CN103937689A - Pseudozyma fusiformata for inhibiting diseases after fresh fruit collection and application thereof - Google Patents
Pseudozyma fusiformata for inhibiting diseases after fresh fruit collection and application thereof Download PDFInfo
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Abstract
The invention discloses pseudozyma fusiformata for inhibiting diseases after fresh fruit collection and application thereof, belonging to the technical field of microorganism application. The pseudozyma fusiformata SF6 disclosed by the invention has the preservation number of CGMCC No.8577, has inhibiting effects on monilinia after peach collection and botrytis cinerea after tomato collection, has inhibiting effects on the brown rot after peach collection and the grey mould after tomato collection, and has the most obvious inhibiting effect on the monilia, and the diameter of the inhibition zone is more than 15mm. The pseudozyma fusiformata has stable, efficient and broad-spectrum antimicrobial properties, and can be applied to prevention and treatment of the germs or diseases.
Description
Technical field
The present invention relates to a kind of yeast-like fungus and application thereof that suppresses fresh fruit post-harvest diseases, belong to technical field of microbe application.
Background technology
After Fruit, fungal disease is the important disease of a class, has caused a large amount of underproduction of fruit, has had a strong impact on edibleness and the marketable value of fruit, has caused orchard worker's income to reduce.In developed country, the loss amount that postharvest diseases of fruit causes accounts for the more than 15% of fruit total amount, and in developing country, owing to lacking necessary prevention and control technology and equipment, the loss amount that postharvest diseases of fruit causes accounts for the more than 30% of total amount.The generation of postharvest diseases of fruit is produced with processing to the especially high-end fruit and vegetable food of modern agricultural production and has been caused and had a strong impact on.
The peach brown rot being caused by Monilinia spp. pathogenic fungi is all worldwide the first disease of peach at present, is also the first problem that affects peach industry development.According to statistics, in developed country, as the U.S., Israel, Italy, France etc., adopt the 10%-30% that loss amount that rear brown heart causes just accounts for peach ultimate production; In developing country, owing to lacking its loss amount of necessary Prevention Technique and relevant device, reach 30%-50%, even higher; Some area, the big area of peach brown rot seriously occurs, and almost causes peach really to be had no harvest.Therefore, the control of peach brown rot and control are seemed to more urgent and important.China's peach garden area surpasses 9,000,000 mu, is the effective cultivated area of peach the first big country in the world; Producing peach per year and surpass 400,000 tons, is peach output the first big country in the world.Beijing peach output occupies domestic last eight, Pinggu County, beijing one ground only, and peach cultivated area just reaches more than 220,000 mu (1.47 ten thousand hectares); Peach industry has become the pillar of the economy industry in Pinggu county, and some peaches and relevant converted products have exported to European Union, Japan and other countries.Since 2000, peach brown rot big area in Beijing increases, and increases the weight of year by year; The heavier peach garden of morbidity in 2005 wherein, peach is really injured, and comes off in a large number, has no the fruit of aobvious disease during harvesting, and after plucking 2-3 days, more than 50% fruit rots because brown heart infects, and this causes serious financial loss to orchard worker.Within 2012, Beijing plant protection unit peach brown rot investigation result shows, each suburban county peach producing region, Beijing all has peach brown rot to occur, and the transportation of peach after adopting and duration of storage sickness rate are all more than 30%.Edibleness and marketable value that this greatly reduces fruit, had a strong impact on the development of Beijing peach industry.Therefore effective prevention and control and the capital Economic development of peach being adopted to rear brown heart are especially closely connected with orchard worker's income.
Tomato gray mold is to affect grape storage and transport heavy is wanted one of disease, and the gray mold being caused by ash arrhizus bacteria (Botrytis cinerea) every year all can cause the marketable value of tomato fruit and edibleness greatly to reduce.Therefore effective prevention and control of tomato gray mold and Economic development are also seemed to outbalance is with urgent.
Applying bactericidal agent control post-harvest diseases is the important means that reduces fruit loss; Domestic application is mainly SO in the chemical agent of postharvest diseases of fruit control at present
2and Top Form Wormer (Thiabendazole); And SO
2be usually widely used and can bring environmental pollution, and in normal distance transportation and long-term fruit storage, its sterilization effect can be lost in time and rapidly; Although Top Form Wormer to Fruit after fungal disease have good prevention effect, but pathogenetic bacteria is not had to effect, and it uses the rear longevity of residure longer, be difficult for degraded, can affect the export abroad of some high-end fruits, the more important thing is, the widespread adoption of Top Form Wormer easily makes some pathogenic bacterias develop immunity to drugs.Therefore, need the novel fresh fruit post-harvest diseases of development and application sterilant badly.
Biological bactericide has inherent advantage for chemical pesticide, has avoided the problems such as pesticide residue, environmental pollution, resistance, and low toxicity, environmental protection, holding effect and nature are ecological better compatible.At present, domestic biological pesticide exploitation mainly concentrates on several aspects such as Bt sterilant, agricultural antibiotic, bacterial origin biological prevention and control agent, and controlling object focuses mostly in field crop disease.But for fruit disease, especially postharvest diseases of fruit is prevented and treated aspect, it is relatively less that biological and ecological methods to prevent plant disease, pests, and erosion applied research is carried out, and the at present domestic biocontrol fungicide product that also not can be used in postharvest diseases of fruit control.Different from the research and development of field crop disease biocontrol fungicide and biocontrol bacteria in soil-borne diseases agent, development and application for postharvest diseases of fruit biocontrol microorganisms microbial inoculum has higher safety requirements, because be different from field crop, after picking fruit, do not need processing or only through simple, process, we just eat.Than producing antibiotics bacterium and having a Mycophyta biocontrol microorganisms, take yeast-like fungi or yeast be the sterilant of main body exploitation on control fruit disease, safer, environmental protection seems.
Yeast and yeast-like fungus source biological bactericide have unique advantage: compare with some biocontrol bacterias (1), does not produce microbiotic, less to the negative impact of environment; (2) there is a Mycophyta biocontrol microorganisms to compare with some, do not produce sensitization spore; (3) growth requires simply, is easy to large scale fermentation and produces.Therefore, exploitation post-harvest diseases yeast source bactericide becomes in international fruit disease control in recent years and fruit food safety research field a new direction and trend.
On world market, the yeast source bactericide one for postharvest diseases of fruit control of having registered has 3 kinds of products at present.They are respectively the sterilant based on black yeast bacterium Aureobasidium pullulans exploitation
(Germany), the sterilant based on the strange yeast Metschnikowia of plum fructicola exploitation
(Israel), the sterilant based on candidiasis Candida sake exploitation
(Spain); And domesticly also do not have yeast source biological bactericide product to occur.Therefore, for us, development of new fresh fruit post-harvest diseases yeast source bactericide be necessary be also important.
Yeast-like fungus Pseudozyma fusiformata ubiquity in physical environment, is mainly present in sugar degree compared with the fruit surface of horn of plenty and takes, and is one of superior microorganism population in plant microecosystem.This bacteria growing speed is fast, and nutrition is simple, and growing ability is strong.As biocontrol microorganisms application aspect, it has own unique advantage: 1) of reduced contamination to environment of this bacterium, does not produce toxin; 2) this bacterium does not produce microbiotic, relatively safe to the mankind; 3), for other fungies, this bacterium does not produce sensitization spore; 4) this bacterium desired nutritional is fairly simple; 5) this bacterium is applicable to commercialization scale operation.Therefore, it has good application prospect.
Yeast-like fungus (Pseudozyma fusiformata) distributes very wide at occurring in nature, and the current domestic report of preventing and treating peach brown rot yeast-like fungus (Pseudozyma fusiformata) that also do not have.
Summary of the invention
The object of the present invention is to provide a kind of yeast-like fungus (Pseudozyma fusiformata), bacterial strain number is SF6, and preserving number is: CGMCC No.8577.
Object of the present invention is also to provide a kind of pathogenic bacteria inhibitor, and its activeconstituents is above-mentioned yeast-like fungus and/or its metabolite.
Yeast-like fungus of the present invention (Pseudozyma fusiformata), bacterial strain number is SF6, registering on the books of Qi China Committee for Culture Collection of Microorganisms common micro-organisms center is numbered CGMCC No.8577.
Described yeast-like fungus (Pseudozyma fusiformata) SF6 is for to obtain from fruit surface separation.
The 5.8S rDNA gene of described yeast-like fungus (Pseudozyma fusiformata) SF6, ITS1/ITS2 sequence, 26S rDNA sequence are respectively as shown in SEQ ID No.1 and No.2.
The morphological feature of described yeast-like fungus (Pseudozyma fusiformata) SF6: thalline vegetative cell is spherical in shape, avette or subsphaeroidal, monolateral or polygon gemmation; On YPD Agar substratum and on PDA substratum, at 28 ℃, grow 48 hours, bacterium colony is that butteriness, oyster white are opaque, flat or circular, neat in edge, bacterium colony corrugationless.
The physiological and biochemical property of described yeast-like fungus (Pseudozyma fusiformata) SF6: can carry out glucose fermentation, can not carry out raffinose and lactose fermentation; Carbon assimilation aspect: sorbose, sucrose, maltose, trehalose, PEARLITOL 25C are positive, semi-lactosi, lactose, rhamnosyl, ribitol, erythritol are negative.
Described yeast-like fungus (Pseudozyma fusiformata) SF6 all can normal growth in the substratum that contains 5% (mass content) sodium-chlor, in the substratum that contains 10% (mass content) sodium-chlor, can not grow, all can normal growth in the substratum of pH4.0-5.5, in envrionment temperature, be at 4 ℃ a little less than growth potential, more than 40 ℃ can not growing; Best suitable growth temperature is 25-30 ℃.Described yeast-like fungus (Pseudozyma fusiformata) SF6 produces organic acid.
The present invention also aims to provide above-mentioned yeast-like fungus (Pseudozyma fusiformata) SF6 and/or its metabolite suppressing pathogenic bacteria, preparation pathogenic bacteria inhibitor, suppress disease, preparing the application in disease suppression agent.
In the purposes of above-mentioned yeast-like fungus (Pseudozyma fusiformata) SF6, described pathogenic bacteria be following at least one:
A, peach are adopted rear brown rot germ;
B, tomato are adopted rear ash arrhizus bacteria.
Described disease be following at least one:
A, peach are adopted rear brown heart;
B, tomato are adopted rear gray mold.
Wherein, described peach is adopted rear brown rot germ and can be Monilinia fructicola or Monilinia laxa or Monilinia fructigena; It is Botrytis cinerea that described tomato is adopted rear ash arrhizus bacteria.
The metabolite of described yeast-like fungus (Pseudozyma fusiformata) SF6 can obtain from the fermented liquid of described yeast-like fungus (Pseudozyma fusiformata) SF6.
The metabolite of described yeast-like fungus (Pseudozyma fusiformata) SF6 specifically can be prepared as follows: in liquid medium within, cultivate described yeast-like fungus (Pseudozyma fusiformata) SF6, described yeast-like fungus (Pseudozyma fusiformata) SF6 removing in liquid culture (fermented liquid) obtains the metabolite of described yeast-like fungus.
Yeast-like fungus in the present invention (Pseudozyma fusiformata) SF6 to peach adopt rear brown rot germ, that tomato is adopted rear ash arrhizus bacteria is inhibited; To peach adopt rear brown heart, that tomato is adopted rear gray mold is inhibited.In selected plant pathogenic fungi, to Monilinia fructicola, antibacterial circle diameter is more than 15mm, illustrates that yeast-like fungus (Pseudozyma fusiformata) SF6 has anti-microbial property stable, efficient, wide spectrum.
Biomaterial preservation explanation
Classification And Nomenclature: yeast-like fungus (Pseudozyma fusiformata)
Strain number: SF6
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on December 13rd, 2013
The preservation center numbering of registering on the books: CGMCC No.8577.
Accompanying drawing explanation
Fig. 1. yeast-like fungus (Pseudozyma fusiformata) SF6 cellular form under the microscope.
Fig. 2 .a) yeast-like fungus (Pseudozyma fusiformata) SF6 inhibition to Monilinia fructicola (Monilinia fructicola) on PDA substratum; B) yeast-like fungus (Pseudozyma fusiformata) SF6 inhibition to tomato ash arrhizus bacteria (Botrytis cinerea) on PDA substratum.
Fig. 3 .a) yeast-like fungus (Pseudozyma fusiformata) SF6 inhibition to peach brown rot spot on peach fruit; B) yeast-like fungus (Pseudozyma fusiformata) SF6 inhibition to tomato grey mold scab on tomato fruit.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material and reagent used in following embodiment, if no special instructions, all can obtain from commercial channels.
Pathogenic bacteria used in following embodiment, the public can also can obtain from Beijing City Agriculture and Forestry Institute from field acquisition, to repeat the application's experiment:
Monilinia fructicola---chain sclerotinia sclerotiorum (Monilinia fructicola (wint.) Rehm) (Wang Fei etc. the generation of peach brown rot and control. fruit tree flowers .2012 the 5th phase p58).
Tomato is adopted rear ash arrhizus bacteria--Botrytis cinerea (Botrytis cinerea Per.ex Fr).
Separation and the identification of strains of embodiment 1, yeast-like fungus of the present invention (Pseudozyma fusiformata) SF6
1, sample collecting
From having the organic orchard of north of China, gather apple.
2, the separation screening of bacterial strain and antagonism screening
Isolated strains from organic fruits, adopt conventional gradient dilution coating separated, with PDA (containing Vetstrep) and YPD Agar (containing Vetstrep) totally 2 kinds of substratum under 28 ℃ of conditions, cultivate respectively, large and the bacterium that can pure culture of picking colony morphological differences, purification storage on YPD Agar substratum, and take Monilinia fructicola as target pathogens carry out the primary dcreening operation of Antagonistic Fungi and repeatedly again sieve, finally obtain the yeast-like fungi bacterial strain that a strain bacteriostatic activity is strong, naming it is SF6.
3, the evaluation of bacterial strain
Bacterial strain SF6 is carried out to the analysis of morphology, physio-biochemical characteristics and part conserved sequence.With reference to < < The Yeasts, a Taxonomic Study > > (Kurtzman, C.P. (1998a) .Williopsis Zender.In The Yeasts, a Taxonomic Study, 4th edn, pp.529-920.Edited by C.P.Kurtzman & J.W.Fell.Amsterdam:Elsevier.) in, described method carries out Physiology and biochemistry mensuration and molecular biology identification.
Result shows, the morphological feature of SF6: thalline vegetative cell is avette, or subsphaeroidal, mostly is monolateral gemmation or less for polygon gemmation; On YPD Agar substratum and on PDA substratum, at 28 ℃, grow 48 hours, bacterium colony is that butteriness, oyster white are opaque, flat or circular, neat in edge, and bacterium colony corrugationless, as shown in Figure 1.
The physiological and biochemical property of SF6: can carry out glucose fermentation, can not carry out raffinose and lactose fermentation; Carbon assimilation aspect: sorbose, sucrose, maltose, trehalose, PEARLITOL 25C are positive, semi-lactosi, lactose, rhamnosyl, ribitol, erythritol are negative.SF6 all can normal growth in the substratum that contains 5% (mass content) sodium-chlor, in the substratum that contains 10% (mass content) sodium-chlor, can not grow, all can normal growth in the substratum of pH4.0-5.5, in envrionment temperature, be at 4 ℃ a little less than growth potential, more than 40 ℃ can not growing; Best suitable growth temperature is 25-30 ℃.The 5.8S rDNA gene of bacterial strain SF6, ITS1/ITS2 sequence and 26S rDNA D1/D2 nucleotide sequence are as shown in SEQ ID No.1 and No.2.The mensuration of embodiment 2, yeast-like fungus of the present invention (Pseudozyma fusiformata) SF6 antimicrobial spectrum
For trying pathogenic bacteria:
Monilinia fructicola---chain sclerotinia sclerotiorum (Monilinia fructicola (wint.) Rehm);
Tomato is adopted rear ash arrhizus bacteria--Botrytis cinerea (Botrytis cinerea Per.ex Fr).
Selecting peach to adopt rear brown heart germ, tomato, to adopt rear ash arrhizus bacteria etc. be indication fungi, adopts bioassay in dull and stereotyped face-off culture method and body to screen.
Concrete operations are as follows: yeast-like fungus (Pseudozyma fusiformata) SF6 bacterial strain 28 ℃ of constant temperature culture 48h on PDA substratum.Plant pathogenic fungi is 25 ℃ of constant temperature culture 3-4 days on PDA flat board, from colony edge, with the aseptic stainless steel punch tool of diameter 0.7cm, make the agar block that carries disease germs; With aseptic inoculation pin picking pathogenic bacteria bacterium sheet, mycelia faces down and is inoculated in the dull and stereotyped center of PDA, simultaneously at equal aseptic inoculation ring yeast-like fungus (Pseudozyma fusiformata) SF6 that rules for about 3.00cm place apart from pathogenic fungi bacterium sheet both sides, the flat board that only connects pathogenic bacteria of take is contrast, each is processed in triplicate, 25 ℃ of constant temperature culture 5-7 days, measure the antibacterial bandwidth between pathogenic fungi edge and the bacterium colony bandwidth center of yeast-like fungus (Pseudozyma fusiformata) SF6.
The raw screening method of surveying in body: artificial punching inoculation method concrete operations in sporocarp (take prevent and treat peach adopt rear brown heart describe as example) are as follows:
Yeast-like fungus (Pseudozyma fusiformata) SF6 bacterial strain 28 ℃ of constant temperature culture 48h on YPD liquid nutrient medium, with distilled water, being adjusted to bacterial concentration is 5 * 10
7cfu/ml.Monilinia fructicola M.laxa is 25 ℃ of constant temperature culture 5-7 days on PDA flat board, collect botrytis cinerea spore, with distilled water, make 5 * 10
4the spore suspension of cfu/ml; Ripe peach, first with after 0.5% clorox sterilization, with sterilized pipettor gun head, along peach waist jack, each fruit is pricked 3, hole, and each hole is 30mm * 30mm.30 μ l KB bacterium liquid are inoculated in every hole, and room temperature is dried, and then the pathogenic bacteria spore suspension of inoculation 30 μ l.Finally, the fruit of processing is positioned at 25 ℃, after 5-7 days, measures fruit rot spot size.Rotten spot is less than 10mm and is regarded as not falling ill.According to the scab that rots account for total inoculation hole count number, calculate sickness rate, not the scab number of morbidity account for total inoculation hole count number, calculate scab inhibiting rate.
The extracorporeal extracorporeal suppression of (Pseudozyma fusiformata) SF6 of yeast-like fungus described in table 1. to 2 kinds of pathogenic bacterias
Yeast-like fungus (Pseudozyma fusiformata) SF6 is in selected plant pathogenic fungi, restraining effect to Monilinia fructicola is the most obvious, antibacterial circle diameter is more than 15mm, illustrates that yeast-like fungus (Pseudozyma fusiformata) SF6 has anti-microbial property stable, efficient, wide spectrum as shown in table 1 and Fig. 2.
The inhibition of the rotten scab that embodiment 3 yeast-like funguses (Pseudozyma fusiformata) SF6 causes the 2 kind of plant pathogenic fungies for examination.
After peach is plucked, through SF6 bacterium liquid, (bacterial concentration is respectively 10
7cfu/ml, 10
8cfu/ml, 10
9cfu/ml) immersion treatment 30-60 second, then under room temperature condition, air-dry, in temperature, be then 1 ℃, relative humidity is under 95% condition, dark storage.Preserve after 21 days, compare with control group, when bacterial concentration is 10
7cfu/ml, its preventive effect is 50%, when bacterial concentration reaches 10
8during cfu/ml, its preventive effect is 60%, when bacterial concentration reaches 10
9during cfu/ml, its preventive effect reaches 70%.
After tomato picking, through SF6 bacterium liquid, (bacterial concentration is respectively 10
7cfu/ml, 10
8cfu/ml, 10
9cfu/ml) immersion treatment 30-60 second, then under room temperature condition, air-dry, in temperature, be then 4 ℃, relative humidity is under 95% condition, dark storage.Preserve after 14 days, compare with control group, when bacterial concentration is 10
7cfu/ml, its preventive effect is 45%, when bacterial concentration is 10
8during cfu/ml, its preventive effect to gray mold is respectively 55%, when bacterial concentration reaches 10
9during cfu/ml, its preventive effect 65% to gray mold.
Under holding conditions, yeast-like fungus (Pseudozyma fusiformata) SF6 all has good inhibition as shown in table 2 to the rotten scab that causes of 2 kind of plant pathogenic fungies for examination.
(application concentration is 10 to table 2.Pseudozyma fusiformata SF6
8during cfu/ml) different postharvest diseases of fruit preventive effects under different condition
Claims (7)
1. a yeast-like fungus (Pseudozyma fusiformata), bacterial strain is SF6, preserving number is: CGMCC No.8577.
2. a pathogenic bacteria inhibitor, its activeconstituents is the metabolite of yeast-like fungus described in yeast-like fungus and/or claim 1 described in claim 1.
3. pathogenic bacteria inhibitor according to claim 2, is characterized in that, described pathogenic bacteria inhibitor is inhibited to following at least one pathogenic bacteria:
A, peach are adopted rear brown rot germ;
B, tomato are adopted rear ash arrhizus bacteria.
4. a disease suppression agent, its activeconstituents is the metabolite of yeast-like fungus described in yeast-like fungus and/or claim 1 described in claim 1.
5. disease suppression agent according to claim 4, is characterized in that, described disease be following at least one:
A, peach are adopted rear brown heart;
B, tomato are adopted rear gray mold.
6. the following arbitrary application of the metabolite of yeast-like fungus described in yeast-like fungus and/or claim 1 described in claim 1:
1) application of the metabolite of yeast-like fungus in suppressing pathogenic bacteria described in yeast-like fungus and/or claim 1 described in claim 1;
2) application of the metabolite of yeast-like fungus in preparation pathogenic bacteria inhibitor described in yeast-like fungus and/or claim 1 described in claim 1;
3) application of the metabolite of yeast-like fungus in suppressing disease described in yeast-like fungus and/or claim 1 described in claim 1;
4) application of the metabolite of yeast-like fungus in preparing disease suppression agent described in yeast-like fungus and/or claim 1 described in claim 1.
7. application according to claim 6, is characterized in that, described pathogenic bacteria be following at least one:
A, peach are adopted rear brown rot germ;
B, tomato are adopted rear ash arrhizus bacteria;
Described disease be following at least one:
A, peach are adopted rear brown heart;
B, tomato are adopted rear gray mold.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543116A (en) * | 2016-02-03 | 2016-05-04 | 北京市农林科学院 | Microzyme strain and application thereof in disease control after fresh fruit picking |
| CN106259467A (en) * | 2016-07-20 | 2017-01-04 | 北京市农林科学院 | The application of yeast-like fungus suppression downy mildew |
-
2014
- 2014-04-25 CN CN201410171177.8A patent/CN103937689B/en not_active Expired - Fee Related
Non-Patent Citations (2)
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| DIANPENG ZHANG 等: "Selection and evaluation of new antagonists for their efficacy against postharvest brown rot of peaches", 《POSTHARVEST BIOLOGY AND TECHNOLOGY》, vol. 55, no. 3, 31 March 2010 (2010-03-31), XP026824573 * |
| 许皎姣: "拮抗酵母菌的筛选、鉴定及对葡萄冷藏品质的影响", 《中国优秀硕士学位论文全文数据库》, 15 June 2013 (2013-06-15) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543116A (en) * | 2016-02-03 | 2016-05-04 | 北京市农林科学院 | Microzyme strain and application thereof in disease control after fresh fruit picking |
| CN105543116B (en) * | 2016-02-03 | 2021-06-04 | 北京市农林科学院 | Yeast and application thereof in disease control after fresh fruit picking |
| CN106259467A (en) * | 2016-07-20 | 2017-01-04 | 北京市农林科学院 | The application of yeast-like fungus suppression downy mildew |
| CN106259467B (en) * | 2016-07-20 | 2019-03-15 | 北京市农林科学院 | The application of yeast-like fungus inhibition downy mildew |
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