CN105861340A - Pichia manshurica for controlling postharvest diseases of oranges and grapes - Google Patents
Pichia manshurica for controlling postharvest diseases of oranges and grapes Download PDFInfo
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- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
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Abstract
The invention discloses a Pichia manshurica for controlling postharvest diseases of oranges and grapes, and belongs to the field of biological control of postharvest diseases of fruits. A strain is identified as Pichia manshurica, and is named as Pichia manshurica Y4, and the preservation number of the Pichia manshurica Y4 is CGMCC 2.5415. When the Pichia manshurica is used, the Pichia manshurica is activated, cultured and centrifuged to obtain thalli, and the thalli are diluted with sterile water to prepare a thallus suspension with the concentration of 1.0*10<8>/mL. The suspension is sprinkled to the exocuticle of the fruits through using a watering pot, the fruits are air-dried, the dried fruits are put in a plastic basket, the plastic basket is sealed with a preservative film, and the sealed fruits are stored under room temperature conditions. The incidence of orange Penicillium digitatum, grape Aspergillus niger and gray molds is substantially reduced after treatment with the Pichia manshurica. The Pichia manshurica can substitute chemical bactericides to prevent and control postharvest diseases of oranges and grapes, avoids harms of the chemical bactericides to people, and has substantial economic and social benefits.
Description
Technical field
The present invention relates to strain helmet shape Pichia pastoris (Pichia manshurica) Y4, this bacterial strain can control oranges and tangerines and adopt rear green mold
After disease and grape harvest, aspergillus niger disease, gray mold, belong to biologically controlling postharvest disease of fruit field.
Background technology
The heavy losses that fruit causes because of postharvest decay become global problem already, according to conservative estimation, have in developed country
The fresh fruit of 10-20% is lost in rotting after adopting, and lacks the developing country of storing refrigerating equipment, the height that its rotten loss rate has
Reaching 40-50%, the fruit of China rots general at 20-30%.
China is Production of fruit maximum in the world and sells big country, and types of fruits is various, and yield accounts for the 80% of world's fruits output.
Due to Production of fruit away from city, and maturity period Relatively centralized again, therefore during storage and transport owing to rotting, dehydration
Cause with the phenomenon such as nutrition leak and lose after adopting in a large number.Showing according to data in the early time, the annual fruit of China rots more than 80,000,000
Ton, the economic loss thus resulted in reaches 75,000,000,000 yuan, accounts for more than the 30% of the whole output value, and present numerical value should be able to be higher.
After causing fruit to be adopted, loss and deterioration is a lot of because have, including the weightlessness caused due to moisture and nutrient consumption with wither
Fission etc. quality；Owing to adopting the physiological metabolism imbalance that rear adverse circumstance causes；Owing to adopting fruit tissue's structure that after ripening aging causes
Collapse and due to pathogen infect cause rot etc..Wherein pathogen infection cause rotten be considered as cause fruit to be adopted after damage
The main cause lost.
Cause the fungal species rotted during fruit storage a lot, but every kind of dominant pathogenic bacteria of fruit typically have 2-3 kind.Example
As, after (1) grape harvest by fungus-caused disease be mainly aspergillus niger belong to (Aspergillus sp) aspergillus niger of causing sick and by
The gray mold that Botrytis cinerea (Botrytis cinerea) causes, the gray mold wherein caused by light grey and pinkish mould (B.cinerea) is
Grape endangers the disease of maximum in producing, the underproduction 20%～40% is caused in the whole world every year, serious up to more than 60%.Light grey and pinkish
Mould not only can cause field to lose, and remains in the pathogen on grape at storage period equally latent infection, causes Portugal
Rotten, threshing in grape storage and brown stain, make storage period shorten, and quality reduces, and is the important pathogen in Grape During Storage
One of.(2) citrus postharvest decay mainly has the penicilliosis caused with Italy's mould (Penicillium italicum) and finger-like blue or green
The green mould that mould (Penicillium digitatum) causes, wherein rotten is particularly acute with what Penicillium digitatum caused, this type of cause of disease
Bacterium is widely distributed, by saprophytic on organic matter in the way of, produced a large amount of conidiums are diffused among air, thus expand
Being scattered on fruit, cause fruit rot, loss is greatly.The reason that fruit postharvest diseases occurs is extremely complex, before adopting in addition
Field in spite of illness, adopt rear mechanical damage, the storage disorder etc. that causes of temperature and humidity conditions miscarriage is all the factor facilitating morbidity.
The harm adopting rear fruit is not only in that and causes fruit causing fruit to rot in a large number to lose by disease fungus, and due to many
Disease fungus can be secreted the multiple secondary metabolite of generation thus cause serious food-safety problem, as two kinds on grape are main
Ochratoxin A (OTA), Botrytis cinerea (B.cinerea) that pathogenic bacteria aspergillus (Aspergillus sp) produces produce
Botrydial (botrydial) etc..Wherein OTA has multiple toxicity, especially has a renal toxicity, and have certain teratogenesis,
Animal and health, it is considered to be one of the principal element of Balkan region ephrosis, therefore can be made by carcinogenic, mutagenicity
Become the biggest potential hazard.
For a long time, the major measure controlling postharvest disease of fruits and vegetables is to use bactericide.Adding up according to latest data, bactericide exists
Crop disease control market accounts for 26% share, and within 2011, consumption sum is up to 13,300,000,000 dollars.Chemical bactericide has cost
The advantage such as low, simple and convenient, the mechanism of action is clear and definite, effect is obvious.The conventional chemical bactericide kind controlling fruit postharvest diseases
Class is a lot.As control the chemical bactericide of grape disease mainly have dimethomorph, carbendazim, section is fixed, section is good, pyrimethanil, 20%
Difenoconazole water dispersible granule etc..During citrus storage the preventing and treating of green mould rely primarily on chemical agent press down mould sound of crying or vomiting, probenazole,
Fluoxastrobin, TBZ, adjacent sodium phenate, sec-butylamine and benzimidazole germicide etc..
But more than ten years in past, the public and scientific circles are for food and the concern day of Environmental Pesticide (especially bactericide) residual hazard problem
Benefit increases.Long-term and a large amount of use of bactericide, serious environment pollution, it is harmful to human health.NAS (NAS)
Report (1987) point out in whole agricultural chemicals of food, the tumorigenesis that bactericide constitutes 60% is dangerous.Additionally, high-frequency
Ground uses the bactericide of single kind that pathogen can be made to develop immunity to drugs problem, makes the prevention effect of bactericide be substantially reduced.According to early
The investigation of front California, USA, press down mould (Imaziall) as benzo miaow class, the regeneration product of sec-butylamine medicine, continuously mandarin orange
After using 5 years on tangerine, occur in that the anti-of Penicillium digitatum presses down mould bacterial strain.And according to national survey, it is used alone carbendazim
Area for many years, Penicillium digitatum, to which creating serious resistance, makes oranges and tangerines rotting rate go up, and drug-fast strain is to methyl simultaneously
Three kinds of benzimidazole germicides such as thiophanate, benomyl, Apl-Luster have cross tolerance.Therefore, in a lot of developed countries and
Area, some chemical bactericides have begun to be limited use, and e.g., captan (Captan) and benomyl (benomyl) etc. are several to kill
Microbial inoculum is disabled by Environmental Protection in America mechanism.
After calendar year 2001, China's entry into the World Trade Organization, between the world, fruit trade will be the most active, and the competition in fruit market also can
Fiercer, Systems in Certain Developed Countries utilizes " green barrier " to be that agricultural products in China (including grape and oranges and tangerines) outlet is provided with heavy
Obstacle.Therefore, study and utilize safer, more environmentally friendly, the material of chemical bactericide or biology can be substituted to prevent and treat postharvest fruit and vegetable
Disease, is a new research direction of postharvest fruit and vegetable preservation and freshness.Current the most increasingly attention beneficial microbe is carried out
The research of biologically controlling postharvest disease of fruit, and it is thought that one of direction being hopeful substituted chemistry bactericide most., these grind
Study carefully the residual reducing fruit and vegetable food Pesticides, protection environment, health and the economic benefit of increase fruit and vegetable food, all have
Deep meaning.
In recent years, biological control has caused the concern of countries in the world, and the application of antagonistic microbe is proved to be a kind of effective
Method.Wherein saccharomycete owing to antagonistic effect is good, do not produce toxin, can advantage becomes with chemical bactericide is used in conjunction with etc.
Study hotspot.Chalitz etc. from the lemon surface isolated one inferior Dbaly yeast of the strain Chinese (Debaryomyces hansenii),
Citrus common green mold, penicilliosis etc. are had significant prevention effect.The report Pantoea agglomerans such as Torres is to oranges and tangerines
Penicillium digitatum has good prevention effect.Arras reports Candida famata to be had significantly citrus common green mold
Prevention effect, and inquired into its mechanism of action.Partial agonist bacterium commercialization at present or half commercial applications, mainly wrap
Include addicted to oil Candida Canida oleophila, monilia guilliermondii Canida guilliermondi, season also cover pichia
Pichia guilliermondi and A Bidasi Cryptococcus Cryptococcus albidus is several.Product is concentrated mainly on west and sends out
Reach country, as the nineteen ninety-five U.S. develop first be applied to the biological control series of products BioSave100 of fruit postharvest diseases, 110
And Aspire；Subsequently, South Africa is also researched and developed and has been passed through two kinds of biological prevention and control agents of Avogreen and Yield Plus, and current China also could not
Realize biodegradable commercial applications.Main cause is a lack of efficient biocontrol bacterial strain.
Helmet shape Pichia pastoris in the present invention is considered as one of safest yeast, and research proves, it can significantly reduce oranges and tangerines and adopt
The generation of rear disease, and then reach the purpose that fruit storage is fresh-keeping.
Summary of the invention
It is an object of the invention to provide that a strain is isolatable from vineyard can efficiently control black song after oranges and tangerines adopt rear green mould and grape harvest
Mildew, helmet shape Pichia pastoris (Pichia manshurica) Y4 of gray mold, sick after oranges and tangerines and grape harvest can be widely used in
The biological control of evil, reduces the loss that postharvest disease causes, has potential using value.
The technical solution used in the present invention
After preventing and treating oranges and tangerines provided by the present invention adopt rear green mould and grape harvest, the sick yeast strain of gray mold and aspergillus niger is the Congjiang
The vineyard of Jurong, Su Sheng Zhengjiang City is separated to, on NYDA solid medium flat board and in malt juice liquid medium
28 DEG C of cultivations, carry out morphological observation；The 5.8S rDNA-ITS region sequence of this bacterial strain is analyzed, carries out molecular biology identification,
And it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Helmet shape Pichia pastoris (Pichia manshurica) Y4, is now preserved in the BeiChen West Road, Chaoyang District, BeiJing City 1 being positioned at China
The Institute of Microorganism, Academia Sinica of number institute 3, China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), deposit number is: CGMCC No.2.5415, and preservation date is on March 31st, 2016, it is proposed that classification life
Entitled helmet shape Pichia pastoris Pichia manshurica.
Helmet shape Pichia pastoris is utilized to carry out fruit postharvest diseases preventing and treating and preservation and freshness: by the activation of helmet shape Pichia pastoris, fermented and cultured,
It is centrifuged and obtains thalline；The dilution of thalline sterilized water is prepared as 1.0 × 108The bacteria suspension of CFU/mL；It is sprayed to outside fruit with watering can
Epidermis, then air-dry；It is placed in plastic crate, after sealing with preservative film, puts and at room temperature deposit.
Advantages of the present invention:
(1) helmet shape Pichia pastoris (Pichia manshurica) Y4 used in the present invention is this laboratory screening, its antagonistic potencies
By force, harmless.
(2) helmet shape Pichia pastoris (Pichia manshurica) Y4 antimicrobial spectrum used in the present invention is wide, can control grape and mandarin orange
Tangerine is the multiple disease loss caused by pathogenic bacteria after adopting.
(3) helmet shape Pichia pastoris (Pichia manshurica) Y4 used in the present invention is than the antagonism described in earlier related patents
Bacterium biocontrol effect is good.
(4) present invention uses helmet shape Pichia pastoris (Pichia manshurica) Y4, can substitute for chemical bactericide preventing and treating fruit and adopts
Rear disease, it is to avoid use the chemical bactericide harm to people, there is significant economic benefit and social benefit.
By the present invention being will be explained in more detail by following embodiment.Following example are merely illustrative, and the present invention is also
Do not limited by these embodiments.
Accompanying drawing explanation
Fig. 1 is the 5.8S rDNA-ITS region sequence evolutionary relationship figure of helmet shape Pichia pastoris of the present invention.
Fig. 2 is the helmet shape Pichia pastoris inhibition to citrus common green mold.Note: CK: sterile distilled water, i.e. control group；Pm:
1×108The helmet shape Pichia pastoris bacteria suspension of cells/mL.Different letters represent significant difference (P < 0.05).
Fig. 3 is the inhibition that helmet shape Pichia pastoris is sick to grape grey mould and aspergillus niger.Note: CK: sterile distilled water, the most right
According to group；Pm:1 × 108The helmet shape Pichia pastoris bacteria suspension of cells/mL.Different letters represent significant difference (P < 0.05).
Detailed description of the invention
Embodiment 1: helmet shape Pichia pastoris (Pichia manshurica) Y4 strain microorganism characteristic is as follows:
1. morphological feature
(1) (beef extract 0.8%, YE 0.5%, glucose 1%, agar 2%, 121 DEG C wet for NYDA culture medium
Heat sterilization 20min) upper 28 DEG C cultivate 48h, bacterium colony is rounded, the smooth of the edge, relatively moistening, easily provoke.Cellular morphology is length
Ellipse, single dispersion.
(2) after cultivating 24h in NYDB fluid nutrient medium, being formed without mould, bacterium solution is muddy, has precipitation, and microscopy yeast is thin
Born of the same parents' ovalize, budding.
2. molecular biology identification
The 5.8S rDNA-ITS region sequence of bacterium helmet shape Pichia pastoris is analyzed, GenBank retrieves, is defined as helmet
Shape Pichia pastoris (Pichia manshurica).According to the homologous strain retrieved, the Mege5.1 of application DNAStar software
Program, builds biological evolution relational tree such as Fig. 1.
By this yeast helmet shape Pichia pastoris (Pichia manshurica) Y4, now it is preserved in the north, Chaoyang District, Beijing City being positioned at China
The Institute of Microorganism, Academia Sinica of occasion West Road 1 institute 3, China Committee for Culture Collection of Microorganisms's common micro-organisms
Center (CGMCC), deposit number is: CGMCC No.2.5415, and preservation date is on March 31st, 2016, it is proposed that
Classification And Nomenclature is helmet shape Pichia pastoris Pichia manshurica.
Embodiment 2: the helmet shape Pichia pastoris control effect to citrus common green mold
One) testing program
For the fruit of examination through fine selection, selecting fruit uniformity, surface is without scratch or mechanical damage and disease pest injury as far as possible.With
3 holes made a call at fruit equator by aseptic card punch, and the superficial cut of oranges and tangerines is 5mm (diameter) × 3mm (deeply).Each wound etc.
The amount addition 30 following treatment fluids of μ L: (1) 1 × 108The helmet shape Pichia pastoris bacteria suspension of cells/mL；(2) sterile distilled water.After 2h,
30 μ L 1 × 10 are inoculated again respectively to oranges and tangerines wound5Penicillium digitatum (P.digitatum) spore suspension of spores/mL.From
After so drying, fruit put into plastic crate and seals with preservative film, constant incubator cultivating (humidity 95%), is placed in room temperature (20 DEG C).
Often process and be repeated 3 times, repeat 12 fruits every time.Whole test is repeated 2 times.If through the cultivation of 7 days, record fruit was sent out
Sick rate, evaluates the fungistatic effect of helmet shape Pichia pastoris with this.
The computing formula of the incidence of disease is as follows:
The fruit of the incidence of disease (%)=morbidity/fruit sum × 100%
Two) result of the test
Testing according to above-mentioned steps, the rotting rate result of statistics fruit is as follows:
Inhibition to citrus common green mold
As shown in Figure 2, the incidence of disease of control group fruit is about 99.6%, and the oranges and tangerines incidence of disease processed through helmet shape Pichia pastoris is
45.3%.Apply helmet shape Pichia pastoris before i.e. Penicillium digitatum spore suspension artificially inoculated by oranges and tangerines, compared with the control, finish through helmet shape
The red yeast-treated oranges and tangerines incidence of disease is substantially less than comparison (P < 0.05), and therefore explanation helmet shape Pichia pastoris can have at 20 DEG C
Rear green mould adopted by the oranges and tangerines that effect controls to be caused by Penicillium digitatum.
Embodiment 3: the control effect that helmet shape Pichia pastoris is sick to grape grey mould and aspergillus niger
One) testing program
After activating, yeast-inoculated is equipped with 50mL NYDB (yeast extract 5g, beef extract 8g, glucose 10g, distilled water
In triangular flask 1000mL), 28 DEG C, 180r/min shaking table cultivation 20h.Take bacterium solution 8000r/min and be centrifuged 10min, abandon
Clearly, sterilized water cleans 3 times repeatedly, and it is 1 × 10 that blood counting chamber counting is made into concentration8The bacteria suspension of individual/mL, control group is nothing
Bacterium water.The hole of 1 a diameter of 3m (wide) × 3mm (deeply) is made a call to, then the note 15 μ L respectively in every hole at fruit waist card punch
The saccharomycete suspension of concentrations above, comparison fruit injects 15 μ L sterilized waters.After 2h, then inoculate 10 μ L respectively to grape wound
1×105Botrytis cinerea (B.cinerea) spore suspension of spores/mL or 5 × 104Aspergillus niger (the Aspergillus of spores/mL
Niger) spore suspension.Loading in plastic crate by fruit after drying, preservative film sealing keeps the humidity of 95%, at room temperature stores
Hide, after 5d, add up the morbidity rotting rate of fruit.Often process and be repeated 3 times, repeat 36 fruits every time.Whole test is repeated 2 times.
The computing formula of the incidence of disease is as follows:
The fruit of the incidence of disease (%)=morbidity/fruit sum × 100%
Two) result of the test
Testing according to above-mentioned steps, the rotting rate result of statistics fruit is as follows:
1, the inhibition to grape grey mould
As shown in Figure 3, the incidence of disease of control group is 94.4%, and the grape incidence of disease processed through helmet shape Pichia pastoris is 5.6%,
Therefore helmet shape Pichia pastoris at room temperature can effectively control to be adopted rear gray mold disease by what Botrytis cinerea caused grape.
2, the inhibition to vine black aspergillosis
As shown in Figure 3, the incidence of disease of control group is 100%, and the grape incidence of disease processed through helmet shape Pichia pastoris is 11.1%,
Therefore helmet shape Pichia pastoris at room temperature can effectively control to be adopted rear aspergillus niger disease disease by what aspergillus niger caused grape.
This result is substantially good than the Antagonistic Fungi biocontrol effect described in earlier related patents, such as, and Chinese invention patent " uva
Real postharvest disease or the prevention and controls of fruit abscission and special control agent thereof " (application number 200910078858.9) disclose one
Cryptococcus laurentii and Boratex are used in combination the technology of preventing and treating grape disease, the Portugal after Cryptococcus laurentii and Boratex process
After grape place three days at ambient temperature, the rotting rate caused because of gray mold only reduces about 50% than comparison, and does not show
It has inhibitory action to aspergillus niger disease after grape harvest；Chinese invention patent " solves fat Asia sieve yeast, after grape harvest in disease control
Application and using method " (application number CN201410231417.9) provide and a kind of solve after fat Asia sieve yeast prevents and treats grape harvest
Disease and the technology of degraded ochratoxin A, introducing understanding fat Asia sieve yeast in literary composition has significant inhibition to grape Penicillium islandicum,
But do not show that it is to aspergillus niger after grape harvest is sick and gray mold has inhibitory action.
Embodiment 4: the preservation of helmet shape Pichia pastoris
First-selected culture medium is NYDA culture medium, it is possible to use potato agar culture medium: potato 200 grams, glucose
20 grams, agar 15～20 grams, 1000 milliliters of running water, pH 7.0～9.0, cultivates 36h by 26 DEG C～28 DEG C.
Claims (4)
1. helmet shape Pichia pastorisPichia manshurica Y4, deposit number is CGMCC
No. 2.5415。
2. the purposes of the helmet shape Pichi strain described in claims 1, it is characterised in that oranges and tangerines can be controlled with it and adopt that aspergillus niger after rear green mould and grape harvest is sick, gray mold, for preservation and freshness.
3. the helmet shape Pichia pastoris described in claim 1 carries out disease control and method for storing and refreshing after oranges and tangerines and grape harvest: activated by helmet shape Pichia pastoris, with NYDB medium culture helmet shape Pichia pastoris, is centrifuged and obtains thalline, is prepared as 1 × 10 with sterilized water dilution8 The bacteria suspension of cells/mL；Bacteria suspension is uniformly sprayed on fruit, natural air drying；It is placed in plastic crate, after sealing with preservative film, puts and deposit at ambient temperature.
Helmet shape Pichia pastoris the most according to claim 2 uses the method for preventing and treating fruit postharvest diseases, it is characterised in that wherein said NYDB culture medium is: yeast extract 5 g, glucose 10 g, beef extract 8 g, distilled water 1000 ml, pH are natural, 121 DEG C of sterilizing 30min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937289A (en) * | 2017-12-15 | 2018-04-20 | 北京工商大学 | One plant of antagonistic yeast Pichia galeiformis and its preparation and application method |
| CN107988088A (en) * | 2017-12-15 | 2018-05-04 | 北京工商大学 | One plant of antagonistic yeast Pichia deserticola and its preparation and application method |
| CN108118004A (en) * | 2017-12-15 | 2018-06-05 | 北京工商大学 | One plant of happiness cactus Pichia pastoris that can effectively prevent fruit postharvest diseases and its preparation and application method |
| CN113801799A (en) * | 2021-08-13 | 2021-12-17 | 西南大学 | Yeast SLL12 and application thereof in preparing biological control agent for controlling postharvest diseases of jujube fruits |
-
2016
- 2016-04-12 CN CN201610225320.6A patent/CN105861340B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937289A (en) * | 2017-12-15 | 2018-04-20 | 北京工商大学 | One plant of antagonistic yeast Pichia galeiformis and its preparation and application method |
| CN107988088A (en) * | 2017-12-15 | 2018-05-04 | 北京工商大学 | One plant of antagonistic yeast Pichia deserticola and its preparation and application method |
| CN108118004A (en) * | 2017-12-15 | 2018-06-05 | 北京工商大学 | One plant of happiness cactus Pichia pastoris that can effectively prevent fruit postharvest diseases and its preparation and application method |
| CN107937289B (en) * | 2017-12-15 | 2021-06-25 | 北京工商大学 | Application of pichia pastoris BY27 strain in prevention and treatment of postharvest diseases of fruits |
| CN107988088B (en) * | 2017-12-15 | 2021-06-25 | 北京工商大学 | Application of pichia deserticola in prevention and treatment of postharvest diseases of fruits |
| CN113801799A (en) * | 2021-08-13 | 2021-12-17 | 西南大学 | Yeast SLL12 and application thereof in preparing biological control agent for controlling postharvest diseases of jujube fruits |
| CN113801799B (en) * | 2021-08-13 | 2023-05-02 | 西南大学 | Yeast SLL12 and application thereof in preparation of biological control agent for controlling postharvest diseases of jujube fruits |
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| CN105861340B (en) | 2020-02-21 |
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