CN103642700A - Aureobasidium pullulans and application thereof - Google Patents
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Abstract
The invention discloses aureobasidium pullulans and an application thereof. The class of the strain is named aureobasidium pullulans, the full name of the strain is aureobasidium pullulans PA-2, the strain is preserved in China General Microbiological Culture Collection Center No.3 building No.1 Beichen West Road Changyang District Beijing China on October 28, 2013, and the preservation serial number is CGMCC No.8413. The aureobasidium pullulans is used for biologically removing broad-leaf weeds such as cleavers, goosefoots, malva verticillata and the like as well as oat grasses in wheat, rape and pea field lands, and is capable of effectively controlling and preventing the broad-leaf weeds and the oat grasses.
Description
Technical field
The present invention relates to field of ecology, in particular strain aureobasidium pullulans and an application thereof.
Background technology
Farmland weed is controlled and mainly be take chemical herbicide as main body promotion Derived from Agricultural Modernization.Along with being widely used of chemical herbicide, the negative impact of generation is also day by day obvious.As the increase of Herbicide-resistant weeds population, soil compaction and water pollution etc., had a strong impact on agriculture Sustainable development.Therefore develop wide spectrum, efficient, low toxicity novel herbicide and development biological weed control technology and become the task of top priority.This development trend is complied with in the appearance of campelyco.Up to now, investigator has carried out large quantity research to the nearly 100 kinds of pathogenic fungies with weeding potentiality on more than 50 kinds of weeds.Wherein, there is the fungi that weeding activity is expected to be developed to weedicide and mainly concentrate on Colletotrichum Colletotrichum, fusarium Fusarium, Alternaria Alternaria, Staphlosporonites Botrytis, Cercospora Cercospora, Puccinia Puccinia, leaf Ustilago Entyloma, Ascochyta Ascochyta and Sclerotinia Sclerotinia etc.At present, the campelyco product that the whole world has been developed has more than 20 to plant, and wherein Biochon, StumpOut, Camperico, bialaphos sodium, Myco-TechPaste and MBI-005 have become international product.
Except " dodder parasitic fungus preparation ", it is less that China's campelyco product is registered, but much work is being done aspect campelyco candidate biological screening by China.In recent years, Agricultural University Of Nanjing weeds research department is with the curved spore of lady's-grass-Herba Eragrostidis pilosae (Digitaria sanguinalis-Curvularia eragrostidis), barnyard grass-Xin long-radius elbow spore (Culvularia lunata), barnyard grass-helminthosporium sporangium (Helminthosporium gramineum f.sp.Echinochloa) and Goosegrass bipolaris strain (Bipolaris eleusines), solidago canadesis-microsolerotium bacterium (Solidago canadensis-Sclerotium rolfsii), alternanthera philoxeroides-vacation is every alternaric bacteria (Alternantera phyloxeroides-Nimbya alternantherae), the research of the bacterium cursive script systems such as Hemp Eupatorium-alternaric bacteria (Eupatorium adenophorum-Alternaria alternata) has obtained progress, formed a collection of campelyco technological achievement with independent intellectual property right.
Microbial herbicide compares chemical herbicide and has that selectivity by force, does not injure natural enemy, is difficult for developing immunity to drugs, unit surface consumption is few, on people and animals' high safety, on plurality of advantages such as micro-ecological environment impact are little.Evaluation to microbial herbicide, two standards of Main Basis are validity (drug effect) and specificity (security) in theory.Weeds biocontrol microorganisms should possess not only target weeds are had strong pathogenic but also to the large feature of crop comparatively safe two.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, and strain aureobasidium pullulans and an application thereof is provided.
Technical scheme of the present invention is as follows:
One strain aureobasidium pullulans, its Classification And Nomenclature is Aureobasidium pullulans, Aureobasidium pullulans, complete called after Aureobasidium pullulans PA-2, on October 28th, 2013, be deposited in No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers of No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, deposit number is CGMCC No.8413.
The application of described aureobasidium pullulans, is that the ferment filtrate of aureobasidium pullulans is sprayed in the field of wheat, rape or pea, is applied to field weeding.
The application of described aureobasidium pullulans, the preparation of the ferment filtrate of its aureobasidium pullulans, that colony edge after cultivating 7d is got inoculated by hypha block to having in the culturing bottle of 200mLPD nutrient solution, 5 of every bottle grafts, 25 ℃ of 150rmin-1 cultivate 14d, nutrient solution vacuum filtration, filtrate is used 0.45 μ m filtering with microporous membrane again, obtains ferment filtrate.
The application of described aureobasidium pullulans, the amount of spraying of its ferment filtrate is 200mL/4m
2.
Described grass be Tender Catchweed Bedstraw Herb, lamb's-quarters, winter certain herbaceous plants with big flowers and wild avena sativa.
The present invention is directed to Tender Catchweed Bedstraw Herb, lamb's-quarters, the winter broadleaf weeds such as certain herbaceous plants with big flowers in wheat, rape, pea field and wild avena sativa etc., screen a kind of Aureobasidium pullulans bacteria strain, for biological weed control.Energy effective and safe of the present invention ground is controlled and control broadleaf weeds and wild avena sativa, and cost is low, pollution-free, and low residue is environmentally friendly.
Accompanying drawing explanation
Fig. 1 is PA-2 mycelia piece Pathogenicity (symptom during 5d); ; A is the front of leaf, the back side that B is leaf.
Fig. 2 is the Pathogenicity of PA-2 strain fermentation filtrate; A wherein: Tender Catchweed Bedstraw Herb; B: lamb's-quarters; C: the winter certain herbaceous plants with big flowers; D: pale persicaria; E: wild avena sativa.
Fig. 3 is that Aureobasidium pullulans is cultivated proterties; A wherein: colonial morphology (25 ℃ of PDA); B: beads shape mycelia; C: chlamydospore.
Fig. 4 is the phylogenetic tree of the bacterial strain PA-2 based on ITS rDNA sequence B LAST result structure.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
1, experiment material and method
The collection of 1.1 sick samples and the separation and purification of pathogenic bacteria
From Pingan County, Qinghai Province, gather the poplar leaf of morbidity, sample is sub-packed in valve bag, in laboratory, disease symptom is recorded, gathers photo, standby in 4 ℃ of Refrigerator stores.Separation purification method, with reference to the separation method of Fang Zhongda (1998) relevant pathogenic fungi in planting disease research method, carries out separation and cultivation to poplar leaf disease sample.Be the tissue of the sick strong intersection of clip poplar leaf, be placed in 3% clorox surface sterilization 3min, then rinsing 3 times in sterilized water, and blot tissue surface moisture with sterilizing thieving paper, be placed in 25 ℃ of thermostat container dark culturing on PDA culture medium flat plate.On each culture medium flat plate, place 5 tissue block.Cultivate 4d and treat that mycelia appears in diseased tissues surface, the mycelia at inoculating needle picking colony edge carries out purifying the numbering of pathogenic bacteria.
The Pathogenicity of 1.2 pathogenic bacteria PA-2 bacterial strains to several weeds
1.2.1 the Pathogenicity of mycelia piece
Gather the fresh weeds spire of field health, 3 rear natural airings of aseptic water washing for blade, then be positioned in the culture dish (Φ=9cm) that is lined with aseptic filter paper, at the colony edge of cultivating 7d, get mycelia piece (Φ=8mm) and receive face of blade, take that to inoculate aseptic PDA culture block be contrast, be placed in (25 ± 1) ℃ illumination box moisturizing and cultivate, observe blade incidence.
1.2.2 the Pathogenicity of meta-bolites
Colony edge after cultivating 7d is got inoculated by hypha block to PD nutrient solution (200mL/ bottle), 5 of every bottle grafts, 25 ℃ of 150rmin
-1cultivate 14d, nutrient solution vacuum filtration, filtrate is used millipore filtration (Φ=0.45 μ m) to filter again and is obtained ferment filtrate, hand-held little watering can spray inoculation is on 7～10 leaf phases healthy weeds plant, inoculum size is 20mL/ basin, and postvaccinal weeds plant is placed in 28 ℃, the growth cabinet of 12h alternation of light and darkness and cultivates with the moisturizing of plastics bag bagging.Each process to repeat 4 times, using only inoculate aseptic PD nutrient solution plant as blank.After 7d, observe the incidence of inoculation weeds.
1.3PA-2 bacterial strain is to crop safety preliminary examination
By PA-2 strain fermentation filtrate respectively spray inoculation (20mL/ basin) in wheat (youth 587), rape (blue or green assorted No. 5), broad bean (No. 9, Qinghai), pea (grassland 224), the highland barley (blue or green No. 8 of north) of 3～4 leaf phases, the same 1.2.2 of cultural method.Every processing repeats 4 times, using only inoculate aseptic PD nutrient solution plant as blank.7d " Invest, Then Investigate " incidence.By following standard, record occurring degree: NS and represent asymptomatic (without scab, plant normal growth); LS indicates minor response (the fragmentary spot of Leaf positional distribution is grown slightly suppressed); MS represents medium susceptible (scab appears in 1/5～1/4 leaf area, grows suppressed); SS represents seriously susceptible (scab appears in 1/4 above blade area, and growth is subject to severe inhibition).
The evaluation of 1.4PA-2 bacterial strain
1.4.1 identification of morphology
PA-2 is cultivated on PDA substratum to its mycelia of optical microphotograph Microscopic observation, spore shape.By diameter be 8mm inoculated by hypha block in substratum plate (Φ=9cm), 25 ℃ of cultivations, its colony growth rate of routine observation, produce spore speed, sporulation quantity, colonial morphology and color and luster and change.
1.4.2 Molecular Identification
DNA extraction: select universal primer PCR amplifying genom DNA ITS district: by universal primer ITS4 and the ITS5 of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthetic amplification fungi ribosomal gene.ITS4 and ITS5 sequence are respectively: ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '; ITS5:5 '-GGAAGTAAAAGTCGTAACAAGG-3 '.
(50 μ L) is as follows for pcr amplification reaction system: every kind of composition of 4 μ L dNTP Mixture(is 2.5mmol/L), ITS4(20 μ mol/L) and ITS5(20 μ mol/L) each 1.0 μ L, 10 * Taq reaction buffer of 6.0 μ L, 1.0 μ L Taq DNA polymerase(2.5U/ μ L), template DNA is 2 μ L, 35.0 μ L aseptic deionized waters.PCR circulation arranges as follows: 94 ℃ of denaturation 5min; 94 ℃ of sex change 1min, 51 ℃ of annealing 1min, 72 ℃ are extended 1min, establish altogether 35 circulations; Last 72 ℃ are extended 10min.Amplified production is in 1.0% sepharose after electrophoretic separation, and dyeing 15min in ethidium bromide solution (0.5 μ g/mL), by Bio-Rad Gel Doc2000 imaging system displayed record electrophoresis result.Collect PCR product and check order, examining order is completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Sequencing result carries out after sequence correction with chromas.exe software, from the NCBI Blast retrieval DNA sequence dna high with target sequence homology, checks order to be listed as carry out respectively comparing in bacterial strain kind with this test.Utilize MEGA5.0 software neighbour to return group method (Neighbor-Joining, N-J) phylogenetic tree construction, the degree of confidence bootstrapping of each branch detects (bootstrap) 1000 times.
2, result
The Pathogenicity of 2.1 aureobasidium pullulanses (Aureobasidium pullulans) PA-2 bacterial strain
After PA-2 bacterial strain inoculated by hypha block weeds 3d, can be observed Minimal change on grass cutting blade, mycelia piece surrounding grows a large amount of mycelia, starts gradually to Infectikon, around mycelia piece surrounding, forms a circle brown speckle; After 5d, mycelia piece surrounding brown speckle is expanded gradually, and two speckles connect in flakes.Meanwhile, mycelia penetrates whole blade, is covered with blade back portion, extends to subsequently whole blade, and blackening is rotted.Wherein wild avena sativa inoculation position yellow leaf, expands (Fig. 1) along vein.
Mycelia piece Pathogenicity result shows: PA-2 bacterial strain all shows as pathogenic by force to some weeds excised leafs, for examination weeds, is Elsholtzia densa., lamb's-quarters, winter certain herbaceous plants with big flowers, field thistle, Tender Catchweed Bedstraw Herb, wild avena sativa etc.
Spray after ferment filtrate 3d, occur the symptom of being injured on weeds, Tender Catchweed Bedstraw Herb shows as leaf rolling, and wilting appears in lamb's-quarters, the flavescence of Leaf of Cluster Mallow sheet chlorisis, and pale persicaria blade is wilted, and wild avena sativa blade is wilted, chlorosis; After 5d, Tender Catchweed Bedstraw Herb is cashed as cauline leaf withered, and the plant of lamb's-quarters 2/3 is withered, the winter certain herbaceous plants with big flowers all withered, pale persicaria 2/3 blade is withered, the whole strain of wild avena sativa is withered, as boiling water scalded.Pass in time disease and increase the weight of, last whole basin weeds withered (as Fig. 2).
Table 1 is that PA-2 strain fermentation filtrate is to the comparison of different weeds virulence.As can be seen from Table 1: after spray inoculation 7d, except pale persicaria, sickness rate all reaches more than 88.9%, and wherein Tender Catchweed Bedstraw Herb, winter certain herbaceous plants with big flowers sickness rate are up to 96%; Strain fermentation filtrate is 62.11% to the mean fresh effect of pale persicaria, to Tender Catchweed Bedstraw Herb, lamb's-quarters, winter, the mean fresh effect of certain herbaceous plants with big flowers, wild avena sativa reaches respectively 87.25%, 78.46%, 82.25%, 80.27%, mean fresh effect compared with pale persicaria is all significant difference (P < 0.05), and each is also significant difference between processing; Disease index: the winter certain herbaceous plants with big flowers the highest, be secondly Tender Catchweed Bedstraw Herb, illustrate that strain fermentation filtrate is the most responsive to winter certain herbaceous plants with big flowers, Tender Catchweed Bedstraw Herb.Five kinds of weeds incidences are carried out to follow-up investigation, find that inoculation weeds are without anabiosis, the state of an illness constantly increases the weight of, until whole basin weeds are withered.With five kinds of common weeds of field survey object of making a living, test-results shows: this strain fermentation filtrate to Tender Catchweed Bedstraw Herb, lamb's-quarters, winter certain herbaceous plants with big flowers, wild avena sativa there is good preventive effect.
Table 1PA-2 strain fermentation filtrate is to the comparison of different weeds virulence
Note: in table, data are mean value ± standard error.With the significant difference in 0.05 level of different letter representations after column data.
2.2 biocontrol strains are tested crop safety
Table 2 is for studying the susceptibility of thing to PA-2.As can be seen from Table 2, PA-2 strain fermentation filtrate is to spring wheat (youth 587), spring rape (blue or green assorted No. 5), the equal no pathogenicity of pea (grassland 224), crop growing state and plant height are compared with blank plant, are not affected, and show as not reaction (NS); Broad bean (No. 9, Qinghai) is had slight pathogenic, on inoculation plant top there is a small amount of needle-like blackspot in blade, and later stage scab is not expanded, and shows as minor response (LS); And highland barley (north blue or green No. 8) is also had slight pathogenic, and on blade, produce on a small quantity less scab, plant height is had to slight restraining effect, contrast plant height short by 4.2%, show as minor response (LS), and the later stage state of an illness is not expanded.The common main cultivation crop safety test result in Qinghai Province is shown: PA-2 strain fermentation filtrate is to comparatively safe for studying thing.
Table 2 is for studying the susceptibility of thing to PA-2
The qualification result of 2.3PA-2 bacterial strain
2.3.1 morphological specificity
On PDA flat board, bacterium colony is fine and close, and the initial stage is dirty white, thick, and rear surrounding becomes deep green, until black, positive and negative color and luster is consistent.Edge is irregular, is obvious root shape, has fold, and hide-like is close to substratum growth; Mycelia chocolate, has barrier film, is beads shape (as Fig. 3-B), and branch is rare, and conidium is concatenated; Substrate mycelium is flourishing, and aerial hyphae is undeveloped, is difficult for picking (as Fig. 3-A).According to the literature, this strain growth process cellular form has five kinds of variations: yeast shape, thallospore, swelling spore, mycelioid and chlamydospore, growth conditions is depended in the evolution of these several forms.
Having the transition state thickening through cell walls every cystite to change into has every chlamydospore (as Fig. 3-C).Chlamydospore budding produces yeast shape cell, and mycelium changes beads shape mycelia (as Fig. 3-B) into, and because of the variation of Medium's PH Value, five kinds of Growth of Cells forms of Aureobasidium pullulans can alternately occur.
2.3.2 Molecular Identification
Adopt universal primer ITS4 and ITS5, in the genomic dna of bacterial strain, amplification obtains the ITS fragment that clip size is 560bp, described pcr amplification product is carried out to sequencing, the ITS sequence of this bacterial strain is carried out to homology comparison (BLAST) on NCBI website (http://www.ncbi.nlm.nih.gov), transfer the fungi ITS rDNA sequence that wherein homology is higher with it, utilize software Clustal W to carry out multiple coupling arrangement analysis, with neighbour, return group method phylogenetic tree construction.Find that the ITS of this bacterium and the ITS of Aureobasidium pullulans Pseudomonas are highly similar, all reach more than 99%, wherein with Aureobasidium pullulans(HQ829084.1, HM992505.1, HQ115720.1, AB693902.1) homology reach 100%.Result shows: bacterial strain PA-2 and Aureobasidium pullulans gather on same (as Fig. 4).
Press the method described in embodiment 1, prepare ferment filtrate, tested the outdoor tentative experiment of Aureobasidium pullulans PA-2 Controlling Weeds in Rape Fields field weed.Outdoor test carries out in Plant Protection Inst., Academy of Agriculture and Forest, Qinghai Prov. experimental plot, and weeds mainly be take wild avena sativa (3～4 leaf phase), Tender Catchweed Bedstraw Herb, lamb's-quarters (3～5 wheel blade phase) as main, the about 4m of community area
2, the amount of spraying 200ml, repeats for 3 times, sprays preventive effect and the fresh weight effect of within latter 20 days, 40 days, investigating wild avena sativa and broadleaf weeds, and result is as shown in table 3.
The preventive effect of table 3 Aureobasidium pullulans PA-2 to Rapeseed Field major objective weeds
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (5)
1. a strain aureobasidium pullulans, is characterized in that, deposit number is CGMCC No.8413.
2. the application of aureobasidium pullulans according to claim 1, is characterized in that, the ferment filtrate of aureobasidium pullulans is sprayed in the field of wheat, rape or pea, is applied to field weeding.
3. the application of aureobasidium pullulans according to claim 2, it is characterized in that the preparation of the ferment filtrate of aureobasidium pullulans is that the colony edge after cultivating 7d is got inoculated by hypha block to having in the culturing bottle of 200mLPD nutrient solution, 5 of every bottle grafts, 25 ℃ of 150rmin
-1cultivate 14d, nutrient solution vacuum filtration, filtrate is used 0.45 μ m filtering with microporous membrane again, obtains ferment filtrate.
4. the application of aureobasidium pullulans according to claim 2, is characterized in that, the amount of spraying of ferment filtrate is 200mL/4m
2.
5. the application of aureobasidium pullulans according to claim 2, is characterized in that, described grass be Tender Catchweed Bedstraw Herb, lamb's-quarters, winter certain herbaceous plants with big flowers and wild avena sativa.
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Cited By (2)
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| CN110373335A (en) * | 2019-08-28 | 2019-10-25 | 青海省农林科学院 | Aureobasidium pullulans PA-2 fermentation medium and fermentation condition optimization method |
| CN112852908A (en) * | 2021-01-11 | 2021-05-28 | 青海省农林科学院 | Aureobasidin with weeding effect and preparation method and determination method thereof |
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| CN112852908A (en) * | 2021-01-11 | 2021-05-28 | 青海省农林科学院 | Aureobasidin with weeding effect and preparation method and determination method thereof |
| CN112852908B (en) * | 2021-01-11 | 2023-05-19 | 青海省农林科学院 | Aureobasidin with weeding effect, and preparation method and determination method thereof |
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