction with being stripped in the both sides of liquid film while carrying out,
So as to the usage amount for avoiding the limitation of carrier load, reducing organic phase, solve the emulsion stability condition of emulsion liquid membrane and break
The problems such as newborn.The present invention uses supported liquid membrane.
Studied in the biological control of plant disease and mainly have bacterium class, Mycophyta and unwrapping wire using more microorganism
Bacterium.Fungi mainly has Trichoderma (Trichoderma) (such as Trichoderma harzianum (T.harzianum), koning trichoderma
(T.koningii), Trichoderma viride (T.viride) etc.), mould, rhizoctonia, saccharomycete, viscous broom is mould, shield shell is mould etc..There is research
Report from the penicillium purpurogenum of peach branch separation there is fine preventive effect to peach brown rot opportunistic pathogen；Paecilomyces lilacinus pair
Meloidogyne incognita (Meloidogyne incognita), cyst roundworm (Heteridera), white ball packing nematode
(Globoder pallida), puncture nematode (Tylenchulus), golden nematode, golden nematode and pearl nematode
(Nacobbus) there is very strong parasitic ability, by improving plant itself resistance against diseases come antibacterial (Jatala P.Biological
control of plant-parasitic nematodes[J].Ann Rev Phytopathol,1986,24:452-489)；
Shen radiance etc. screens Penicillium griseofulvum (Penicillium griseofulvum) and Aspergillus terreus (Aspergillus terreus)
Two kinds of Antagonistic Fungus have stronger antagonism (Shen radiance, Xue Quanhong, crystalline substance etc., Strawberry Root Rot antagonism to Strawberry Root Rot
Fungi screening is identified and its disease prevention growth-promoting effect [J] Scientia Agricultura Sinicas, 2012 (22)：4612-4626)；Kingdom's equality is from south
Square Chinese yew isolates a kind of new interior fungi Trichoderma taxi bacterial strain, and for treating rice sheath blight disease, (kingdom puts down, Lu Shuling, Zheng Bi
Strong effect [J] China for waiting endogenetic fungus Trichoderma taxi ZJUF0986 bacterial strains and its active metabolite preventing and treating rice sheath blight disease
Biology controls 2009 (01)：30-34)；Also there is study on prevention (king of the biological pesticide penicillium oxalicum aqua to corn southern leaf blight simultaneously
Bravely, the such as Zhang Wenge, He Lu biological pesticide penicillium oxalicum aquas to prevention effect [J] Agriculture of Anhui science of corn southern leaf blight,
2007,35 (7)：1965-1966), preventing and treating of the penicillium oxalicum to soybean cyst nematode Heterodera glycines also has been reported that.(Li Ting, Huang Wenkun, Peng De
It is good etc..3 kinds of anti-fungal zymotic fluids are to prevention effect [J] the Hua Zhong Agriculture University journal of soybean packing nematode, and 2017,01
(007)：42-46).However, on penicillium oxalicum in fruits and vegetables Major Diseases Prospect on Kiwifruit Bacterial Canker, Bacterial septicaemia syndrome, apple wheel
Line disease, the study on prevention in peach brown rot and application have no report.Pollen pini is pinaceae plant masson pine Pinus
Massoniana Lamb., Pinus tabulaeformis tabuliformis Carr. or the dry pollen for belonging to several plants together, containing rich
Rich protein, lipoid, amino acid, various trace elements, vitamin, enzyme, pigment and antibiotic etc., trained by nutrition of pollen pini
The research for supporting penicillium oxalicum enhancing sterilizing ability has no report.
The content of the invention
It is an object of the invention to provide a kind of preparation method of penicillium oxalicum bacteria agent, this method by seed culture,
Expand culture, liquid film extraction obtains the penicillium oxalicum bacteria agent can with preventing and treating fruits and vegetables Major Diseases.
Another object of the present invention is to provide a kind of penicillium oxalicum bacteria agent prepared using the above method.
Third object of the present invention is the application for providing above-mentioned penicillium oxalicum bacteria agent.
To achieve these goals, the present invention provides a kind of preparation method of penicillium oxalicum bacteria agent, comprises the following steps：
1) seed culture：Penicillium oxalicum is inoculated into plus taste PDA liquid medium, 25-28 DEG C of shaking table 180r/min are trained
Foster 7d obtains penicillium oxalicum bacteria culture fluid；
2) the penicillium oxalicum bacteria culture fluid that step 1) is cultivated is inoculated into by 10% inoculum concentration and adds taste PDA liquid medium,
Penicillium oxalicum suspension is made in 25-28 DEG C of shaking table 180r/min cultures 24h；
3) culture is expanded：By penicillium oxalicum suspension by volume 1：25~30 are inoculated into production tank culture medium, ventilation
2L/min is measured, 25-28 DEG C of fermentation tank 200r/min fermentations 72h obtains zymotic fluid；
4) 2% 200 mesh roots of Chinese trichosanthes are added in zymotic fluid, adsorb 15-30min, then with filtered through gauze, filter mycelia
With spore and other insoluble matters, fermentation pretreatment fluid is obtained；
Root of Chinese trichosanthes carries out a preliminary absorption, primary attachment impurity, through smallpox during filtered through gauze to zymotic fluid in this step
Powder absorption is removed into branch by filtered through gauze.
5) liquid film extraction：Fermentation pretreatment fluid is diluted into U=500mg/L with distilled water, system uses Celgard-2500-
30%TFA liquid films, pH=6-8 (preferably pH=7), 30 DEG C, rotating speed 400-500r/min, 28 ± 3 DEG C of temperature, add Na2CO3It is molten
Liquid is to final concentration of 15%Na2CO3, obtain extract.
Celgard-2500-30%TFA liquid films are made in Celgard-2500 coated on both sides 30%TFA (trifluoroacetic acid)
Into.
Wherein, described plus taste PDA liquid medium formula is：Peeled potatoes 200g, glucose 20g, pollen pini 20g, dimension
Raw plain C 0.5g, with distilled water constant volume to 1000mL.
It will add 110-121 DEG C of sterilizing 25min of composition in taste PDA liquid medium in addition to vitamin C, cooling during sterilizing
To room temperature, it is added to after vitamin C filtration sterilization in culture medium.
Taste PDA liquid medium is added to add pollen pini and vitamin C, wherein preserved egg on the basis of existing PDA culture medium
Powder is the spermatid of pine tree pistil, containing multiple proteins, amino acid, mineral matter, enzyme and coenzyme, nucleic acid, monose, polysaccharide etc. 200
Remaining kind of nutrition, and there is antibacterial effect, nutrition needed for growth is provided for penicillium oxalicum, vitamin C promotes penicillium oxalicum
Growth.
Wherein, the production tank culture medium prescription is：Maize flour 1500g, 40 mesh corn stalk powder 12000g, pollen pini
620g、K2HPO4 600g、FeSO41.5g, add water to 30000mL, 121 DEG C of sterilizing 30min.
Pollen pini is added in the culture medium for expanding culture, wherein pollen pini has antibacterial effect, while is penicillium oxalicum
Bacterium provides nutrition needed for growth.
Wherein, the penicillium oxalicum bacteria agent adds auxiliary material and spray, smoke agent, aqua, emulsifying agent, wettable powder is made
Agent, pulvis, granule, tablet or effervescent agent.
The present invention also provides penicillium oxalicum bacteria agent prepared by above-mentioned preparation method.
The present invention also provides above-mentioned penicillium oxalicum bacteria agent and is suppressing peach brown rot, Bacterial septicaemia syndrome, ring rot of apple, horse
Application in bell potato dry rot, Prospect on Kiwifruit Bacterial Canker.
Penicillium oxalicum (Penicillium oxalicum), the bacterium conidiophore is upright, be highly 100-200 μm,
It is colourless, have separation, have brush stigma, stigma grow thickly in branch top in bouquet shape, conidium in chain-like growth, it is spherical extremely
It is oval, colourless extremely to colour, be smooth, it is big 4.5-6.5 × 3.0-4.0 μm；Bacterium colony is in first scatterplot shape, after growth in flakes；Bacterium colony side
It is edge pale, rough, it is yellowish green to blackish green after sporogenesis.Penicillium oxalicum is fermented with the culture medium containing pollen pini, hair
Zymotic fluid using liquid film extraction handle, prepare tool preventing and treating peach brown rot, Bacterial septicaemia syndrome, ring rot of apple, dry rot of potato,
The bactericide of Prospect on Kiwifruit Bacterial Canker.
The beneficial effects of the present invention are：
Penicillium oxalicum microbial inoculum prepared by a kind of penicillium oxalicum microbial inoculum preparation method provided by the invention has selectivity high, easy
Degraded, dosage is few, pollution is small, to people and animals' small toxicity, environment compatibility is good, is not likely to produce the advantages that resistance.Meanwhile biological liquid film
The penicillium oxalicum fermentation liquor preparation for the treatment of by extraction is water base property environmental protection preparation, is had to environment and operator safety, without organic
Solvent and dust, reduce the pollution to environment.This kind of penicillium oxalicum preparation can prevent and treat a variety of diseases of garden stuff by single bacterial strain,
To a variety of diseases of garden stuff such as Prospect on Kiwifruit Bacterial Canker, Bacterial septicaemia syndrome, ring rot of apple, dry rot of potato and peach brown rot
Bacteriostasis rate has a broad antifungal spectrum, can effectively reduce cost accounting up to more than 90%.
Embodiment
Material and reagent：
1. penicillium oxalicum (Penicillium oxalicum) is given by Beijing University of Chemical Technology professor Tian Pingfang.
2.Celgard-2500 purchase is from Celgard companies of the U.S., in Celgard-2500 coated on both sides 30% during use
TFA (trifluoroacetic acid), is made Celgard-2500-30%TFA liquid films.
3. primer is synthesized by Beijing Bioisystech Co., Ltd of farsighted Boxing section.
4. sequencing is carried out by the rich Deco skill Development Co., Ltd advanced in years in Beijing.
Culture medium：
1. add taste PDA liquid medium
Peeled potatoes 200g, glucose 20g, pollen pini 20g, vitamin C 0.5g, distilled water constant volume to 1000mL, sterilizing
When will add 121 DEG C of composition sterilizing 25min in taste PDA liquid medium in addition to vitamin C, be cooled to room temperature, vitamin C mistake
It is added to after filter sterilization in culture medium.
2. add taste PDA solid mediums
Peeled potatoes 200g, glucose 20g, agar 20g, pollen pini 20g, vitamin C 0.5g, distilled water constant volume arrive
1000mL。
It will add 121 DEG C of sterilizing 25min of composition in taste PDA solid mediums in addition to vitamin C during sterilizing, be cooled to room
Temperature, it is added to after vitamin C filtration sterilization in culture medium and mixes before agar solidification.
3. production tank culture medium prescription is：Maize flour 1500g, 40 mesh corn stalk powder 12000g, pollen pini 620g,
K2HPO4 600g、FeSO41.5g, add water to 30000mL, 121 DEG C of sterilizing 30min.
4.PDA solid mediums
Peeled potatoes 200g, glucose 20g, agar powder 15g distilled water constant volume to 1000mL.
By 121 DEG C of sterilizing 25min of PDA solid mediums during sterilizing.
5. sodium carboxymethylcellulose (CMC-Na) differentiates solid medium
CMC-Na 20g；Na2HPO42.5g；KH2PO41.5g；Peptone 2.5g；Agar 2g；Distilled water 1000mL；pH
7.2~7.4.
Penicillium oxalicum used in the present invention (Penicillium oxalicum) teaches favour by Beijing University of Chemical Technology Tian Pingfang
Give, and forensic re-examination is carried out to bacterial strain before use.
The forensic re-examination of the penicillium oxalicum of embodiment 1
First, Morphological Identification
By colony inoculation sodium carboxymethylcellulose (CMC-Na) differentiate solid medium on carry out colony morphological observation and
Sediments microscope inspection.The bacterial strain is after sodium carboxymethylcellulose (CMC-Na) differentiates solid medium culture, bacterium colony originally celadon,
Media surface is evenly distributed on, in wheel line shape, edge semi-transparent clear, bacterium colony back side dark green or blackish green.Microscope
Under visible hyphae colorless, conidiophore is scattered, is vertically born from mycelia, conidiophore long column shape, conidiophore bottom
It is non-limbed；There is diaphragm, the life of conidiophore top is arranged in branch between broom shape, and a branch top layer is stigma, and stigma tool bottle obstructs 3
Individual, basidixed is in the catenulate conidia of string not branch, the smooth subcircular of conidium, blue-green.According to《Fungal identification handbook》
It is accredited as Deuteromycotina (Deuteromyeotina), Hyphomycetes (Hypholnseetes), hyphomycetales (Moniliales), clump
Geng Bao sections (Moniliaceae), Penicillium (Penicillium) bacterial strain.
2nd, ITS is identified
The acquisition of 1.PCR templates：Genomic DNA is extracted as pcr template
Genomic DNA is extracted, it is specific as follows using CTAB methods：
1) the thalline 50-100mg that liquid nitrogen frozen is crossed is taken, 100mg is used in the present embodiment, 2.0mL is added after mortar grinder
In centrifuge tube；
2) 65 DEG C of preheated μ L of CTAB 800 (2%CTAB adds 0.5-1% beta -mercaptoethanols before using) are rapidly added,
65 DEG C of water-baths one hour are put into after well mixed.Shake once within every ten minutes, CTAB is fully mixed with thalline；
3) centrifuge tube is taken out to normal temperature, and 800mL chloroforms/isoamyl alcohol (24 is added after placing 2min：1) mixed liquor, will mix
Liquid slowly shakes 1-2min after being mixed with CTAB, CTAB is fully contacted with chloroform；
4) 12000rpm centrifuges 5-10min, and careful Aspirate supernatant adds 700 μ L again into new 2.0mL centrifuge tubes
Chloroform/isoamyl alcohol mixed liquor, slowly mixes 1-2min, centrifuges again, and Aspirate supernatant is into 1.5mL centrifuge tubes；
5) -20 DEG C of isometric precooled isopropanols are added into 1.5mL centrifuge tubes rapidly, are mixed, -20 DEG C of placement 2h
Left and right；
6) centrifuge tube of -20 DEG C of preservations is taken out, 12000rpm centrifugation 5-10min, discards liquid；
7) 1mL70% ethanol washing DNA precipitations are added, are air-dried；
8) 30-60 μ L ddH are used after air-drying2O dissolves, stand-by.
2.PCR
PCR primer uses fungi ITS universal primers
ITS1(SEQ ID No.1)：5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4(SEQ ID No.2)：5’-TCCTCCGCTTATTGATATGC-3’
The μ L of PCR reaction systems 25：2 × Taq PCR Mix 12.5mL, primer I TS1 1 μ L, primer I TS4 1 μ L, ddH2O
8.5 μ L, the μ L of DNA profiling 2.
PCR programs：94℃5min；94 DEG C of 40s, 55 DEG C of 40s, 72 DEG C of 90s, 30 circulations；72℃10min.
PCR uses 1% agarose gel electrophoresis after terminating, and PCR primer 606bp, is sequenced after purified recovery, sequencing knot
Fruit carries out sequence analysis with the sequence in DNAMAN version5.2.2 and BLAST software and GenBank, it was demonstrated that the bacterium is
Penicillium oxalicum.
Penicillium oxalicum (Penicillium oxalicum), by the use of 50% (V/V) glycerine as protective agent, -80 DEG C permanent
Preservation.
The Screening of Media of the penicillium oxalicum of embodiment 2
Penicillium oxalicum uses 8mm card punch under aseptic technique, selects the more single neat place's punching of bacterium colony, obtains
Bacteria cake 5 of the same size, is added to 100mL and adds in taste PDA liquid medium, cultivated at 25 DEG C on 180r/min shaking table
7d, it is transferred to next group by 10% inoculum concentration and is added in taste PDA liquid medium, is continued to cultivate 7d, press 10% again afterwards
Inoculum concentration be transferred to next group and added in taste PDA culture medium, continue to cultivate 7d, after according to said method transferring 5 times, take nutrient solution
0.1mL is transferred to plus taste PDA solid culture wares, spread plate, is placed in 25 DEG C of constant incubators and is cultivated 48h, choose bacterium colony compared with
It is inoculated in add for single neat single bacterium colony and is cultivated on taste PDA solid mediums.
Repeat to operate above with PDA culture medium simultaneously.
Embodiment 3：Penicillium oxalicum and various plants cause of disease thalline flat board dual test (common PDA culture medium)
Diameter 8mm cause of disease bacteria cake and penicillium oxalicum bacteria cake is prepared, cause of disease bacteria cake (8mm) and antagonism bacteria cake (8mm) are equidistant
From common PDA culture medium culture medium center both sides are placed on, straight line is formed；Do not put Antagonistic Fungi for blank control.Often locate
Reason is repeated 5 times, and puts 25 DEG C of constant incubator cultures, and inhibition zone size is observed after 5 days, and colony diameter is measured with crossing method.
It the results are shown in Table 1.As a result show, penicillium oxalicum to Monilinia fructicola, Botryosphaeria berengeriana f. sp, dry rot of potato bacterium, bacillary wear
The antagonism of hole disease is between 35-52%, hence it is evident that less than culture medium plus the inhibiting rate of pollen pini.
The penicillium oxalicum of table 1 is stood facing each other with phytopathogen (culture medium is not added with pollen pini) and inhibitory action
Note：Stand facing each other diameter (diameter of disease fungus when disease fungus co-cultures with penicillium oxalicum)
The disease fungus is specially Monilinia fructicola, Botryosphaeria berengeriana f. sp Bacterial septicaemia syndrome, Prospect on Kiwifruit Bacterial Canker.Suppression
Bacterium rate %=[(blank colony diameter -8)-(face-off colony diameter -8)] × 100%/(blank colony diameter -8)
Embodiment 4：Add taste PDA Liquid Cultures
Penicillium oxalicum Liquid Culture：100mL penicillium oxalicum bacterial strains are inoculated into sterilizing plus taste PDA liquid medium
In 1000mL, 28 DEG C, 180r/min, shaken cultivation 24h, penicillium oxalicum suspension is made.
Production tank culture medium each component formula includes：Maize flour 1500g, corn stalk powder (40 mesh) 12000g, pollen pini
620g、K2HPO4600g、FeSO41.5g, vitamin C 1.5g, add water to 30000mL.30000mL fluid nutrient mediums are poured into
50000mL fermentation tanks, sterilize 30min in 121 DEG C, is cooled to room temperature after taking-up, connects penicillium oxalicum suspension 100mL, initially
PH5, in 28 DEG C, 200r/min fermentations 72h.
PO1 liquid film extractions liquid is prepared：Zymotic fluid adds root of Chinese trichosanthes 600g to adsorb.Using Celgard-2500-30%TFA liquid
Film, PO1 zymotic fluid filtered through gauze, mycelia and spore and other insoluble matters are filtered, fermentation pretreatment fluid is obtained, is diluted with distilled water
U=500mg/L, pH=7,30 DEG C, rotating speed 400r/min, using 15%Na2CO3Solution extracts, and obtains extract (active compound).
Embodiment 5：The preparation of penicillium oxalicum aqua
Extract and each 300ml mixing of 50% sodium hydroxide are taken, 45 DEG C is heated to, reacts 1h, adjust pH value to add anti-to 7.0
Freeze liquid glycerine 5ml, preservative ethanol 5ml mixing, obtain aqua.
Water quality selects：The distilled water and well water of different zones, running water and distilled water, result of the test show, different quality
Had no adverse effects to preparing penicillium oxalicum aqua.
By increasing the expanding area on auxiliary agent increase target organismses, promote preparation quickly to absorb, improve resistance of rainwater washing against
Ability, small toxicity, it is environmentally safe the features such as.
Embodiment 6：Prevention effect of the liquid film extraction zymotic fluid to Bacterial septicaemia syndrome
2 groups of experiment point：Compare and be not added with pollen pini, without root of Chinese trichosanthes adsorbent, fermentation for PDA culture medium, production medium
Zymotic fluid of the liquid without EXTRACTION BY SUPPORTED LIQUID MEMBRANES；Test group adds taste PDA culture medium, production medium to add pollen pini, inhaled with root of Chinese trichosanthes
Attached dose, zymotic fluid use the OP1 zymotic fluids through EXTRACTION BY SUPPORTED LIQUID MEMBRANES；500 times of liquid are respectively prepared in two groups of controls, in Beijing 14
Number peach initial bloom stage (the flowers are in blossom about 20% when) spray once, after fallen flowers spray in 10 days once, 30 days observation bacteriums after being sprayed at second
Property shothole disease prevention effect, the results showed that, control group zymotic fluid preventive effect 61.2%, the test group zymotic fluid after EXTRACTION BY SUPPORTED LIQUID MEMBRANES
Preventive effect 94.5%.
Embodiment 7：Prevention effect of the liquid film extraction zymotic fluid to ring rot of apple
2 groups of experiment point：Compare and be not added with pollen pini, without root of Chinese trichosanthes adsorbent, fermentation for PDA culture medium, production medium
Zymotic fluid of the liquid without EXTRACTION BY SUPPORTED LIQUID MEMBRANES；Test group is plus taste PDA culture medium, production medium add pollen pini, use root of Chinese trichosanthes
Adsorbent, zymotic fluid use OP1 zymotic fluids.500 times of liquid are respectively prepared in two groups of controls, in Fuji apple spray on October 20 one
It is secondary, sprayed after 10 days once, 10 days observation ring rot of apple prevention effects after being sprayed at second, the results showed that, control group fermentation
Liquid preventive effect 72.6%, the test group zymotic fluid preventive effect 96.7% after EXTRACTION BY SUPPORTED LIQUID MEMBRANES.
Embodiment 8：Prevention effect of the liquid film extraction zymotic fluid to peach brown rot
Experiment is divided to two groups：Compare and be not added with pollen pini, without root of Chinese trichosanthes adsorbent, fermentation for PDA culture medium, production medium
Zymotic fluid of the liquid without EXTRACTION BY SUPPORTED LIQUID MEMBRANES；Test group is plus taste PDA culture medium, production medium add pollen pini, use root of Chinese trichosanthes
Adsorbent, zymotic fluid use OP1 zymotic fluids.1000 times of liquid are respectively prepared in two groups of controls, in big Kubo peach spray on July 13
Once, sprayed once after 10 days, observation in 10 days is to peach brown rot prevention effect after being sprayed at second, the results showed that, control group hair
Zymotic fluid preventive effect 75.8%, the zymotic fluid preventive effect 91.5% after EXTRACTION BY SUPPORTED LIQUID MEMBRANES.
Embodiment 9：Prevention effect of the liquid film extraction zymotic fluid to Prospect on Kiwifruit Bacterial Canker
Experiment is divided to two groups：Compare and be not added with pollen pini, without root of Chinese trichosanthes adsorbent, fermentation for PDA culture medium, production medium
Zymotic fluid of the liquid without EXTRACTION BY SUPPORTED LIQUID MEMBRANES；Test group adds taste PDA culture medium, production medium to add pollen pini, inhaled with root of Chinese trichosanthes
Attached dose, zymotic fluid use OP1 zymotic fluids.500 times of liquid are respectively prepared in two groups of controls, in long U.S. No. 2 actinidia tree initial bloom stages
(the flowers are in blossom about 20% when) spray once, after fallen flowers spray in 10 days once, observation Prospect on Kiwifruit Bacterial Canker preventing and treating in 30 days after being sprayed at second
Effect, the results showed that, without the control group zymotic fluid preventive effect 66.1% of EXTRACTION BY SUPPORTED LIQUID MEMBRANES, the experiment after EXTRACTION BY SUPPORTED LIQUID MEMBRANES
Group zymotic fluid preventive effect 93.7%.
Embodiment 10：Prevention effect of the liquid film extraction zymotic fluid to dry rot of potato
Experiment is divided to two groups：Compare and be not added with pollen pini, without root of Chinese trichosanthes adsorbent, fermentation for PDA culture medium, production medium
Zymotic fluid of the liquid without EXTRACTION BY SUPPORTED LIQUID MEMBRANES；Test group is plus taste PDA culture medium, production medium add pollen pini, use root of Chinese trichosanthes
Adsorbent, zymotic fluid use OP1 zymotic fluids.1000 times of liquid are respectively prepared in two groups of controls, in Favorita Ma Ling on April 12
Potato sprays once, is sprayed after 10 days once, and observation in 10 days is to dry rot of potato prevention effect after being sprayed at second, the results showed that,
Control group zymotic fluid preventive effect 73.8%, the zymotic fluid preventive effect 94.7% after EXTRACTION BY SUPPORTED LIQUID MEMBRANES.
Test period：2014.3-2016 March in year.
Penicillium oxalicum potted plant experiment and field experiment shown in the present invention show the penicillium oxalicum handled by supported liquid membrane
Zymotic fluid can reach more than 92% to the pathogenic bacteria inhibition of fruits and vegetables Common Diseases.
Described above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment
Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within
Enclose.
SEQUENCE LISTING
<110>Beijing Union University
<120>A kind of penicillium oxalicum microbial inoculum and its preparation method and application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Penicillium oxalicum
<400> 1
tccgtaggtg aacctgcgg 19
<210> 2
<211> 20
<212> DNA
<213> Penicillium oxalicum
<400> 2
tcctccgctt attgatatgc 20
Claims (10)
1. a kind of preparation method of penicillium oxalicum bacteria agent, it is characterised in that comprise the following steps：
1) seed culture：Penicillium oxalicum is inoculated into plus taste PDA liquid medium, 25-28 DEG C of shaking table 180r/min cultivate 7d
Obtain penicillium oxalicum bacteria culture fluid；
2) the penicillium oxalicum bacteria culture fluid that step 1) is cultivated is inoculated into by 10% inoculum concentration and adds taste PDA liquid medium, 25-28
Penicillium oxalicum suspension is made in DEG C shaking table 180r/min culture 24h；
3) culture is expanded：By penicillium oxalicum suspension by volume 1：25~30 are inoculated into production tank culture medium, throughput 2L/
Min, 25-28 DEG C of fermentation tank 200r/min fermentations 72h obtain zymotic fluid；
4) 2% 200 mesh roots of Chinese trichosanthes absorption 15-30min is added in zymotic fluid, then with filtered through gauze, must ferment pretreatment
Liquid；
5) liquid film extraction：Fermentation pretreatment fluid is diluted into U=500mg/L with distilled water, system uses Celgard-2500-30%
TFA liquid films, pH=6-8,30 DEG C, rotating speed 400-500r/min, 28 ± 3 DEG C of temperature, add Na2CO3Solution is to final concentration of 15%
Na2CO3, obtain extract.
2. the preparation method of penicillium oxalicum bacteria agent as claimed in claim 1, it is characterised in that further comprise：Step 7) is pressed
Extract：50% sodium hydroxide=1：1 mixing, stirring, is heated to 35-50 DEG C, reacts 1h, adjusts pH value 6-8, is filtered to remove impurity
And insoluble matter.
3. the preparation method of penicillium oxalicum bacteria agent as claimed in claim 1 or 2, it is characterised in that described plus taste PDA liquid training
Foster based formulas is：Peeled potatoes 200g, glucose 20g, pollen pini 20g, vitamin C 0.5g, arrived with distilled water constant volume
1000mL。
4. the preparation method of penicillium oxalicum bacteria agent as claimed in claim 1 or 2, it is characterised in that the production tank culture
Based formulas is：Maize flour 3-9%, 40 mesh corn stalk powder 30-50%, pollen pini 2-8%, K2HPO41.9-2.4%, FeSO4
0.005-0.1%, vitamin C 0.005-0.1%, surplus are water, 110-121 DEG C of sterilizing 30min.
5. such as the preparation method of penicillium oxalicum bacteria agent as claimed in claim 1 or 2, it is characterised in that the step 5) body
It is pH=7.
6. the formulation of penicillium oxalicum bacteria agent prepared by the preparation method as described in claim any one of 1-5 is spray, smog
Agent, aqua, emulsifying agent, wettable powder, pulvis, granule, tablet or effervescent agent.
7. application of the penicillium oxalicum bacteria agent as claimed in claim 6 in fruits and vegetables Major Diseases are prevented and treated, increase soil fertility.
8. application as claimed in claim 7, it is characterised in that the fruits and vegetables Major Diseases selection peach brown rot, bacillary wear
Hole disease, ring rot of apple, dry rot of potato or Prospect on Kiwifruit Bacterial Canker.
9. a kind of add taste PDA liquid medium for penicillium oxalicum seed culture, it is characterised in that adds taste PDA liquid to train
Foster based formulas is：Peeled potatoes 200g, glucose 20g, pollen pini 20g, vitamin C 0.5g, arrived with distilled water constant volume
1000mL。
A kind of 10. production tank culture medium for expanding culture for penicillium oxalicum, it is characterised in that production tank culture medium prescription
For：Maize flour 1500g, 40 mesh corn stalk powder 12000g, pollen pini 620g, K2HPO4 600g、FeSO41.5g, vitamin C
1.5g, add water to 30000mL, 121 DEG C of sterilizing 30min.
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| CN109224364A (en) * | 2018-09-27 | 2019-01-18 | 中南大学 | A method of Bayer process red mud alkalinity is reduced using penicillium oxalicum |
| CN110964644A (en) * | 2019-10-21 | 2020-04-07 | 西北农林科技大学 | Penicillium, and fermentation method and application thereof |
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|---|---|---|---|---|
| CN109224364A (en) * | 2018-09-27 | 2019-01-18 | 中南大学 | A method of Bayer process red mud alkalinity is reduced using penicillium oxalicum |
| CN110964644A (en) * | 2019-10-21 | 2020-04-07 | 西北农林科技大学 | Penicillium, and fermentation method and application thereof |
| CN110964644B (en) * | 2019-10-21 | 2023-09-19 | 西北农林科技大学 | Penicillium and fermentation method and application thereof |
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Application publication date: 20180216 |