CN107043726A - One plant of actinomyces SVFJ 07 and its application in preventing and treating cymbidium anthracnose - Google Patents

Abstract

Application the present invention relates to one plant of actinomyces SVFJ 07 and its in preventing and treating cymbidium anthracnose disease, belongs to microbial technology field.The actinomyces are the wine red streptomyces of streptomyces（Streptomyces vinaceus）SVFJ 07, preserving number is：CGMCC No.14020.The wine red streptomyces（Streptomyces vinaceus）SVFJ 07 can prevent and treat plant disease caused by cymbidium anthracnose bacterium etc., can especially stablize in orchid plant body with orchid symbiosis and survive and effectively prevent and treat cymbidium anthracnose.

Description

One plant of actinomyces SVFJ-07 and its application in preventing and treating cymbidium anthracnose

Technical field

Application the present invention relates to one plant of actinomyces SVFJ-07 and its in preventing and treating cymbidium anthracnose disease, belongs to micro- life Thing prevention and control field.

Background technology

The current preventing and treating to plant disease is main based on chemical prevention, but chemical pesticide is long-term a large amount of using often The problems such as environmental pollution, residues of pesticides and pathogenic bacteria can be caused to develop immunity to drugs, and biocontrol microorganisms with its low toxicity, it is pollution-free, It is not likely to produce the resistance to the action of a drug and the characteristic such as raw material is easy to get is by it has been recognized that be preferable microorganism.It is important that actinomyces are that a class has The microorganism of economic value and practical value, species is enriched, and is distributed widely in nature.The life found at present from microorganism In active substances, nearly 70% is produced by actinomyces, and the antibiotic that particularly streptomycete produces extensively should in agricultural production With, such as AVM, Zhongshengmycin etc..Soil actinomycete has the advantages that effect is lasting, with strong points and environmentally friendly, should With having good prospects.

Orchid is（Cymbidium spp.）It is orchid family（Orchidaceae）Cymbidium（Cymbidium）Perennial unifacial leaf Herbaceous plant, cultivation history is long, and kind is more, with higher ornamental values and the economic values.With prolonging for implantation time Long and cultivation intensive degree lifting, colletotrichum gloeosporioides Penz（Colletotrichum gloeosporioides）Caused anthrax Disease to the growth of orchid, view and admire or even survival causes to have a strong impact on.The disease main harm blade, surface production at aggrieved blade initial stage Raw light brown depression dot, the later stage expands as the scab of irregular shape or ellipse, and sick portion's depression is fairly obvious, and middle ash is brown Color, edge dark brown produces the pore of many colyliform arrangements, multiple scabs are connected when serious, and sick leaf is gradually withered, and blue strain is not Even whole strain of blooming is dead.Effectively to control the pathogen infection to cause harm, existing scholar is to its biological characteristics and pesticide control Have made intensive studies, it is proposed that some feasible Control Technology schemes.At present, the main policies of anthracnose are prevented and treated anti-for chemistry Disease-resistant variety, chemical prevention pollution environment and harm human health are controlled and cultivate, disease-resistant variety, which cultivates difficulty and there is resistance, to be subtracted Weak risk, therefore, biological control is because its is safe efficient, noresidue turns into the method that people most favor.Soil is various micro- lifes The good habitat of thing, 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 to be screened from plant root surface or rhizosphere soil, this There is good colonization ability in plant root surface after the microbial inoculant that sample is filtered out.

The content of the invention

Answering it is an object of the invention to provide one plant of actinomyces SVFJ-07 and its in preventing and treating cymbidium anthracnose disease With available for preventing and treating plant disease as caused by cymbidium anthracnose.

The actinomyces that the present invention is provided are wine red streptomyces（Streptomyces vinaceus）SVFJ-07, in 2017 On April 13, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center（Abbreviation CGMCC）, preserving number For：CGMCC No.14020, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

The invention provides applications of the described actinomyces SVFJ-07 in preventing and treating cymbidium anthracnose disease.The plant Disease is the microbial plant disease of cymbidium anthracnose.

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

Protection domain, when needing, in the microbial inoculum also comprising microbial inoculum prepare in commonly use carrier and auxiliary material.

The red streptomyces that relieve the effect of alcohol（Streptomyces vinaceus）SVFJ-07 is trained in Gause I culture medium, PDA 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.Light microscope Lower its fibrillae of spores of observation is straight, flexible, end buckle.Spore oval, Long Circle, surface are smooth.Bacterial strain SVFJ-07 is to D- grapes The carbon source such as sugar, D- xyloses, PEARLITOL 25C and D-Fructose has different degrees of Utilization ability, do not utilize L-arabinose, sucrose, Gossypose；Starch is not hydrolyzed；Do not produce H₂S and Melanoidins；Milk does not solidify, but peptonizes；Gelatin liquefaction is slow.In multiple features Upper and contrast bacterial strain（Streptomyces vinaceus）It is similar.

The remarkable advantage of the present invention：The wine red streptomyces of the present invention（Streptomyces vinaceus）SVFJ-07 is to orchid Flower anthracnose has very strong inhibitory action, with the potentiality that preventive and therapeutic effect is played to its caused fungal disease.

The wine red streptomyces of the present invention（Streptomyces vinaceus）SVFJ-07 is isolated from orchid plant rhizosphere week Soil is enclosed, experiment can effectively prevent and treat cymbidium anthracnose after proving inoculation orchid plant, be provided for the preventing and treating of cymbidium anthracnose One environmental protection, simple, effective approach.

Brief description of the drawings

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

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

Fig. 3 is bacterial strain wine red streptomyces（Streptomyces vinaceus）SVFJ-07 spore suspensions are to orchid stem rot The preventive effect result of disease, and it is control only to connect Pathogen spore suspension（CK）Experimental result.

Embodiment

Method in following embodiments, is conventional method unless otherwise instructed.

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

Wherein cymbidium anthracnose bacteria strain carries out tissue separation according to a conventional method from from the typical leaf portion of orchid disease symptom Culture is obtained, and monospore verifies that its moves into the preservation of PDA culture medium test tube slant after pathogenic by Koch's Postulates after purification.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 is isolated from soil layer soil；It is blue Flower anthrax bacteria（C. gloeosporioides）、Orchid Pathogen（Fusarium oxysporum）, Rhizoctonia solani Kuhn （Rhizoctonia solani）, banyan alternaria（Alternaria alternata）, banyan anthrax bacteria （Colletotrichum gloeosporioides）、Banana blight bacteria（Fusarium oxysporum）, early blight of tomato Bacterium（Alternaria solani）Preserved by this laboratory.

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

Method

1.2.1 actinomyces are separately cultured

It will collect after the sieving of taxi earth sample natural air drying, be separated using plate dilution method：The g of pedotheque 1 is weighed, is added to In the centrifuge tube for having 9 mL sterilized waters, 30 min are vibrated, 10 are then configured to^{- 3}、10^{- 4} 、10^{- 5} Suspension.Take difference Each 0.1 mL of suspension of concentration is in improvement HVA culture medium flat plates, and even spread, each ware of degree of putting culture 3 is placed in 28 DEG C Insulating box in be inverted culture.The single bacterium colony grown on culture 7d picking culture mediums carries out switching purifying.According to the form of bacterial strain Feature and cultural characteristic, remove the bacterial strain repeated.4 DEG C of ice are placed in after being cultivated in the actinomycetes strain inoculation slant medium of purifying Case is saved backup.

The screening of Antagonistic Actinomycetes

It is new or deer connects bright etc. method with reference to Du Yi, flat board opposite culture method is respectively adopted and actinomyces culture filtered fluid suppresses Rate method primary dcreening operation and secondary screening have the actinomyces of antagonistic activity to cymbidium anthracnose bacterium, and the actinomyces for separating acquisition are inoculated in into Gao Shi mono- In number culture medium flat plate, the mm of diameter 5 bacterium dish is taken to be positioned over PDA plate edge, centre inoculation orchid after 28 DEG C of 7 d of culture Anthrax bacteria, 28 DEG C of 5 d of culture, observes the presence or absence of antibacterial band, screens the wider bacterial strain of antibacterial band.Flat board face-off is filtered out into suppression Cingula width is inoculated in fermentation medium in 10 more than mm Actinomycetes species.Fermentation is with 500 mL triangular flasks, per bottled Liquid measure is 200 mL, and 7 d are cultivated in 28 DEG C of 180 r/min.After zymotic fluid centrifuges 10 min through 10000 g, supernatant is taken With 0.22 μm of filtering with microporous membrane, the mL of filtrate 0.5 is taken to be added in the cm of diameter 9 culture dish, 9. 5 with being cooled to 50 DEG C ML PDA culture mediums mix and the flat board that carries disease germs are made, and are beaten and are taken for examination pathogen bacteria cake with 5 mm aseptic card punch, are placed in the flat board that carries disease germs Center, the flat board being made using sterilized water and PDA is compares, 3 repetitions.After 28 DEG C of 7 d of culture, measured with crossing method Colony diameter, calculates growth inhibition ratio, growth inhibition ratio is calculated as follows：Growth inhibition ratio=（Compare flat-plate bacterial colony straight Footpath-processing flat-plate bacterial colony diameter）/（Compare flat-plate bacterial colony diameter-bacteria cake diameter）×100% .

The antimicrobial spectrum of Antagonistic Actinomycetes is determined

The antimicrobial spectrum of the actinomyces filtered out is determined using Odontothrips loti, target bacterium mycelia and spore are scraped with transfer needle and are made The mL of bacteria suspension 1 is poured into 9 cm culture dish after being mixed with the mL of PDA culture mediums 10 and flat board is made, and sterilizing Oxford cup is put The 200 sterile ferment filtrates of μ L are added in the PDA flat boards center of mixed target bacterium, cup, are determined after 28 DEG C of 3 d of culture are put respectively Antibacterial circle diameter, experiment sets 3 repetitions.

The identification of Antagonistic Actinomycetes

（1）Morphological feature is determined：The Antagonistic Actinomycetes SVFJ-07 obtained will be screened in using aseptic inoculation on Gause I culture medium Ring streak inoculation, then by the cover glass after sterilizing（1 cm×1 cm）Oblique cutting enters in culture medium, in cultivating 5 in 28 DEG C of incubators After d, spore and hypha form that cover glass is placed in optical microphotograph Microscopic observation bacterial strain are taken out.

（2）Cultural characteristic is determined：Reference《The strepto- dientification of bacteria is in volume》In method carry out.

（3）Physiological and biochemical property is determined：Reference《Actinomyces Rapid identification and genealogical classification》With《Streptomycete identification handbook》 Described in method, bacterial strain SVFJ-07 physio-biochemical characteristics such as utilization of carbon source, melanin are produced, H₂S is produced, aesculin Utilize the index such as, cellulose utilization, gelatin liquefaction, Starch Hydrolysis, milk solidification and jellyization, cellulose hydrolysis, nitrate reduction It is measured.

（4）16S rDNA sequence analyses：Bacterial strain SVFJ-07 cultivates 4-5 d bacterium solution in Gause I fluid nutrient medium, Put collected after centrifugation bacterial sediment in sterile centrifugation tube.0.1 ~ 0.2 g thalline are weighed in sterile mortar, are added after a small amount of PVP With liquid nitrogen grinding into powder, thalline STb gene is extracted using actinomyces genome extracts kit.With universal primer 27F：5′- AGAGTTTGATCCTGGCTCAG-3 ', 1541R：5 '-AAGGAGGTGATCCAGCCGCA-3 ' amplification actinomyces 16S rDNA.Will PCR primer is splined in the Ago-Gel containing GelRed dyestuffs and reclaims purpose band after electrophoresis, delivers outstanding Lee in Shanghai biological Company is sequenced.Row be sequenced are compared using NCBI Blast, the higher bacterium of homology therewith in GenBank is chosen The 16S rDNA sequences of strain, multiple alignment, analysis bacterial strain SVFJ-07 and reference strains are carried out to it using Clustal X softwares Between sequence similarity, and pass through the Neighbor joining method phylogenetic tree constructions in Mega4.1 softwares.

Data analysis

Sorting-out in statistics is carried out to test data using Excel2003, the DuncanShi duncan's new multiple range methods of the softwares of SAS 9.1 are to each Processing carries out significance difference analysis.

As a result with analysis

The separation and screening of 2.1 Antagonistic Actinomycetes

Will separation obtain 25 plants of actinomyces, by flat board opposite culture method filter out 6 plants it is equal to cymbidium anthracnose bacterium antagonistic effect Preferable actinomycetes strain.Inhibiting rate of 6 plants of bacterial strains to cymbidium anthracnose bacterium is determined using actinomyces culturing filtrate respectively, as a result Show（Table 1）, there is certain inhibitory action to cymbidium anthracnose bacterium after 100 times of 6 plants of actinomyces filtered fluids dilution of primary dcreening operation, Wherein numbering be SVFJ-8, SVFJ-15 and SVFJ-18 bacterial strain filtered fluid inhibitory action it is stronger, inhibiting rate is all higher than 70%, volume Number for SVFJ-07 bacterial strain inhibitory action it is most strong, inhibiting rate reaches nearly 85%.

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

Examined with different lowercase letters are indicated after column data through DuncanShi duncan's new multiple range methodsPThe level differences of ＜ 0.05 are notable.

Bacteriostatic activity of the Antagonistic Actinomycetes to phytopathogen

By flat board face-off method, screening obtains bacterial strain one plant stronger to cymbidium anthracnose bacterium bacteriostatic activity, is named as SVFJ-07.The bacterial strain for 7 plants of phytopathogens of examination to being respectively provided with different degrees of inhibitory action（Table 2）, wherein, it is blue to bacterial strain Flower anthrax bacteria（C. gloeosporioides）With banyan anthrax bacteria（C. gloeosporioides）Inhibitory action is most strong, And to Rhizoctonia solani Kuhn（R. solani）Bacteriostatic activity it is weaker.To orchid Pathogen（F. oxysporum）, banyan it is black Pinta bacterium（A. alternata）, banana blight bacteria（F. oxysporum）, tomato early blight bacterium（A. solani）Etc. having Stronger inhibitory action.

Inhibitory action of the bacterial strain SVFJ-07 of table 2 to test plant pathogen

Examined with different lowercase letters are indicated after column data through DuncanShi duncan's new multiple range methodsPThe level differences of ＜ 0.05 show Write.

Bacterial strain SVFJ-07 identification

2.3.1 morphological feature and cultural character

Bacterial strain SVFJ-07 well-growns 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：Orange brown, the base of gas silk Silk is brown. Isp5：Gas silk orange is brown, and base silk is brown.Its fibrillae of spores of optical microphotograph Microscopic observation is straight, flexible, end buckle.Spore oval Shape, Long Circle, surface are smooth.

Physiological and biochemical property

Bacterial strain SVFJ-07 has different degrees of utilization energy to carbon sources such as D-Glucose, D- xyloses, PEARLITOL 25C and D-Fructoses Power, L-arabinose, sucrose, gossypose are not utilized；Starch is not hydrolyzed；Do not produce H₂S and Melanoidins；Milk does not solidify, but peptone Change；Gelatin liquefaction is slow.With contrast bacterial strain in multiple features（Streptomyces vinaceus）It is similar.

Sequence analysis

Analysis is compared through NCBI Blast to find, the 16S rDNA sequences of separated actinomyces（Sequence such as SEQ ID No.1 institutes Show）Multiple kinds belonged to GenBank Streptomyces are respectively provided with higher homology, choose the 16S rDNA sequences of 10 typical strains The row software component phylogenetic trees of MEGA 5.0（Fig. 2）.As a result show, bacterial strain SVFJ-07 with（S. vinaceus）Belong to same One branch, wherein with bacterial strain（S. vinaceus）Similitude is up to more than 99%, and combining form and cultural characteristic identify bacterial strain For wine red streptomyces（S. vinaceus）.

Embodiment 2：Bacterial strain SVFJ-07 greenhouse pot culture efficiency tests

Cymbidium variety is potted orchids.Every plant is poured 50ml SVFJ-07 bacterial strain fermentation liquors (10⁶Individual spore/mL).Pin after 24h Thorn inoculation anthrax bacteria, inoculum density 1 × 10⁶Second of fermentation liquor treatment is carried out after individual spore/mL, inoculation pathogen within the 4th day. Compareed using the plant of 72% agricultural streptomycin root irrigation as agricultural chemicals, sky is used as using the plant that fermentation medium and clear water are handled White control.5 young plants of each processing, often handle 3 repetitions.Investigation incidence daily after being ill pending, the statistics incidence of disease and the state of an illness Index, until the clear water control incidence of disease terminates experiment when reaching more than 90%.

Severity Scaling standard is as follows：

0 grade：Disease-free spot；

1 grade：Per 1~3, leaf disease spot or lesion area below 5.0%；

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

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

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

9 grades：Full leaf area more than 25.1% is accounted for per more than 20, leaf disease spot or lesion area.

The incidence of disease (%)=morbidity strain number/total strain number × 100%

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

Prevention effect (%)=(control refers to clearly [(the control processing of disease index one disease index)/control disease index] × 100% Water control treatment)

Test result indicates that (table 3), for examination wine red streptomyces（S. vinaceus）SVFJ-07 zymotic fluids can significantly reduce orchid The disease index of flower anthracnose, prevention effect is up to 70.06%, is significantly higher than the prevention effect of SC1500 times of 45% Prochloraz 54.56%.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 both differences are not notable.Greenhouse pot culture experimental result illustrates that SVFJ-07 bacterial strains can effectively prevent and treat cymbidium anthracnose, Show good application potential.

Potted plant prevention effects of the actinomyces SVFJ-07 of table 3 to cymbidium anthracnose

Presently preferred embodiments of the present invention is the foregoing is only, all equivalent changes done according to scope of the present invention patent are with repairing Decorations, should all belong to the covering scope of the present invention.

SEQUENCE LISTING

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

<120>One plant of actinomyces SVFJ-07 and its application in preventing and treating cymbidium anthracnose

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taggcggctt gtcgcgtcgg ttgtgaaagc ccggggctta accccgggtc tgcagtcgat 600

acgggcaggc tagagttcgg taggggagat cggaattcct ggtgtagcgg tgaaatgcgc 660

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gcgaaagcgt gggagcgaac aggattagat accctggtag tccacgccgt aacggtgggc 780

actaggtgtg ggcgacattc cacgtcgtcc gtgccgcagc taacgcatta agtgccccgc 840

ctggggagta cggccgcaag gctaaaactc aaaggaattg acgggggccc gcacaagcgg 900

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Claims (3)

1. one plant of actinomyces, it is characterised in that：The actinomyces are wine red streptomyces（Streptomyces vinaceus） SVFJ-07, is preserved in Chinese microorganism strain on April 13rd, 2017 and is deposited in administration committee's common micro-organisms center, protects Tibetan number is：CGMCC No.14020.

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

3. application of the actinomyces as claimed in claim 1 in preventing and treating cymbidium anthracnose.