CN103952362A - Citrus endophytic actinomycetes with antibacterial activity on various plant pathogens - Google Patents
Citrus endophytic actinomycetes with antibacterial activity on various plant pathogens Download PDFInfo
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Abstract
The invention relates to citrus endophytic actinomycetes with antibacterial activity on various plant pathogens, belonging to the technical field of microbes. The endophytic actinomycetes are Streptomyces sp. R1020 which was registered and collected in China General Microbiological Culture Collection Center on March 17, 2014 with the collection number of CGMCC No. 8923. The strain R1020 has relatively good inhibitory effect on citrus anthrax bacteria, citrus bacterial canker disease bacteria, citrus penicillium italicum, citrus penicillium digitatum, bayberry deadwood disease bacteria, bayberry acicola, bayberry anthrax bacteria, loquat anthrax bacteria, loquat leaf spot fungi, loquat ringspot pathogenic bacteria, tomato alternaria solani, botrytis cinerea, colletotrichum orbiculare, tea tree anthrax bacteria and elsinoeleucospila and has the potential of being developed into biological pesticides.
Description
Technical field
The present invention relates to raw actinomycetes in a strain has bacteriostatic activity oranges and tangerines to various plants pathogenic bacteria, belong to microbial technology field.
Background technology
Endophyte of plant is the Biocontrol microorganism resource that a class is new, and it is that one can surely be grown and running in plant tissue, and to the harmless even useful microorganism of plant.Because it has stable living space in plant materials, and can produce and the same or analogous physiologically active substance of host plant metabolism, thereby can effectively suppress the disease resistance that infects or improve host plant of pathogenic bacteria.Endophyte is distributed in host plant organization internal, has more stable living environment than the epiphyte that is exposed to outside atmosphere, is easier to play a role.In addition, plant endogenesis actinomycetes have certain host specificity, except drawing nutriment from its host plant and to infecting other biology of host plant works, host plant itself and other are not biologically almost affected, and interior raw actinomycetes can be imported into different plants and can be carried out heredity by plant seed by artificial inoculation.Therefore endophyte of plant has the good characteristic as biological pesticide exploitation.
At present, mainly taking chemical prevention as main, well-known to the control of corps diseases, chemical pesticide long-term a large amount of use and tend to cause the problems such as resistance of environmental pollution, pesticide residue and pathogenic bacteria to be difficult to solve.The antibacterial substance that utilizes endophyte of plant to produce carries out the biological control of Plant diseases, have efficient, low toxicity, low residue, pollution-free, be difficult for developing immunity to drugs and the raw material advantage such as be easy to get.
, as the first ancestor ground of the wide skin oranges and tangerines in the world, there is the citriculture history of more than 2300 year Huangyan District, Taizhou plain.Wherein Man tangerine is a kind of ancient local variety of Huangyan area plantation, thinks in its plant body and maybe can have the beneficial microorganism not possessing in other Citrus Cultivars or plant.For this reason, we have carried out a large amount of separation screenings to the microorganism in Man tangerine body, finally separate and obtain the interior raw actinomycetes of a strain to the strong bacteriostatic activity of various plants pathogenic bacteria tool from Man tangerine root, after qualification by its called after streptomycete (
streptomycessp.) R1020, further development and exploitation that this invention is microbial pesticide provide good starting strain.
Summary of the invention
The object of the present invention is to provide a strain to suppress raw actinomycetes in active oranges and tangerines to various plants pathogenic bacteria tool, this bacterial strain has obvious antagonistic action to various plants pathogenic bacteria, has the potential that is developed to biological pesticide.
The object of the invention is to be achieved through the following technical solutions:
Raw actinomycetes in strain oranges and tangerines provided by the invention, for streptomycete (
streptomycessp.) R1020, has registered preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 17th, 2014, deposit number is CGMCC No. 8923.
The invention provides raw actinomycetic application in described oranges and tangerines, streptomycete (
streptomycessp.) application of R1020 in Suppressing phytopathogens, described phytopathogen is citrus anthracnose bacterium, c itrus canker germ, Penicillium italicum bacterium, citrus common green mold bacterium, red bayberry deadwood germ, red bayberry brown patch germ, red bayberry anthrax bacteria, loquat anthrax bacteria, loquat leaf spot disease bacterium, loquat zonate spot bacterium, tomato early blight bacterium, botrytis cinerea pers, watermelon anthrax bacteria, tea tree anthrax bacteria and tea white star germ.
More specifically, described streptomycete (
streptomycessp.) application of the tunning crude extract of R1020 in Suppressing phytopathogens, described phytopathogen is citrus anthracnose bacterium, c itrus canker germ, Penicillium italicum bacterium, citrus common green mold bacterium, red bayberry deadwood germ, red bayberry brown patch germ, red bayberry anthrax bacteria, loquat anthrax bacteria, loquat leaf spot disease bacterium, loquat zonate spot bacterium, tomato early blight bacterium, botrytis cinerea pers, watermelon anthrax bacteria, tea tree anthrax bacteria and tea white star germ.
The preparation process of described tunning crude extract is as follows: (1) get the streptomycete that activates on synthetic No. 1 substratum of Gao Shi (
streptomycessp.) R1020, is inoculated in synthetic No. 1 liquid fermentation medium of Gao Shi, shaking table 200 rpm/min shaking culture 4 d at 28 DEG C;
(2) preparation of tunning crude extract: get fermented liquid via aseptic filter paper suction filtration, obtain respectively supernatant liquor and mycelium.Get supernatant liquor, be extracted with ethyl acetate 3 times, combining extraction liquid, obtains the acetic acid ethyl acetate extract of supernatant liquor; Mycelium, with after 80% aqueous acetone solution soaked overnight, with ultrasonic disruption extraction 3 times, united extraction liquid, and is evaporated to extracting solution without after acetone, and gained water with isopyknic ethyl acetate extraction 3 times, obtains mycelial acetic acid ethyl acetate extract again.Supernatant liquor and mycelial acetic acid ethyl acetate extract are merged, be evaporated to dryly, obtain the tunning crude extract of this bacterial strain.
Raw actinomycetes streptomycete in oranges and tangerines described in the present invention also provides (
streptomycessp.) R1020 is in the application of preparing in biological pesticide.
Described streptomycete (
streptomycessp.) R1020 adopts the separation of endophyte microbe separation technology to obtain from Huangyan District, City of Taizhou Man tangerine root, and cultural characteristic, morphological specificity, physiological and biochemical property, 16S rDNA sequence and the bacterium classification result of this bacterial strain are as follows:
1, the cultural characteristic of bacterial strain R1020 and morphological specificity: bacterial strain R1020 is well-grown on Gao Shi synthetic No. 1 substratum, yeastex maltose agar (ISP-2) substratum, oatmeal agar (ISP-3) substratum and inorganic salt Starch Agar (ISP-4) substratum, can produce more rich aerial hyphae.And it is poor to grow on glucose asparagine agar (ISP-5) substratum and peptone yeast iron agar (ISP-6) substratum.Wherein bacterial strain R1020 can produce melanochrome on ISP-6 substratum, and produces without soluble pigment on above-mentioned other substratum.The concrete cultural characteristic of bacterial strain R1020 refers to table 1, wherein bacterial strain R1020 well-grown on synthetic No. 1 substratum of Gao Shi, and aerial hyphae is lightpink, substrate mycelium is milk yellow, produces (as Fig. 1 shows) without soluble pigment.Under opticmicroscope, observe fibrillae of spores straight (as Fig. 2 shows), spore circle is to cylindricality, smooth surface (as Fig. 3 shows).
The cultural characteristic of table 1 bacterial strain R1020
2, the physiological and biochemical property of bacterial strain R1020: bacterial strain R1020 can utilize D-sucrose, D-semi-lactosi, D-Glucose and L-arabinose as carbon source, gelatine liquefication is strong, not hydrolyzed starch and Mierocrystalline cellulose, on Mierocrystalline cellulose, do not grow, can utilize Vitamin C2, can make milk solidify, can produce melanochrome, concrete physiological and biochemical property refers to table 2.
The physiological and biochemical property of table 2 bacterial strain R1020
Note: in form, "+" represents that measurement result is positive, "-" represents that measurement result is negative
3, the 16S rDNA sequence of bacterial strain R1020: the 16S rDNA sequence of bacterial strain R1020 is as shown in SEQ ID NO:1.Institute's calling sequence is carried out to the discovery of Blast compare of analysis on state-run biotechnology information center of the U.S. (NCBI) website, in this sequence and GenBank, the sequence of multiple species of listed streptomyces has higher homology, its sequence similarity reach 99% and more than, as
streptomyces sporoverrucosus(KF554239, KF554180),
streptomyces cinnamonensis(KB484681),
streptomyces flaveus(JX293177),
streptomyces virginiae(JN999924, JN999923),
streptomyces lavendulae(JN609386),
streptomyces goshikiensis(HQ844511, HQ853024) etc.Its with the previous other strain separating in this laboratory the interior raw actinomycetes-streptomycete to various plants pathogenic bacteria tool bacteriostatic activity (
streptomycessp.) sequence similarity of CR20 is 99%, has base difference.
4, bacterium classification result: according to the morphological specificity of bacterial strain R1020, cultural characteristic, physiological and biochemical property and 16S rDNA sequence, can be accredited as the bacterial classification of actinomycetic streptomyces.16S rDNA sequence and the multiple different strains of GenBank Streptomyces of the bacterial strain R1020 that surveys all have higher sequence similarity.But the cultural characteristic of bacterial strain R1020 and physiological and biochemical property (in table 1 and table 2) with
streptomyces sporoverrucosus,
streptomyces cinnamonensis,
streptomyces flaveus,
streptomyces virginiaeall be not quite similar Deng bacterial classification.Its 16S rDNA sequence and morphological specificity, cultural characteristic and physicochemical property also there are differences with the streptomycete CR20 that this laboratory separates before.Therefore, according to above result of study, think that bacterial strain R1020 of the present invention is a new bacterial strain in above-mentioned certain bacterial classification, be specially which bacterial classification as for it, wait further to determine.
Therefore by this bacterial strain called after streptomycete (
streptomycessp.) R1020, has registered preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 17th, 2014, be called for short CGMCC, and address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No. 8923.
Compared with prior art, advantage of the present invention and beneficial effect are: (1) streptomycete provided by the invention (
streptomycessp.) R1020 separates and obtains from the Man tangerine root in Huangyan tangerine district, Taizhou plain, and the Antagonistic Streptomyces about separation in Man tangerine plant with bacteriostatic activity rarely has report; (2) streptomycete of the present invention (
streptomycessp.) R1020 is a kind of plant endogenesis actinomycetes, and to various plants pathogenic bacteria tool bacteriostatic activity, more current disease control chemical agent used is compared, and has efficient, low toxicity, low residue, the pollution-free and feature that is difficult for developing immunity to drugs; (3) streptomycete (
streptomycessp.) R1020 all has good inhibition to citrus anthracnose bacterium, c itrus canker germ, Penicillium italicum bacterium, citrus common green mold bacterium, red bayberry deadwood germ, red bayberry brown patch germ, red bayberry anthrax bacteria, loquat anthrax bacteria, loquat leaf spot disease bacterium, loquat zonate spot bacterium, tomato early blight bacterium, botrytis cinerea pers, watermelon anthrax bacteria, tea tree anthrax bacteria and tea white star germ, being developed to Biological agents for the biological control to Plant diseases, there is good market application foreground.
Brief description of the drawings
Fig. 1 be streptomycete (
streptomycessp.) cultural characteristic of R1020 on synthetic No. 1 substratum of Gao Shi.
Fig. 2 be the streptomycete of optical microphotograph Microscopic observation (
streptomycessp.) mycelia of R1020 and fibrillae of spores feature.
Fig. 3 be the streptomycete of optical microphotograph Microscopic observation (
streptomycessp.) the spore feature of R1020.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this.
One strain, to raw actinomycetes in the oranges and tangerines of various plants pathogenic bacteria tool bacteriostatic activity, is characterized in that: bacterial classification called after R1020, and Classification And Nomenclature: streptomycete (
streptomycessp.), its deposit number: CGMCC No. 8923, preservation date: on 03 17th, 2014, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment 1: raw actinomycetic separation and Culture in oranges and tangerines
1, biomaterial and substratum: the Man tangerine root sample that picks up from the different orangeries in Huangyan District, City of Taizhou for separating of the actinomycetic biomaterial of interior life.In separating, raw actinomycetes isolation medium used is that (formula is tap water yeast powder agar: yeast decoction 0.25 g, K
2hPO
40.5 g, agar 18 g, tap water 1000 mL).
2, sample preparation: the sample of above-mentioned fresh collection is rinsed out under tap water to the earth of sample surfaces, then use ultrasonic cleaning; Then on aseptic operating platform with 99% Ethanol Treatment 1 min, 3% clorox is processed 5 min, finally processes 30 s with 99% ethanol again.Sample after sterilizing is cut into the fritter of 1 cm × 1 cm left and right, is placed in separation and Culture primary surface., the scavenging solution of last processing sample is coated on isolation medium, in order to detect the effect of surface sterilization meanwhile.Isolation medium is placed in 28 DEG C of incubators and cultivates 2 ~ 6 weeks, treats that actinomycetes grow.
3, actinomycetes are cultivated: single bacterium colony of growing on picking substratum purifying of transferring, according to the morphological specificity of bacterial strain and cultural characteristic, remove the bacterial strain repeating, and finally amount to separate and obtain raw actinomycetes strain in 20 strains.To the bacterial strain of purifying, being inoculated in Gao Shi, synthetic No. 1 (formula is: Zulkovsky starch 20 g, KNO
31 g, K
2hPO
40.5 g, MgSO
47H
2o 0.5 g, FeSO
47H
2o 0.01 g, NaCl 0.5 g, agar 20 g, distilled water 1000 mL, pH7.2-7.4) in slant medium, in 28 DEG C of constant incubators, cultivate after 5 ~ 7 d, be placed in 4 DEG C of Refrigerator stores for subsequent use.
Embodiment 2: the screening of raw actinomycetes antagonistic strain in oranges and tangerines
1, the preparation of indicator flat board: (compound method is: potato 200 g that take fresh peeling to prepare potato dextrose agar (PDA) culture medium flat plate, shred 1000 mL that add water and boil half hour, filtered through gauze, get filtrate and be settled to 1000 mL, add again 20 g glucose and 20 g agar, heating divides and is filled in Erlenmeyer flask or glass test tube after fully dissolving, 121 DEG C, autoclaving 20 min), beat the citrus anthracnose bacterium of getting fresh culture with the aseptic punch tool of internal diameter 5 mm, the bacterium colony of red bayberry deadwood germ and 3 kinds of pathogenic bacterias of loquat zonate spot bacterium (these 3 kinds of pathogenic bacterias are the indicator for antagonistic strain screening), transfer on PDA culture medium flat plate, surrounding at each flat board approximately 3 cm apart from center is placed 6 identical bacterium cakes, make indicator flat board.
2, tool suppresses the screening of active bacterial strain: be inoculated on synthetic No. 1 culture medium flat plate of Gao Shi separating raw actinomycetes in the oranges and tangerines that obtain in embodiment 1, cultivate after 5 d for 28 DEG C, buy the bacterium cake of cut-off footpath 5 mm there being bacteria growing place punch tool, be placed in the flat board central authorities that inoculation has indicator, each actinomycetes strain does 3 repetitions, set up simultaneously only add phytopathogen bacterium cake and do not add actinomycetes bacterium cake flat board in contrast, being placed in 28 DEG C of constant incubators cultivates, when phytopathogen covers with whole culture dish in contrasting, measure actinomycetic antibacterial bandwidth (actinomycetes colony edge is to the distance of phytopathogen colony edge), the size of the antibacterial band of more each actinomycetes strain, therefrom filter out the strongest bacterial strain of phytopathogen bacteriostasis.
By above method, in separating 20 strains that obtain, raw actinomycetes, filtering out 1 strain all has the bacterial strain compared with strong inhibitory activity to citrus anthracnose bacterium, red bayberry deadwood germ and loquat zonate spot bacterium, by its called after R1020.
Embodiment 3: streptomycete (
streptomycessp.) preparation of R1020 tunning crude extract
1, the activation culture of bacterial strain: the bacterial strain R1020 that separates preservation is moved on synthetic No. 1 culture medium flat plate of Gao Shi and cultivates 7 d in 28 DEG C of incubators.
2, the fermentation culture of bacterial strain: get the bacterial strain R1020 activating on synthetic No. 1 substratum of Gao Shi, be inoculated into synthetic No. 1 liquid fermentation medium (the 100 mL substratum/250 mL Erlenmeyer flasks of Gao Shi, formula is with synthetic No. 1 solid medium of Gao Shi, containing agar) in, shaking table 200 rpm/min shaking culture 4 d at 28 DEG C.
3, the preparation of tunning crude extract: get fermented liquid via aseptic filter paper suction filtration, obtain respectively supernatant liquor and mycelium.Get supernatant liquor, be extracted with ethyl acetate 3 times, combining extraction liquid, obtains the acetic acid ethyl acetate extract of supernatant liquor.Mycelium, with after 80% aqueous acetone solution soaked overnight, with ultrasonic disruption extraction 3 times, united extraction liquid, and is evaporated to extracting solution without after acetone, and gained water with isopyknic ethyl acetate extraction 3 times, obtains mycelial acetic acid ethyl acetate extract again.Supernatant liquor and mycelial acetic acid ethyl acetate extract are merged, be evaporated to dryly, obtain the acetic acid ethyl ester extract of this bacterial strain.
Embodiment 4: streptomycete (
streptomycessp.) bacteriostatic activity of R1020 tunning crude extract to phytopathogen.
1, strains tested and substratum: test plant pathogenic fungi comprises citrus anthracnose bacterium, Penicillium italicum bacterium, citrus common green mold bacterium, red bayberry deadwood germ, red bayberry brown patch germ, red bayberry anthrax bacteria, loquat anthrax bacteria, loquat leaf spot disease bacterium, loquat zonate spot bacterium, tomato early blight bacterium, botrytis cinerea pers, watermelon anthrax bacteria, tea tree anthrax bacteria and tea white star germ; Test plant pathogenetic bacteria is c itrus canker germ.Cultivating used medium for plant pathogenic fungi is potato dextrose agar (PDA) substratum, and its concrete formula as described in example 2 above; Cultivating used medium for plant pathogenetic bacteria is that (formula is beef extract-peptone agar (NA) substratum: extractum carnis 3 g, peptone 10 g, NaCl 5 g, agar 15 g, distilled water 1000 mL, pH7.0-7.2,121 DEG C, autoclaving 20 min).
2, the activation of strains tested: PDA and NA substratum are poured in the sterile petri dish that diameter is 9 cm after heating for dissolving respectively, prepare culture medium flat plate.The bacterium piece that is stored in 4 DEG C of each fungies in refrigerator slant medium with aseptic inoculation pin picking is inoculated on PDA culture medium flat plate,, on NA culture medium flat plate, be placed in 28 DEG C of constant incubators and cultivate activation with the streak inoculation of aseptic inoculation ring picking c itrus canker germ.Each fungus culture 5 d left and right, c itrus canker germ is cultivated 40 d, stand-by.
3, the mensuration of bacteriostatic activity: take in embodiment 3 streptomycete that obtains (
streptomycessp.) tunning crude extract 1 g of R1020, adds the sterilized water of 100 mL, and ultrasonic oscillation fully dissolves, and final wiring solution-forming concentration is 10 g/L.Inhale crude extract solution 1 mL in diameter 9 cm culture dish with pipettor, add the PDA substratum (substratum temperature 50 C left and right) of 9 mL to mix rapidly and make band medicine flat board, make streptomycete (
streptomycessp.) the fermentation runic thing solution ultimate density of R1020 is 1 g/L.By citrus anthracnose bacterium good above-mentioned activation, Penicillium italicum bacterium, citrus common green mold bacterium, red bayberry deadwood germ, red bayberry brown patch germ, red bayberry anthrax bacteria, loquat anthrax bacteria, loquat leaf spot disease bacterium, loquat zonate spot bacterium, tomato early blight bacterium, botrytis cinerea pers, watermelon anthrax bacteria, tea tree anthrax bacteria and tea white star germ, beat and get bacterium piece and be placed in the central authorities with medicine flat board with the punch tool of diameter 5 mm, compare with sterilized water, repeat 3 times, after inoculation, band medicine flat board is placed in to 28 DEG C of constant incubators and cultivates 5 d.Adopt right-angled intersection to measure colony diameter, calculate colony diameter mean value and mycelial growth inhibition rate, mycelial growth inhibition rate calculation formula is as follows:
Mycelial growth inhibition rate (%)=[1-(processing colony diameter-bacterium cake diameter)/(contrast colony diameter-bacterium cake diameter)] × 100.
C itrus canker germ is cultivated after 40 h, add 10 mL sterilized waters, scrape lawn with triangle rod and make bacterial suspension, draw with pipettor that bacterium liquid 1 mL adds in diameter 9 cm culture dish, NA substratum (40 DEG C ~ 50 DEG C of liquid temperatures) 9 mL that pour thawing into mix and make the flat board that carries disease germs; Beat and get aseptic filter paper dish with the punch tool of diameter 1 cm, drop into the middle absorption of solution (concentration is 1 g/L) 15 s of bacterial strain R1020 tunning, take out filter paper dish and be positioned over the flat board that carries disease germs, 4 of each culture dish diagonal angle discharges, and make blank with absorption sterilized water, culture dish moves into 28 DEG C of constant incubators and cultivates 40 h, measures antibacterial circle diameter (mm), calculates the mean diameter of inhibition zone in culture dish.
Test-results is in table 3.As seen from Table 3, streptomycete (
streptomycessp.) when the strength of solution of R1020 fermented liquid runic thing preparation is 1 g/L, to the 14 kind of plant pathogenic fungi inhibiting rates for examination all more than 60%, particularly to colletotrichum (
colletotrichum) citrus anthracnose bacterium, red bayberry anthrax bacteria, watermelon anthrax bacteria and tea tree anthrax bacteria and intend Pestalotia (
pestalotiopsis) the inhibiting rate of red bayberry deadwood germ, loquat zonate spot bacterium all more than 80%, fungistatic effect is remarkable.And find by simultaneous test, this laboratory from oranges and tangerines root separate another strain Antagonistic Fungi-streptomycete (
streptomycessp.) CR20 to intend Pestalotia (
pestalotiopsis) red bayberry deadwood germ, the inhibiting rate of loquat zonate spot bacterium be only 62% and 65%, and the streptomycete separating from marine organisms squama large bamboo hat with a conical crown and broad brim barnacle (
streptomycessp.) A3202 is only respectively also 67% and 70% to the inhibiting rate of these two kinds of bacterium, and streptomycete (
streptomycessp.) CR20 and streptomycete (
streptomycessp.) A3202 to the inhibiting rate of red bayberry brown patch germ all lower than 65%.Visible streptomycete (
streptomycessp.) R1020 has stronger inhibition to germs such as plan dish steys.In addition, streptomycete (
streptomycessp.) R1020 also can reach 18.8 mm to the antibacterial circle diameter of oranges and tangerines pathogenetic bacteria-c itrus canker germ.
Table 3 streptomycete (
streptomycessp.) inhibiting rate of R1020 fermented liquid runic thing to various plants pathogenic bacteria
Note: for citrus ulcer bacteria, in form, numerical value is antibacterial circle diameter, and for other fungies, in form, numerical value is fungal colony diameter.
Embodiment 5: cultural characteristic, morphological specificity and the physiological and biochemical property of streptomycete (Streptomyces sp.) R1020
1, the cultural characteristic of bacterial strain R1020: adopt 5 kinds of international substratum, (ISP-4, filling a prescription is: Zulkovsky starch 10 g, K to be respectively inorganic salt Starch Agar
2hPO
41 g, MgSO
47H
2o 1 g, NaCl 1 g, (NH
4)
2sO
42 g, CaCO
32 g, FeSO
47H
2o 0.001g, MnCl
27H
2o 0.001 g, ZnSO47H
2o 0.001g, agar 20 g, distilled water 1000 mL, pH7.0-7.4), (ISP-5, fill a prescription is glucose asparagine agar: ASPARTIC ACID 1 g, glycerine 10 g, K
2hPO
41 g, trace element solution 1 mL, agar 20 g, distilled water 1000 mL, pH 7.2, trace element solution: FeSO
47H
2o 0.1 g, MnCl
24H
2o 0.1 g, ZnSO
47H
2o 0.1 g), (ISP-3, fill a prescription is oatmeal agar: oatmeal 20 g, trace element solution 1 mL, pH 7.2, trace element: FeSO
47H
2o 0.1 g, MnCl
24H
2o 0.1 g, ZnSO
47H
2o 0.1 g), yeastex maltose agar (ISP-2, formula is: yeast extract paste 4 g, wort 10 g, glucose 4 g, agar 20 g, distilled water 1000 mL, pH 7.0), (ISP-6, fill a prescription is peptone-yeast-iron nutrient agar: peptone 20 g, ironic citrate 0.5 g, K
2hPO
41 g, yeast soak powder 1 g, agar powder 20 g, distilled water 1000 mL, pH7.2), and PDA substratum (formula as described in Example 2) and synthetic No. 1 substratum of Gao Shi (formula as described in Example 1), bacterial strain R1020 is inoculated on above substratum, then be placed in 28 DEG C of constant temperature and be inverted cultivation, after 1 ~ 3 week, observe aerial hyphae, the growing state of substrate mycelium and the production of soluble pigment of bacterial strain R1020.
Make discovery from observation, bacterial strain R1020 is well-grown on Gao Shi synthetic No. 1 substratum, yeastex maltose agar (ISP-2) substratum, oatmeal agar (ISP-3) substratum and inorganic salt Starch Agar (ISP-4) substratum, can produce more rich aerial hyphae.And it is poor to grow on glucose asparagine agar (ISP-5) substratum and peptone yeast iron agar (ISP-6) substratum.Wherein bacterial strain R1020 can produce melanochrome on ISP-6 substratum, and produces without soluble pigment on above-mentioned other substratum.The concrete cultural characteristic of bacterial strain R1020 refers to table 1, wherein bacterial strain R1020 well-grown on synthetic No. 1 substratum of Gao Shi, and aerial hyphae is lightpink, substrate mycelium is milk yellow, produces (as Fig. 1 shows) without soluble pigment.
2, the morphological specificity of bacterial strain R1020: bacterial strain R1020 is synthesized on No. 1 substratum with the streak inoculation of aseptic inoculation ring at Gao Shi, then the cover glass after sterilizing (1 cm × 1 cm) oblique cutting is entered in substratum, in 28 DEG C of incubators, cultivate after 7 d, taking-up cover glass is placed in spore and the mycelia form of optical microphotograph Microscopic observation bacterial strain.Under opticmicroscope, can be observed the fibrillae of spores straight (as Fig. 2 shows) of bacterial classification R1020, spore circle is to cylindricality, smooth surface (as Fig. 3 shows).
3, the physiological and biochemical property of bacterial strain R1020: with reference to " actinomycetes Rapid identification and phylogenetic systematics " (Ruan Jisheng, Huang Ying. actinomycetes Rapid identification and phylogenetic systematics [M]. Beijing: Science Press, 2011) and " streptomycete identification handbook " (Institute of Microorganism, Academia Sinica's classification of actinomycetes group. streptomycete identification handbook [M]. Beijing: Science Press, 1975) method described in, to the physio-biochemical characteristics of bacterial strain R1020 as utilization of carbon source, melanochrome produce, H
2the indexs such as S generation, Vitamin C2 utilization, cellulose utilization, gelatine liquefication, Starch Hydrolysis, milk solidify, cellulose hydrolysis, nitrate reduction are measured.
Measurement result shows, bacterial strain R1020 can utilize D-sucrose, D-semi-lactosi, D-Glucose and L-arabinose as carbon source, gelatine liquefication is strong, not hydrolyzed starch and Mierocrystalline cellulose, on Mierocrystalline cellulose, do not grow, can utilize Vitamin C2, can make milk solidify, can produce melanochrome, concrete physiological and biochemical property refers to table 2.
<110> Zhejiang Citrus Research Institute
<120> mono-strain is to raw actinomycetes in the oranges and tangerines of various plants pathogenic bacteria tool bacteriostatic activity
<160> 1
<170> PatentIn Version 3.3
<210> 1
<211> 1334
<212> DNA
<213> streptomycete (
streptomycessp.)
<400> 1
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tgatgcagcg acgccgcgtg agggatgacg gccttcgggt tgtaaacctc tttcagcagg 360
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ggggagtacg gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcggcg 840
gagcatgtgg cttaattcga cgcaacgcga agaaccttac caaggcttga catataccgg 900
aaagcattag agatagtgcc ccccttgtgg tcggtataca ggtggtgcat ggctgtcgtc 960
agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtcctgtgtt 1020
gccagcatgc ccttcggggt gatggggact cacaggagac cgccggggtc aactcggagg 1080
aaggtgggga cgacgtcaag tcatcatgcc ccttatgtct tgggctgcac acgtgctaca 1140
atggccggta caatgagctg cgataccgtg aggcggagcg aatctcaaaa agccggtctc 1200
agttcggatt ggggtctgca actcgacccc atgaagtcgg agtcgctagt aatcgcagat 1260
cagcattgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc acgtcacgaa 1320
agtcggtaac accc 1334
Claims (3)
1. raw actinomycetes in strain oranges and tangerines, for streptomycete (
streptomycessp.) R1020, has registered preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 17th, 2014, deposit number is CGMCC No. 8923.
2. the application of raw actinomycetes in Suppressing phytopathogens in oranges and tangerines as claimed in claim 1, is characterized in that: described phytopathogen is citrus anthracnose bacterium, c itrus canker germ, Penicillium italicum bacterium, citrus common green mold bacterium, red bayberry deadwood germ, red bayberry brown patch germ, red bayberry anthrax bacteria, loquat anthrax bacteria, loquat leaf spot disease bacterium, loquat zonate spot bacterium, tomato early blight bacterium, botrytis cinerea pers, watermelon anthrax bacteria, tea tree anthrax bacteria and tea white star germ.
3. interior raw actinomycetes as claimed in claim 1 are in the application of preparing in biological pesticide.
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| CN201410202696.6A CN103952362B (en) | 2014-05-14 | 2014-05-14 | One strain is to the oranges and tangerines endogeny rayungus of various plants pathogenic bacteria tool bacteriostatic activity |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651278A (en) * | 2015-02-09 | 2015-05-27 | 山东省林业科学研究院 | Streptomyces misionensis strain and application thereof in controlling walnut and winter jujube anthracnose |
| CN107907535A (en) * | 2017-10-20 | 2018-04-13 | 浙江省柑桔研究所 | A kind of method for diagnosing red bayberry dead-arm |
| CN108419828A (en) * | 2018-03-21 | 2018-08-21 | 江西农业大学 | A kind of citrus comprising loquat-leaf extract adopts rear plant antistaling agent |
| CN108728378A (en) * | 2018-05-30 | 2018-11-02 | 四川大学 | One plants endogenetic actinomyces and its application |
| CN111096337A (en) * | 2020-01-16 | 2020-05-05 | 中国科学院南海海洋研究所 | Application of deep sea actinomycete biological preparation in preparing medicine for preventing citrus canker disease |
| CN112359003A (en) * | 2020-12-16 | 2021-02-12 | 西南林业大学 | Streptomyces thalictriformis strain and application thereof |
| CN112725228A (en) * | 2020-12-30 | 2021-04-30 | 西南大学 | Virginia streptomycete capable of preventing and treating peach brown rot and application thereof |
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| CN113930355A (en) * | 2021-09-15 | 2022-01-14 | 中国热带农业科学院热带生物技术研究所 | Biocontrol streptomyces weinmannii W7 from termites and application thereof |
| CN115029268A (en) * | 2022-06-21 | 2022-09-09 | 安徽农业大学 | Streptomyces venezuelae and application thereof in preventing and treating crop diseases |
| CN116064334A (en) * | 2023-02-17 | 2023-05-05 | 华南农业大学 | Streptomyces malachite for improving content of tea branch citrus monoterpenes and application thereof |
| CN117165495A (en) * | 2023-10-26 | 2023-12-05 | 广东省农业科学院植物保护研究所 | Streptomyces virginiae Sv1 and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845412A (en) * | 2010-04-27 | 2010-09-29 | 华中农业大学 | Bio-control streptomyces globisporus strain and application in prevention and control of penicilliosis of citrus |
| CN103013852A (en) * | 2012-10-10 | 2013-04-03 | 湖南农业大学 | Actinomycete capable of inhibiting phytophthoramelonis and screening method thereof |
| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
| CN103222479A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum |
-
2014
- 2014-05-14 CN CN201410202696.6A patent/CN103952362B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845412A (en) * | 2010-04-27 | 2010-09-29 | 华中农业大学 | Bio-control streptomyces globisporus strain and application in prevention and control of penicilliosis of citrus |
| CN103013852A (en) * | 2012-10-10 | 2013-04-03 | 湖南农业大学 | Actinomycete capable of inhibiting phytophthoramelonis and screening method thereof |
| CN103222479A (en) * | 2013-01-07 | 2013-07-31 | 中国计量学院 | Use of Streptomyces sioyaensis metabolite in prevention and control of Colletotrichum lindemuthianum |
| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
Non-Patent Citations (2)
| Title |
|---|
| 李辉 等: "柑橘绿霉病菌拮抗放线菌MY-5的筛选与鉴定", 《中国农学通报》 * |
| 胡秀荣 等: "柑橘内生放线菌CR20的鉴定与拮抗活性", 《浙江农业科学》 * |
Cited By (17)
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| CN107907535A (en) * | 2017-10-20 | 2018-04-13 | 浙江省柑桔研究所 | A kind of method for diagnosing red bayberry dead-arm |
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| CN113930355A (en) * | 2021-09-15 | 2022-01-14 | 中国热带农业科学院热带生物技术研究所 | Biocontrol streptomyces weinmannii W7 from termites and application thereof |
| CN115029268A (en) * | 2022-06-21 | 2022-09-09 | 安徽农业大学 | Streptomyces venezuelae and application thereof in preventing and treating crop diseases |
| CN115029268B (en) * | 2022-06-21 | 2023-08-11 | 安徽农业大学 | Streptomyces venezuelae and application thereof in crop disease control |
| CN116064334A (en) * | 2023-02-17 | 2023-05-05 | 华南农业大学 | Streptomyces malachite for improving content of tea branch citrus monoterpenes and application thereof |
| CN116064334B (en) * | 2023-02-17 | 2024-04-12 | 华南农业大学 | Streptomyces malachite for improving content of tea branch citrus monoterpenes and application thereof |
| CN117165495A (en) * | 2023-10-26 | 2023-12-05 | 广东省农业科学院植物保护研究所 | Streptomyces virginiae Sv1 and application thereof |
| CN117165495B (en) * | 2023-10-26 | 2024-01-23 | 广东省农业科学院植物保护研究所 | Streptomyces virginiae Sv1 and application thereof |
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