CN107043726B - One plant of actinomyces SVFJ-07 and its application in prevention and treatment cymbidium anthracnose - Google Patents

Abstract

The present invention relates to one plant of actinomyces SVFJ-07 and its applications in prevention and treatment cymbidium anthracnose disease, belong to microorganisms technical field.The actinomyces be streptomyces wine red streptomyces (Streptomyces vinaceus) SVFJ-07, deposit number are as follows: CGMCC No.14020.The wine red streptomyces (Streptomyces vinaceus) SVFJ-07 can prevent and treat plant disease caused by cymbidium anthracnose bacterium etc., especially it can stablize in orchid plant body with orchid symbiosis and survive and effectively prevent cymbidium anthracnose.

Description

One plant of actinomyces SVFJ-07 and its application in prevention and treatment cymbidium anthracnose

Technical field

The present invention relates to one plant of actinomyces SVFJ-07 and its applications in prevention and treatment cymbidium anthracnose disease, belong to micro- life Object prevention and control field.

Background technique

Currently the prevention and treatment of plant disease is mainly controlled by chemical pesticides, however chemical pesticide is long-term a large amount of using often Will lead to environmental pollution, pesticide residue and pathogenic bacteria the problems such as to develop drug resistance, and biocontrol microorganisms it is less toxic, pollution-free with it, It is not likely to produce drug resistance and the characteristics such as raw material is easy to get is known as by people as ideal microorganism.Actinomyces are a kind of with important The microorganism of economic value and practical value, type is abundant, distributes widely in nature.The life found from microorganism at present In active substances, nearly 70% is generated by actinomyces, and the antibiotic that especially streptomycete generates is answered extensively in agricultural production With such as avermectin, Zhongshengmycin etc..Soil actinomycete has many advantages, such as that effect is lasting, with strong points and environmentally friendly, answers With having good prospects.

Orchid be (Cymbidium Spp.) be orchid family (Orchidaceae) Cymbidium (Cymbidium) perennial unifacial leaf Herbaceous plant, cultivation history is long, and kind is more, ornamental values and the economic values with higher.With prolonging for implantation time Long and cultivation intensive degree promotion, colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides) caused by anthrax Disease causes to seriously affect to growth, the ornamental even survival of orchid.The disease main harm blade, surface of aggrieved blade initial stage produce Raw light brown recess dot, later period expand as the scab of irregular shape or ellipse, and sick portion's recess is fairly obvious, and intermediate ash is brown Color, edge dark brown generate the pore of many colyliform arrangements, and multiple scabs are connected when serious, and sick leaf is gradually withered, and blue strain is not Even whole strain of blooming is dead.Cause harm effectively to control the pathogen infection, has scholar to its biological characteristics and pesticide control It has made intensive studies, proposes some feasible Control Technology schemes.Currently, the main policies of prevention and treatment anthracnose are that chemistry is anti- Disease-resistant variety is controlled and cultivates, chemical prevention pollutes environment and endangers human health, and disease-resistant variety cultivates difficulty and there are resistances to subtract Weak risk, therefore, biological control is because its is safe and efficient, noresidue becomes the method that people most favor.Soil is various micro- lifes The good habitat of object, survive many microorganisms with mutual antagonism.In the screening of biocontrol bacterial strain, it is contemplated that Interaction relationship between plant and microorganism, people lay stress on from plant root surface or rhizosphere soil and screen, this There is good colonization ability in plant root surface after the microbial inoculant that sample filters out.

Summary of the invention

The purpose of the present invention is to provide one plant of actinomyces SVFJ-07 and its answering in prevention and treatment cymbidium anthracnose disease With, can be used for prevent and treat the plant disease as caused by cymbidium anthracnose.

Actinomyces provided by the invention be wine red streptomyces (Streptomyces vinaceus) SVFJ-07, in 2017 On April 13, in is preserved in Chinese microorganism strain and is deposited in administration committee's common micro-organisms center (abbreviation CGMCC), deposit number Are as follows: CGMCC No.14020, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

The present invention provides application of the actinomyces SVFJ-07 in prevention and treatment cymbidium anthracnose disease.The plant Disease is the microbial plant disease of cymbidium anthracnose.

With above-mentioned wine red streptomyces (Streptomyces vinaceus) SVFJ-07 be active constituent bacteria agent Belong to the present invention

Protection scope, when needed, also comprising common carrier and auxiliary material in microbial inoculum preparation in the microbial inoculum.

The red streptomyces that relieve the effect of alcohol (Streptomyces vinaceus) SVFJ-07 Gause I culture medium, PDA train Support well-grown on base.Gao Shi: gas silk is white, and base silk is orange red.Isp2: gas silk is white, base silk orange.Isp3: gas silk is white, base silk orange Huang produces light yellow soluble pigment.Isp4: gas silk orange is brown, and base silk is brown.Isp5: gas silk orange is brown, and base silk is brown.Optical microscopy Lower its fibrillae of spores of observation is straight, flexible, end buckle.Spore oval, oblong, surface are smooth.Bacterial strain SVFJ-07 is to D- grape The carbon sources such as sugar, D- xylose, PEARLITOL 25C and D-Fructose have different degrees of Utilization ability, do not utilize L-arabinose, sucrose, Gossypose；Starch does not hydrolyze；Do not produce H₂S and Melanoidins；Milk does not solidify, but peptonizes；Gelatin liquefaction is slow.In multiple features Upper and comparison bacterial strain (Streptomyces vinaceus) similar.

Remarkable advantage of the invention: wine red streptomyces of the invention (Streptomyces vinaceus) SVFJ-07 is to orchid Flower anthracnose has very strong inhibiting effect, has the potentiality that preventive and therapeutic effect is played to fungal disease caused by it.

Wine red streptomyces of the invention (Streptomyces vinaceus) SVFJ-07 is isolated from orchid plant rhizosphere week Soil is enclosed, experiments have shown that cymbidium anthracnose can be effectively prevented after inoculation orchid plant, is provided for the prevention and treatment of cymbidium anthracnose One environmental protection, simple, effective approach.

Detailed description of the invention

Fig. 1 be bacterial strain wine red streptomyces (Streptomyces vinaceus) culture of the SVFJ-07 in PDA culture medium Feature.

Fig. 2 is phylogenetic tree of the bacterial strain SVFJ-07 according to 16S rDNA sequence construct.

Fig. 3 be bacterial strain wine red streptomyces (Streptomyces vinaceus) SVFJ-07 spore suspension is to orchid anthrax The preventive effect of disease is as a result, and only connecing anthrax bacteria spore suspension is the experimental result for compareing (CK).

Specific embodiment

Method in following embodiments is unless otherwise instructed conventional method.

Percentage (%) in following embodiments indicates mass percent unless otherwise instructed.

Wherein cymbidium anthracnose bacteria strain carries out tissue separation from from the typical leaf portion of orchid disease symptom according to a conventional method Culture obtains, and monospore is verified its pathogenic rear PDA culture medium test tube slant that moves by Koch's Postulates after purification and saved.Bacterial strain is protected It is hidden in Inst. of Plant Protection, fujian Academy of Agricultural Science's ecological regulation and control room.

Embodiment 1: the screening and identification of actinomyces

1 materials and methods

1.1 material

Bacterial strain: Antagonistic Actinomycetes wine red streptomyces (Streptomyces vinaceus) SVFJ-07 be isolated from soil layer soil Earth；Cymbidium anthracnose bacterium (C. gloeosporioides)、Orchid Pathogen (Fusarium oxysporum), rice banded sclerotial blight Germ (Rhizoctonia solani), banyan alternaria (Alternaria alternata), banyan anthrax bacteria (Colletotrichum gloeosporioides)、Banana blight bacteria (Fusarium oxysporum), early blight of tomato Bacterium (Alternaria solani) saved by this laboratory.

Main agents and culture medium: for examination carbon source D-Glucose, D- xylose, PEARLITOL 25C, D-Fructose, gossypose, L- Ah Drawing uncle's sugar, sucrose and L- inositol is Amersco Products；Actinomyces genome extraction kit, Taq PCR Master The related reagents such as Mix are purchased from TaKaRa company；Actinomyces sequence amplification universal primer 16S rDNA universal primer 27F:5 '- AGAGTTTGATCCTGGCTCAG-3 ', 1541R:5 '-AAGGAGGTGATCCAGCCGCA-3 ' are closed by hero Shanghai biotech firm At.Other reagents are that import or domestic analysis are pure.Actinomyces screening and culturing medium is improvement HVA culture medium, actinomycetes strain Activation uses No. 1 culture medium of Gao Shi with preservation；Liquid shake-flask fermentation is No. 1 culture medium of liquid Gao Shi；The PCR such as r-Taq enzyme are related Reagent is purchased from TaKaRa company；Streptomycete culture medium 2 (ISP2), streptomycete culture medium 3 (ISP3), streptomycete culture medium 4 The culture mediums such as number (ISP4) and streptomycete culture medium No. 5 (ISP5) are purchased from Qingdao Hai Bo Bioisystech Co., Ltd.

Method

1.2.1 actinomyces are separately cultured

After the sieving of taxi earth sample natural air drying will be collected, is separated using plate dilution method: weighing 1 g of pedotheque, It is added in the centrifuge tube for having 9 mL sterile waters, vibrates 30 min, be then configured to 10^{- 3}、10^{- 4} 、10^{- 5} Suspension.It takes In improvement HVA culture medium flat plate, even spread, each 3 ware of degree of putting culture is placed in each 0.1 mL of the suspension of various concentration Culture is inverted in 28 DEG C of insulating box.The single colonie grown on culture 7d picking culture medium carries out switching purifying.According to bacterial strain Morphological feature and cultural characteristic remove duplicate bacterial strain.Culture is placed on 4 in the actinomycetes strain inoculation slant medium of purifying DEG C refrigerator saves backup.

The screening of Antagonistic Actinomycetes

It is new with reference to Du Yi or deer connects bright etc. method, plate opposite culture method is respectively adopted and actinomyces cultivate filtered fluid Inhibiting rate method primary dcreening operation and secondary screening have the actinomyces of antagonistic activity to cymbidium anthracnose bacterium, and the actinomyces that separation obtains are inoculated in height In family name's No.1 culture medium flat plate, the bacterium dish of 5 mm of diameter is taken to be placed in PDA plate edge, centre inoculation after 28 DEG C of 7 d of culture Cymbidium anthracnose bacterium, 28 DEG C of 5 d of culture, observes the presence or absence of antibacterial band, screens the wider bacterial strain of antibacterial band.Plate is stood facing each other and is screened Antibacterial bandwidth is inoculated in fermentation medium in the Actinomycetes species of 10 mm or more out.Ferment 500 mL triangular flasks, often Bottled liquid measure is 200 mL, cultivates 7 d in 28 DEG C of 180 r/min.Fermentation liquid takes after 10000 g are centrifuged 10 min 0.22 μm of filtering with microporous membrane of clear liquid, takes 0.5 mL of filtrate to be added in the culture dish of 9 cm of diameter, and is cooled to 50 DEG C 9. the plate that carries disease germs is made in the mixing of 5 mL PDA culture mediums, is beaten and taken for trying pathogen bacteria cake with 5 mm aseptic card punch, be placed in band The center of bacterium plate is compareed with sterile water with plate made of PDA, 3 repetitions.After 28 DEG C of 7 d of culture, right-angled intersection is used Method measures colony diameter, calculates growth inhibition ratio, calculates growth inhibition ratio as follows: growth inhibition ratio=(control plate bacterium Fall diameter-processing flat-plate bacterial colony diameter)/(control flat-plate bacterial colony diameter-bacteria cake diameter) × 100%.

The antimicrobial spectrum of Antagonistic Actinomycetes measures

Using the antimicrobial spectrum for the actinomyces that Odontothrips loti measurement filters out, target bacterium mycelia and spore are scraped with transfer needle It is made in the culture dish for pouring into 9 cm after 1 mL of bacteria suspension is mixed with 10 mL of PDA culture medium and plate is made, by the Oxford that sterilizes Cup is placed in the PDA plate center of mixed target bacterium, the 200 sterile ferment filtrates of μ L is added in cup, after setting 28 DEG C of 3 d of culture respectively Measure antibacterial circle diameter, test 3 repetitions of setting.

The identification of Antagonistic Actinomycetes

(1) morphological feature measure: by screening obtain Antagonistic Actinomycetes SVFJ-07 on Gause I culture medium with sterile Then oese streak inoculation enters coverslip (cm of 1 cm × 1) oblique cutting after sterilizing in culture medium, in 28 DEG C of incubators After cultivating 5 d, spore and hypha form that coverslip is placed in optical microphotograph microscopic observation bacterial strain are taken out.

(2) cultural characteristic measures: carrying out referring to the method in " the strepto- dientification of bacteria is in volume ".

(3) physiological and biochemical property measures: referring to " actinomyces Rapid identification and genealogical classification " and " streptomycete identification handbook " Described in method, the physio-biochemical characteristics of bacterial strain SVFJ-07 such as utilization of carbon source, melanin are generated, H₂S generation, aesculin Utilize the indexs such as, cellulose utilization, gelatin liquefaction, Starch Hydrolysis, milk solidification and jellyization, cellulose hydrolysis, nitrate reduction It is measured.

(4) 16S rDNA sequence is analyzed: bacterial strain SVFJ-07 cultivates the bacterium solution of 4-5 d in Gause I fluid nutrient medium, It sets and bacterial sediment is collected after centrifugation in sterile centrifugation tube.0.1 ~ 0.2 g thallus is weighed in sterile mortar, after a small amount of PVP is added With liquid nitrogen grinding at powder, thallus total DNA is extracted using actinomyces genome extraction kit.With universal primer 27F:5 '- AGAGTTTGATCCTGGCTCAG-3 ', 1541R:5 '-AAGGAGGTGATCCAGCCGCA-3 ' amplification actinomyces 16S rDNA.It will PCR product is splined in the Ago-Gel containing GelRed dyestuff and recycles purpose band after electrophoresis, delivers Shanghai outstanding person Lee biology Company's sequencing.It is compared using NCBI Blast to column are sequenced, chooses the higher bacterium of homology therewith in GenBank The 16S rDNA sequence of strain carries out multiple alignment to it using Clustal X software, analyzes bacterial strain SVFJ-07 and reference strains Between sequence similarity, and pass through the Neighbor joining method phylogenetic tree construction in Mega4.1 software.

Data analysis

Sorting-out in statistics, the DuncanShi duncan's new multiple range method of 9.1 software of SAS are carried out to test data using Excel2003 Significance difference analysis is carried out to each processing.

As a result with analysis

The separation and screening of 2.1 Antagonistic Actinomycetes

Separation is obtained into 25 plants of actinomyces, 6 plants is filtered out by plate opposite culture method and cymbidium anthracnose bacterium antagonism is imitated The preferable actinomycetes strain of fruit.6 plants of bacterial strains are measured to the inhibiting rate of cymbidium anthracnose bacterium using actinomyces culturing filtrate respectively, The result shows that (table 1), 6 plants of actinomyces filtered fluids of primary dcreening operation have certain inhibition to make cymbidium anthracnose bacterium after diluting 100 times With, wherein number be SVFJ-8, SVFJ-15 and SVFJ-18 bacterial strain filtered fluid inhibiting effect it is stronger, inhibiting rate is all larger than 70% , the bacterial strain inhibiting effect that number is SVFJ-07 is most strong, and inhibiting rate reaches nearly 85%.

Bacteriostatic activity of the 16 plants of actinomycetes fermentation liquors of table to cymbidium anthracnose bacterium

It indicates to examine through DuncanShi duncan's new multiple range method with different lowercases are indicated after column dataP0.05 level error of < It is different significant.

Bacteriostatic activity of the Antagonistic Actinomycetes to phytopathogen

By tablet face-off method, screening obtains one plant to the stronger bacterial strain of cymbidium anthracnose bacterium bacteriostatic activity, is named For SVFJ-07.The bacterial strain all has different degrees of inhibiting effect (table 2) to for 7 plants of phytopathogens of examination, wherein to bacterial strain Cymbidium anthracnose bacterium (C. gloeosporioides) and banyan anthrax bacteria (C. gloeosporioides) inhibiting effect is most By force, and to Rhizoctonia solani Kuhn (R. solani) bacteriostatic activity it is weaker.To orchid Pathogen (F. oxysporum), banyan Tree alternaria (A. alternata), banana blight bacteria (F. oxysporum), tomato early blight bacterium (A. solani) etc. With stronger inhibiting effect.

Inhibiting effect of the 2 bacterial strain SVFJ-07 of table to test plant pathogen

It indicates to examine through DuncanShi duncan's new multiple range method with different lowercases are indicated after column dataP0.05 level error of < It is different significant.

The identification of bacterial strain SVFJ-07

2.3.1 morphological feature and cultural character

Bacterial strain SVFJ-07 well-grown in Gause I culture medium, PDA culture medium.Gao Shi: gas silk is white, and base silk is orange red. Isp2: gas silk is white, base silk orange.Isp3: gas silk is white, and base silk is orange, produces light yellow soluble pigment.Isp4: gas silk orange is brown, base Silk is brown.Isp5: gas silk orange is brown, and base silk is brown.Its fibrillae of spores is straight, flexible under the microscope for optical microphotograph, end buckle.Spore oval Shape, oblong, surface are smooth.

Physiological and biochemical property

Bacterial strain SVFJ-07 has different degrees of utilization to carbon sources such as D-Glucose, D- xylose, PEARLITOL 25C and D-Fructoses Ability does not utilize L-arabinose, sucrose, gossypose；Starch does not hydrolyze；Do not produce H₂S and Melanoidins；Milk does not solidify, but It peptonizes；Gelatin liquefaction is slow.In multiple features with comparison bacterial strain (Streptomyces vinaceus) similar.

Sequence analysis

It compares analysis through NCBI Blast to find, 16S rDNA sequence (the sequence such as SEQ ID No.1 of separated actinomyces It is shown) with GenBank Streptomyces belong to multiple kinds all have higher homology, choose the 16S rDNA of 10 typical strains Sequence 5.0 software component phylogenetic tree (Fig. 2) of MEGA.The result shows that bacterial strain SVFJ-07 with (S. vinaceus) belong to Same branch, wherein with bacterial strain (S. vinaceus) for similitude up to 99% or more, combining form and cultural characteristic reflect bacterial strain Be set to wine red streptomyces (S. vinaceus).

Embodiment 2: bacterial strain SVFJ-07 greenhouse pot culture efficiency test

Cymbidium variety is potted orchids.Every plant of pouring 50ml SVFJ-07 bacterial strain fermentation liquor (10⁶A spore/mL).24h Stab inoculation anthrax bacteria afterwards, inoculum density 1 × 10⁶A spore/mL is inoculated with second of the fermentation liquid of progress in the 4th day after pathogen Processing.It is compareed using the plant of SC1500 times of root irrigation of 45% Prochloraz as pesticide, the plant handled with fermentation medium and clear water Strain is used as blank control.5 young plants of each processing, 3 repetitions of every processing.Investigation incidence, statistics are fallen ill daily after the onset Rate and disease index, until clear water control disease incidence terminates to test when reaching 90% or more.

Disease grade standard is as follows:

0 grade: disease-free spot；

1 grade: 1~3, every leaf disease spot or 5.0% or less lesion area；

3 grades: 4~6, every leaf disease spot or lesion area 5.1%~10.0%；

5 grades: 7~10, every leaf disease spot or lesion area 10.1%~15.0%；

7 grades: 11~20, every leaf disease spot or lesion area 15.1%~25.0%；

9 grades: 20, every leaf disease spot or more or lesion area account for 25.1% or more full leaf area.

Disease incidence (%)=morbidity strain number/total strain number × 100%

Disease index (%)=[I: the sick series × disease grade plant number)/(highest disease series × total plant number)] × 100%

Control efficiency (%)=[(control disease index one handles disease index)/control disease index] × 100% (control Refer to clear water control treatment)

The experimental results showed that (table 3), for examination wine red streptomyces (S. vinaceus) SVFJ-07 fermentation liquid can significantly drop The disease index of low cymbidium anthracnose, control efficiency are up to 78.21%, are significantly higher than the control efficiency of SC1500 times of 45% Prochloraz 68.72%.In addition it can also be seen that the disease index of fermentation medium processing is slightly below the state of an illness of clear water control treatment from table Index, but the difference between the two is not significant.Greenhouse pot culture the experiment results show that SVFJ-07 bacterial strain can effectively prevent cymbidium anthracnose, It shows good application potential.

Potting control efficiency of the 3 actinomyces SVFJ-07 of table to cymbidium anthracnose

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>Inst. of Plant Protection, fujian Academy of Agricultural Science

<120>one plants of actinomyces SVFJ-07 and its application in prevention and treatment cymbidium anthracnose

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aatggctcac caaggcgacg acgggtagcc ggcctgagag ggcgaccggc cacactggga 300

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taggcggctt gtcgcgtcgg ttgtgaaagc ccggggctta accccgggtc tgcagtcgat 600

acgggcaggc tagagttcgg taggggagat cggaattcct ggtgtagcgg tgaaatgcgc 660

agatatcaga ggaacaccgg tggcgaaggc ggatctctgg gccgatactg acgctgagga 720

gcgaaagcgt gggagcgaac aggattagat accctggtag tccacgccgt aacggtgggc 780

actaggtgtg ggcgacattc cacgtcgtcc gtgccgcagc taacgcatta agtgccccgc 840

ctggggagta cggccgcaag gctaaaactc aaaggaattg acgggggccc gcacaagcgg 900

cggagcatgt ggcttaattc gacgcaacgc gaagaacctt accaaggctt gacatacacc 960

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tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgtcccgtg 1080

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Claims (3)

1. one plant of actinomyces, it is characterised in that: the actinomyces be wine red streptomyces (Streptomyces vinaceus) SVFJ-07 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation on April 13rd, 2017 Number are as follows: CGMCC No.14020.

2. a kind of bacteria agent comprising actinomyces described in claim 1.

3. application of the actinomyces as described in claim 1 in prevention and treatment cymbidium anthracnose.