CN103642700B - One strain aureobasidium pullulans and application thereof - Google Patents
One strain aureobasidium pullulans and application thereof Download PDFInfo
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- CN103642700B CN103642700B CN201310645649.4A CN201310645649A CN103642700B CN 103642700 B CN103642700 B CN 103642700B CN 201310645649 A CN201310645649 A CN 201310645649A CN 103642700 B CN103642700 B CN 103642700B
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Abstract
The invention discloses strain aureobasidium pullulans and an application thereof, the Classification And Nomenclature of this bacterial strain is Aureobasidium pullulans, Aureobasidium pullulans, complete called after Aureobasidium pullulans PA-2, be deposited in No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers on October 28th, 2013, deposit number is CGMCC No.8413.Aureobasidium pullulans PA-2 provided by the invention wheat, rape, pea Tanaka, for Tender Catchweed Bedstraw Herb, lamb's-quarters, the winter broadleaf weeds and the wild avena sativa etc. such as certain herbaceous plants with big flowers biological weed control, it can control and prevent and treat broadleaf weeds and wild avena sativa effectively.
Description
Technical field
The present invention relates to field of ecology, in particular strain aureobasidium pullulans and an application thereof.
Background technology
It is that main body promotes Derived from Agricultural Modernization with chemical herbicide that farmland weed controls main.Along with widely using 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 has been done in campelyco candidate biological screening by China.In recent years, weeds research department of Agricultural University Of Nanjing 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-Sclerotiumrolfsii), alternanthera philoxeroides-vacation is every alternaric bacteria (Alternantera phyloxeroides-Nimbyaalternantherae), the research of the bacterium cursive script systems such as Hemp Eupatorium-alternaric bacteria (Eupatorium adenophorum-Alternaria alternata) achieves progress, define a collection of campelyco technological achievement with independent intellectual property right.
Microbial herbicide is compared chemical herbicide and is had that selectivity by force, does not injure natural enemy, not easily develops 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.To the evaluation of microbial herbicide, Main Basis two standards and validity (drug effect) and specificity (security) in theory.Weeds biocontrol microorganisms should possess not only have target weeds pathogenic but also to the comparatively safe two major features of crop by force.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, provides strain aureobasidium pullulans and an application thereof.
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, be deposited in No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers on October 28th, 2013, deposit number is CGMCC No.8413.
The application of described aureobasidium pullulans, is the field sprayed by the ferment filtrate of aureobasidium pullulans in 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, get inoculated by hypha block in the culturing bottle having 200mLPD nutrient solution cultivating the colony edge after 7d, every bottle graft 5 pieces, 25 DEG C of 150rmin-1 cultivate 14d, nutrient solution vacuum filtration, filtrate uses 0.45 μm of 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, the winter certain herbaceous plants with big flowers and wild avena sativa.
The present invention is directed to wheat, rape, the Tender Catchweed Bedstraw Herb in pea field, lamb's-quarters, the winter broadleaf weeds and the wild avena sativa etc. such as certain herbaceous plants with big flowers, screen a kind of Aureobasidium pullulans bacteria strain, for biological weed control.Energy effective and safe ground of the present invention controls and prevents and treats 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 block Pathogenicity (symptom during 5d); ; A is the front of leaf, and B is the back side of leaf.
Fig. 2 is the Pathogenicity of PA-2 strain fermentation filtrate; Wherein A: 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 Aureobasidium pullulans cultural colony; Wherein A: colonial morphology (25 DEG C 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
Gather the poplar leaf of morbidity from Pingan County, Qinghai Province, sample is sub-packed in valve bag, in laboratory, disease symptom recorded, gather photo, for subsequent use in 4 DEG C of Refrigerator stores.Separation purification method at the separation method of planting relevant pathogenic fungi in disease research method with reference to Fang Zhongda (1998), is separated poplar leaf disease sample and cultivates.Namely the tissue of the strong intersection of clip poplar leaf disease, is placed in 3% clorox surface sterilization 3min, then rinsing 3 times in sterilized water, and blots tissue surface moisture with sterilizing thieving paper, is placed in 25 DEG C of thermostat container dark culturing on PDA culture medium flat plate.Each culture medium flat plate places 5 tissue block.Cultivate 4d and treat that mycelia appears in diseased tissues surface, the mycelia at inoculating needle picking colony edge carries out the purifying of pathogenic bacteria and numbers.
1.2 pathogenic bacteria PA-2 bacterial strains are to the Pathogenicity of several weeds
1.2.1 the Pathogenicity of mycelia block
Gather the fresh weeds spire of field health, blade aseptic water washing 3 rear natural airings, then be positioned over be lined with aseptic filter paper culture dish (Φ=9cm) in, get mycelia block (Φ=8mm) at the colony edge cultivating 7d and receive face of blade, to inoculate aseptic PDA culture block for contrast, be placed in (25 ± 1) DEG C illumination box moisturizing to cultivate, observe blade incidence.
1.2.2 the Pathogenicity of meta-bolites
Colony edge after cultivating 7d gets inoculated by hypha block to PD nutrient solution (200mL/ bottle), every bottle graft 5 pieces, 25 DEG C of 150rmin
-1cultivate 14d, nutrient solution vacuum filtration, filtrate is used millipore filtration (Φ=0.45 μm) 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 DEG C, the growth cabinet plastics bag bagging moisturizing of 12h alternation of light and darkness cultivates.Each process repetition 4 times, only to inoculate the plant of aseptic PD nutrient solution as blank.The incidence of inoculation weeds is observed after 7d.
1.3PA-2 bacterial strain is to crop safety preliminary examination
By PA-2 strain fermentation filtrate respectively spray inoculation (20mL/ basin) in the wheat (youth 587) of 3 ~ 4 leaf phases, rape (blue or green assorted No. 5), broad bean (No. 9, Qinghai), pea (grassland 224), highland barley (blue or green No. 8 of north), the same 1.2.2 of cultural method.Often process repetition 4 times, only to inoculate the plant of aseptic PD nutrient solution as blank.7d " Invest, Then Investigate " incidence.Record occurring degree: NS by following standard to represent asymptomatic (without scab, normal plants); LS indicates minor response (the fragmentary spot of Leaf positional distribution grows slightly suppressed); MS represents medium susceptible (scab appears in the leaf area of 1/5 ~ 1/4, grows suppressed); SS represents seriously susceptible (scab appears in more than 1/4 blade area, and growth is subject to serious suppression).
The qualification of 1.4PA-2 bacterial strain
1.4.1 identification of morphology
PA-2 is cultivated on PDA substratum, its mycelia of optical microphotograph Microscopic observation, spore shape.By diameter be 8mm inoculated by hypha block in substratum plate (Φ=9cm), 25 DEG C of cultivations, its colony growth rate of routine observation, produce the change of spore speed, sporulation quantity, colonial morphology and color and luster.
1.4.2 Molecular Identification
DNA extraction: the ITS district selecting universal primer PCR amplifying genom DNA: universal primer ITS4 and ITS5 being synthesized amplification fungi ribosomal gene by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.ITS4 and ITS5 sequence is respectively: ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '; ITS5:5 '-GGAAGTAAAAGTCGTAACAAGG-3 '.
Pcr amplification reaction system (50 μ L) is as follows: 4 μ L dNTP Mixture(often plant composition and are 2.5mmol/L), ITS4(20 μm of ol/L) and ITS5(20 μm of ol/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 is circularly set as follows: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 51 DEG C of annealing 1min, 72 DEG C extend 1min, establish 35 circulations altogether; Last 72 DEG C extend 10min.Amplified production is in the sepharose of 1.0% 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 primer to check order, examining order is completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.After sequencing result chromas.exe software carries out sequence correction, retrieve and the high DNA sequence dna of target sequence homology from NCBI Blast, with this test check order to arrange and carry out comparison in bacterial strain kind respectively.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, grass cutting blade can be observed Minimal change, and mycelia block surrounding grows a large amount of mycelia, starts to Infectikon gradually, forms a circle brown speckle around mycelia block surrounding; After 5d, mycelia block 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 whole blade subsequently, and blackening is rotted.Wherein wild avena sativa inoculation position yellow leaf, along vein expansion (Fig. 1).
Mycelia block Pathogenicity result shows: PA-2 bacterial strain all shows as pathogenic by force to some weeds excised leafs, for examination weeds be Elsholtzia densa., lamb's-quarters, the winter certain herbaceous plants with big flowers, field thistle, Tender Catchweed Bedstraw Herb, wild avena sativa etc.
After spraying ferment filtrate 3d, damage symptoms appears in weeds, Tender Catchweed Bedstraw Herb shows as leaf rolling, and wilting appears in lamb's-quarters, and Leaf of Cluster Mallow sheet chlorisis turns yellow, and pale persicaria blade is wilted, and wild avena sativa blade is wilted, chlorosis; After 5d, Tender Catchweed Bedstraw Herb cash for cauline leaf withered, 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 wither, the whole strain of wild avena sativa wither, as boiling water scalded.Pass disease in time to increase the weight of, last whole basin weeds withered (as Fig. 2).
Table 1 is that PA-2 strain fermentation filtrate is compared 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%, wherein Tender Catchweed Bedstraw Herb, winter certain herbaceous plants with big flowers sickness rate up to 96%; The mean fresh effect of strain fermentation filtrate to pale persicaria is 62.11%, to Tender Catchweed Bedstraw Herb, lamb's-quarters, the winter certain herbaceous plants with big flowers, wild avena sativa mean fresh effect reach 87.25%, 78.46%, 82.25%, 80.27% respectively, imitate all in significant difference (P < 0.05) compared with the mean fresh of pale persicaria, also in significant difference between each process; Disease index: the winter certain herbaceous plants with big flowers the highest, be secondly Tender Catchweed Bedstraw Herb, illustrate strain fermentation filtrate to the winter certain herbaceous plants with big flowers, Tender Catchweed Bedstraw Herb is the most responsive.Carry out follow-up investigation to five kinds of weeds incidences, find that inoculation weeds are without anabiosis, the state of an illness constantly increases the weight of, until whole basin weeds are withered.To make a living survey object with five kinds of weeds that field is common, test-results shows: this strain fermentation filtrate to Tender Catchweed Bedstraw Herb, lamb's-quarters, the winter certain herbaceous plants with big flowers, wild avena sativa has good preventive effect.
Table 1PA-2 strain fermentation filtrate is compared different weeds virulence
Note: in table, data are mean value ± standard error.With letter representation different after column data significant difference in 0.05 level.
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), pea (grassland 224) all no pathogenicities, crop growing state and plant height are compared with blank plant, are not affected, and show as not reaction (NS); Have slight pathogenic to broad bean (No. 9, Qinghai), 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 also have slight pathogenic to highland barley (north blue or green No. 8), blade produces a small amount of less scab, have slight restraining effect to plant height, comparatively 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
Compact colonies on PDA flat board, 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, in obvious root shape, has fold, hide-like, is close to substratum growth; Mycelia chocolate, has barrier film, and in beads shape (as Fig. 3-B), branch is rare, and conidium is concatenated; Substrate mycelium is flourishing, and aerial hyphae is undeveloped, not easily picking (as Fig. 3-A).According to the literature, this strain growth Procedure Cell form has five kinds of changes: yeast shape, thallospore, swelling spore, mycelioid and chlamydospore, and this several shape State evolution depends on growth conditions.
Having to change into through the transition state that cell walls thickens every cystite 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 change 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, sequencing is carried out to described pcr amplification product, the ITS sequence of this bacterial strain is carried out tetraploid rice (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, return group method phylogenetic tree construction with neighbour.Find that the ITS of this bacterium is highly similar to the ITS of Aureobasidium pullulans Pseudomonas, 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 gathers on same (as Fig. 4).
Embodiment 2
By the method described in embodiment 1, prepare ferment filtrate, test 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 are mainly based on wild avena sativa (3 ~ 4 leaf phase), Tender Catchweed Bedstraw Herb, lamb's-quarters (3 ~ 5 wheel blade phase), and plot area is about 4m
2, the amount of spraying 200ml, repeats for 3 times, and spray preventive effect and the fresh weight effect of latter 20 days, 40 days investigation wild avena sativas and broadleaf weeds, result is as shown in table 3.
Table 3 Aureobasidium pullulans PA-2 is to the preventive effect of 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 improve and convert the protection domain that all should belong to claims of the present invention.
Claims (2)
1. a strain aureobasidium pullulans (Aureobasidium pullulans), is characterized in that, deposit number is CGMCC No.8413.
2. the application of aureobasidium pullulans according to claim 1 in weeding, is characterized in that, the ferment filtrate of aureobasidium pullulans is sprayed the field in wheat, rape or pea, is applied to field weeding; The preparation of the ferment filtrate of aureobasidium pullulans is that the colony edge after cultivating 7d gets inoculated by hypha block in the culturing bottle having 200mLPD nutrient solution, every bottle graft 5 pieces, 25 DEG C of 150rmin
-1cultivate 14d, nutrient solution vacuum filtration, filtrate uses 0.45 μm of filtering with microporous membrane again, obtains ferment filtrate; The amount of spraying of ferment filtrate is 200mL/4m
2; Described grass be Tender Catchweed Bedstraw Herb, lamb's-quarters, the winter certain herbaceous plants with big flowers and wild avena sativa.
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| 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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| WO2013092224A1 (en) * | 2011-12-21 | 2013-06-27 | Basf Se | Use of strobilurin type compounds for combating phytopathogenic fungi resistant to qo inhibitors |
| CN103451108A (en) * | 2013-08-06 | 2013-12-18 | 红云红河烟草(集团)有限责任公司 | Aureobasidium pullulans OF-01 strain and method for preparing tobacco endogenous derived spice by using strain |
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| WO2013092224A1 (en) * | 2011-12-21 | 2013-06-27 | Basf Se | Use of strobilurin type compounds for combating phytopathogenic fungi resistant to qo inhibitors |
| CN103451108A (en) * | 2013-08-06 | 2013-12-18 | 红云红河烟草(集团)有限责任公司 | Aureobasidium pullulans OF-01 strain and method for preparing tobacco endogenous derived spice by using strain |
Non-Patent Citations (1)
| Title |
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| 出芽短梗霉菌菌株PA-2的除草活性及对作物的安全性;李永龙，等;《中国生物防治学报》;20140430;第30卷(第2期);232-238 * |
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