CN108949607A - A kind of Chinese prickly ash endogeny rayungus HJG-5 and its application - Google Patents
A kind of Chinese prickly ash endogeny rayungus HJG-5 and its application Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N1/20—Bacteria; Culture media therefor
Abstract
A kind of Chinese prickly ash endogeny rayungus HJG-5 and its purposes in terms of production growth promoting activity substance, broad-spectrum antibacterial active material.The invention has the beneficial effects that: one of present invention Chinese prickly ash endogeny rayungus HJG-5 is to isolate and purify that the antimicrobial spectrum screened is wide, the significant bacterial strain of fungistatic effect from each tissue of Chinese prickly ash；Contain growth promoting activity substance and broad-spectrum antibacterial active material in the fermentation liquid of bacterial strain HJG-5, the developmental research of horn of plenty biological pesticide natural resources and microbial pesticide is laid a good foundation.
Description
Technical field
The invention belongs to microbial fermentation and applied technical fields, and in particular to a kind of Chinese prickly ash endogeny rayungus HJG-5 and
It is applied.
Background technique
Plant disease is one of the principal element of threat agricultural production, and at present in the agricultural production in China, chemistry closes
At pesticide due to has a broad antifungal spectrum, preventive effect height, quick feature, main status is occupied always in disease control, but with it
3R problem caused by long-term super use, abuse, misuse is also increasingly severe.With sustainable development tourism be rooted in the hearts of the people and people
Food-safe concern, seek new controlling way that is safe and efficient, meeting sustainable development idea, become plant
Disease control project urgently to be resolved.Using microorganism or its metabolite controlling disease, because its high-efficiency low-toxicity, selectivity are strong,
It is small to eco-environmental impact and the features such as have both increasing both production and income, become current research hotspot.Endophytic actinomycetes in plants is posted with it
Master forms harmonious relationship, host is extensive, the complicated multiplicity of type, the Substance kind of generation during symbiosis
Class is various, has broad application prospects.Verma etc. is separated from the root tissue of Azadirachta indicaA.Juss
It can significantly promote the growth of plant, and can inhibit alternariosis after finding its suspension processing seed to three plants of endogenetic streptomycetes
The growth and breeding of opportunistic pathogen.Misk et al. isolated 11 plants of endogeny rayungus from the various plants of Australia find it
It can inhibit the growth of Phytophthora Root Rot, and bacterial strain WRA1 can significantly promote plant strain growth.Wang Zhenzhen etc. divides from rice plant
It manages and filters out one plant of endogeny rayungus- meter Xiu streptomycete OsiRt-1 that can significantly inhibit Growth of Magnaporthe Grisea, it is right under field condition
Rice seedlings pest, the preventive effect of fringe pest are respectively 7.76%, 25.65%.Therefore, plant endogenesis Antagonistic Actinomycetes are screened, its life is studied
Anti- effect and Biocontrol Mechanism have important practical significance to the production application of microbial pesticide.
Chinese prickly ash can not only make the raw material of fragrance but also can be used as medicine.Its medical value is equal in Shennong's Herbal and Compendium of Material Medica
It is on the books, can dispelling cold and removing dampness, warm in pain.Han Shengnan has found that pepper volatile oil has stronger inhibition to make HeLa, A549, K562
With.One of the compounds extracted from Chinese prickly ash such as Ge, PauL main component can inhibit gelechiid oviposition, and to its larva have compared with
Strong Antifeedant Effects.The researchs such as Singh discovery Japan pepper essential oil not can control the breeding of rice weevil but wheat can be protected from damage.
The discovery Chinese prickly ash stem skin such as Navarrete can reduce the quantity of gutstring worm in sheep body, chloroform extract α-sanshool pair after decocting
Roundworm has acute toxicity.The discovery Chinese prickly ash pericarp volatile materials such as Paik can inhibit HepG2 liver cancer cells.But at present to Chinese prickly ash
The research of endogeny rayungus yet there are no relevant report.
In recent years, the production for the problems such as a large amount of uses of chemical pesticide cause the export of farm produce limited, drug resistance, environmental pollution
It is raw, be badly in need of developing new and effective, wide spectrum, low toxicity, environmental protection biological bacteriostatic agent.
Summary of the invention
The present invention provides a kind of Chinese prickly ash endogeny rayungus HJG-5, contains growth promoting activity object in the fermentation liquid of bacterial strain HJG-5
Matter can promote plant growth, and the Substance containing efficient, wide spectrum, low toxicity, environmental protection can play good suppression
Bacterium effect.
A kind of Chinese prickly ash endogeny rayungus HJG-5, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, preservation day: 2017.11.30, preservation registration number 14969.
Further, it is determined by molecular biology research, HJG-5 bacterial strain can be classified as cortex cinnamomi maroon streptomycete.
Further, Chinese prickly ash endogeny rayungus HJG-5 bacterial strain cultivates 2-3d, bacterium colony table on oatmeal agar medium
Face is smooth white, and the aerial hyphae that white is begun with after cultivating to 6d grows from colony edge, starts to gradually become after 8d
It is light grey；Colonial morphology is cone protuberance in incubation, and the concentric wheel stripe that colony edge has 4-6 to enclose is with dizzy shape earth outside
Side；By microexamination, aerial hyphae is flourishing, branch is more, and substrate mycelium is without tabula, not broken.
Further, the cultural characteristic of Chinese prickly ash endogeny rayungus HJG-5 bacterial strain is, on ISP2, PDA and nutrient agar
For bacterium colony surface elevation in fine and close suede powdery, aerial hyphae is flourishing, for white or canescence, substrate mycelium brown color or yellowish-brown
Color；Aerial hyphae is light gray on ISP5, and substrate mycelium is light light yellow；On ISP4, Cha Shi and Gause I culture medium
Raw mycelia is thin, is in very light lime color, no substrate mycelium more；It is hardly grown on ISP3.
Further, Chinese prickly ash endogeny rayungus HJG-5 bacterial strain can produce melanin, part of gelatin can be made to liquefy, nitrate
It is reduced to the positive, Starch Hydrolysis, milk is solidified and peptonized, and does not generate H2S is not grown on cellulose；Starch, wood cannot be utilized
Sugar, arabinose and fructose, can utilize glucose, maltose, sucrose, lactose, mannitol and glycerol.
A kind of use of Chinese prickly ash endogeny rayungus HJG-5 in terms of production growth promoting activity substance, broad-spectrum antibacterial active material
On the way.
Further, the growth promoting activity substance, broad-spectrum antibacterial active material are trained from aforementioned HJG-5 strain fermentation
Fermentation liquid after supporting.
Further, carbon source is maltose in the fermentation medium of aforementioned HJG-5 strain fermentation culture, and nitrogen source is pancreas egg
White peptone；And maltose: the proportion of tryptone is 2:1.
Further, the condition of culture of aforementioned HJG-5 strain fermentation culture is revolving speed 180rmin-1, pH=8, dress
Liquid measure 125mL/250mL, inoculum concentration 8%, cultivation temperature are 28 DEG C, incubation time 9d.
Further, aforesaid fermentation liquid has preferable thermal stability, and 80 DEG C or less bacteriostasis rates maintain 80% or so,
It is highly stable under neutral and weakly alkaline environment；Do not change bacteriostatic activity through ultraviolet irradiation；Under the conditions of 4 DEG C, storage stability is good
It is good, after placing 90d, the 85.15% of original fermentation liquid bacteriostatic activity can be reached to the inhibiting rate of A.soLani.
Further, when aforesaid fermentation liquid dilutes 20 times, growth promoting function and bacteriostasis are optimal；20 times of diluted hairs
Germination index improves 16.75% relative to CK after zymotic fluid processing tomato seeds；Tomato seedling plant height, root long, fresh weight, dry weight
It has been respectively increased 29.0%, 20.4%, 24.9%, 35.9% compared with CK group, has there is significant growth.
The invention has the beneficial effects that: one of present invention Chinese prickly ash endogeny rayungus HJG-5 is each group from Chinese prickly ash
Isolate and purify that the antimicrobial spectrum screened is wide, the significant bacterial strain of fungistatic effect in knitting；Contain growth promotion in the fermentation liquid of bacterial strain HJG-5
Active material and broad-spectrum antibacterial active material, the developmental research of horn of plenty biological pesticide natural resources and microbial pesticide are established
Basis.
Detailed description of the invention
Fig. 1 is a kind of inhibiting effect figure of Chinese prickly ash endogeny rayungus HJG-5 of the invention to 12 kinds of pathogens；
Fig. 2 is a kind of inhibiting effect figure of Chinese prickly ash endogeny rayungus HJG-5 ferment filtrate of the invention to 12 kinds of pathogens；
Fig. 3 is the gentle raw Mycelial morphological characteristic figure of a kind of Chinese prickly ash endogeny rayungus HJG-5 bacterium colony of the invention；
Fig. 4 is the 16SrDNA amplified production electrophoresis result figure of Chinese prickly ash endogeny rayungus HJG-5 of the invention a kind of；
Fig. 5 is a kind of Chinese prickly ash endogeny rayungus HJG-5 phylogenetic tree of the invention；
A kind of Chinese prickly ash endogeny rayungus HJG-5 growth of Fig. 6 invention and the Yield mapping of Substance；
Influence diagram of Fig. 7 difference basal medium to HJG-5 bacterial strain bacteriostatic activity；
Influence diagram of the source difference C Fig. 8 to HJG-5 strain fermentation filtrate bacteriostatic activity；
Influence diagram of the source difference N Fig. 9 to HJG-5 strain fermentation filtrate bacteriostasis rate；
Figure 10 condition of culture orthogonal test analysis tendency chart；
Inhibiting effect figure of Figure 11 optimization front and back HJG-5 bacterial strain fermentation liquor to A.soLani；
Influence diagram of Figure 12 temperature to bacterial strain HJG-5 ferment filtrate bacteriostatic activity；
Influence diagram of Figure 13 pH value to HJG-5 strain fermentation filtrate bacteriostasis rate；
Figure 14 ultraviolet light irradiates the influence diagram to HJG-5 strain fermentation filtrate bacteriostatic activity；
Influence diagram of Figure 15 period of storage to HJG-5 strain fermentation filtrate bacteriostatic activity；
The influence diagram that Figure 16 HJG-5 strain fermentation filtrate grows A.soLani mycelia；
Influence diagram of Figure 17 various concentration strain fermentation filtrate to A.soLani cell leakage；
Influence diagram of Figure 18 HJG-5 strain fermentation filtrate to tomato seeds GI；
Influence diagram of Figure 19 HJG-5 bacterial strain fermentation liquor to Growth of Tomato Seedling；
Influence diagram of Figure 20 HJG-5 bacterial strain fermentation liquor to Growth of Tomato Seedling；
The in vitro preventive effect figure of Figure 21 HJG-5 strain fermentation filtrate；
Figure 22 silica gel column chromatography separation component bacteriostatic activity testing result figure.
Specific embodiment
1. test material
1.1 plant sample
Root, stem, leaf and the fruit of Chinese prickly ash pick up from Agricultural University Of Shanxi in July, 2015；Tomato seeds improve red general F1
From Xi'an Shuan Feng seed Co., Ltd.
1.2 for trying culture medium
Actinomycetes strain culture Storaged media: PDA culture medium.
Fermentation medium: PD culture medium.
Gause I culture medium: soluble starch 20g, MgSO4·7H2O 1g, KNO31g, NaCl 0.5g, K2HPO4·
3H2O 0.5g, FeSO4·7H2O 0.01g, agar 20g, distilled water 1000mL, pH7.4~7.6.
Soybean powder fluid nutrient medium: soybean powder 10g, peptone 3g, glucose 10g, NaCl 2.5g, CaCO32g, distillation
Water 1000mL, pH7.2~7.4.
Millet fluid nutrient medium: millet 10g, glucose 10g, NaCl 2g, peptone 3g, CaCO32g, NH4NO31g steams
Distilled water 1000mL, pH7.2~7.4.
Soybean, corn powder liquid culture medium: soybean powder 10g, corn flour 10g, soluble starch 5g, K2HPO40.5g, distillation
Water 1000mL, pH7.2~7.4.
1.3 test plant disease fungus
Botrytis cinerea (Bortytis cinerea), tomato early blight bacterium (Alternaria solani), wheat is red
Mildew bacterium (Fusarium graminearum), cotton rhizoctonia solani (Rhizoctonia solani), bean sclerotinia rot bacterium
(Sclerotinia sclerotiorum (lib.) de Bary), bean anthrax bacteria (Colletotrichum
Lindemuthianum), capsicum wilt bacterium (Fusarium oxysporium), withered germ of water-melon (Fusarium
Oxysporumf.sp.Niveum), Valsa mali (Valsa mali), carrot brown rot (leptosphaeria
Libanotis), Rot in walnut bacterium (Monilinia laxa), Monilinia fructicola (Moniliniafructicola), by
Agricultural University Of Shanxi's Pesticide Science laboratory saves.
1.4 for trying chemical reagent
CH4O、C6H14、C4H8O2、CHCl3、CH2Cl2、CH3(CH2)3OH、HCl、NaOH。
1.5 for test instrument
HZQ-QX warm concussion and cultivate case entirely, UV-754 ultraviolet-uisible spectrophotometer, HD-920 superclean bench, ST16R
Refrigerated centrifuge, ALPCL-32L high-temperature sterilization pot, Rotary Evaporators, S-234 assay balance, electro-heating standing-temperature cultivator, DDS-
11A conductivity gauge.
2. experimental method
The separation screening of raw Antagonistic Actinomycetes in 2.1 Chinese prickly ashes
2.1.1 the acquisition of plant sample
Selection is grown fine, and the root, stem and leaf fruit of healthy Chinese prickly ash plant is acquired, and 4 DEG C of refrigerators save simultaneously in freshness protection package
It is marked with label paper, is handled as early as possible in Yu Yizhou.
2.1.2 endogeny rayungus isolates and purifies
The pretreatment of plant sample: thoroughly clear with ultrasonic cleaning instrument after the sample acquired back is rinsed well under a stream of water
The impurity of wash clean sample surfaces remaining.After sample is cut into 2cm segment with sterile knife, in 75% alcohol impregnate 3min,
Aseptic water washing 2 times, impregnate 3min in 3% NaClO, aseptic water washing 3 times, then after impregnating 1min to 75% alcohol, it is sterile
Dry filter paper on piece after water flushing 3 times in sterilizing is dried.
Material after disinfection: being added the grinding of 10mL sterile water by tissue homogenate method isolated strains in mortar, static
15min takes 80 μ L of supernatant to be coated on containing K2Cr2O7750 No. 1 plates of μ g/mL Gao Shi, 28 DEG C are cultivated 2-3 weeks, daily observation note
Record bacterium colony situation, one it is found that there is bacterium colony to grow, immediately picking, according to bacterium colony appearance features, size, soluble pigment whether there is or not etc.
It crosses and cultivates on Gao Shi culture medium, repeatedly number simultaneously slant preservation after purification.
Surface sterilization product test: taking 80 μ L of last time flushing water to be coated on Gause I plate, equal conditions training
It supports, as control for examining surface sterilization whether thorough.
2.1.3 the screening of raw Antagonistic Actinomycetes in
(1) primary dcreening operation
Using 4 tablet face-off methods, the B.cinerea and A.solani of culture 6-7 days are taken, makes Ф 5mm with punch
Bacteria cake, be connected to PDA plate center, away from its 2cm at cross inoculation for examination actinomyces, 28 DEG C cultivate, measure cause of disease after 7 days
Bacterium diameter calculates bacteriostasis rate, and to be inoculated with pathogen only as CK, 3 repetitions are tested in every processing.
(2) secondary screening
Actinomyces to be measured are inoculated in PDA culture medium, 28 DEG C, after cultivating 7d, play the bacteria cake for taking 2 Ф to be 5mm, access
In seed culture fluid (100mL/250mL), 28 DEG C, 160rmin-1Shaking flask culture 48h, obtains seed liquor, it is pressed 10% (V/
V in inoculum concentration access 200mL/500mL fermentation medium), 28 DEG C, 160rmin-1, after shaking flask culture 7d, obtain bacterial strain
Fermentation liquid.Fermentation liquid is through 12000rmin-1Centrifugation, 0.22 μm of filtering with microporous membrane obtain ferment filtrate.
Using growth rate method, ferment filtrate and PDA culture medium are prepared into plate by 1:9 mixing.It, will after plate solidification
The A.solani fungus block of Ф 5mm accesses center, and after cultivating 6-7d at 28 DEG C, crossing method measures germ diameter, calculates antibacterial
Rate.3 repetitions are CK with blank PD culture solution.
The taxonomic identification of 2.2 endogeny rayungus HJG-5
2.2.1 observation of morphological
Using inserted sheet method by HJG-5 strain inoculated in PDA culture medium, 7d-21d is cultivated at 28 DEG C, is during which taken every 5d
Piece is buried in optical microphotograph microscopic observation, is tentatively reflected according to " streptomycete identification handbook " and " classification and identification of actinomyces "
It is fixed, main detection colonial morphology, substrate mycelium, aerial hyphae growthform；Whether there is or not the features such as tabula, fracture for substrate mycelium.
2.2.2 cultural characteristic is observed
Select Gao Shi 1, ISP 2, ISP3, ISP 4, ISP 5, potato agar, nutrient agar, 8 kinds of Czapek's agar trainings
Base is supported, plate is prepared, after being inoculated with HJG-5 bacterial strain, 28 DEG C of cultures observe and record bacterium after 5d, 7d, 10d, 15d, 21d respectively
Silk color and growing state, if generate the color of soluble pigment and generation.
2.2.3 Physiology and biochemistry is identified
Referring to " streptomycete identification handbook " and " actinomyces Rapid identification and genealogical classification " method, to bacterial strain HJG-5 into
The identification of row Physiology and biochemistry.
2.2.4 utilization of carbon source
Based on general dagger-axe Er Shi solid medium, carbon source kind have glucose, multitudinous sugar, lactose, maltose, xylose, Ah
Uncle's sugar, fructose, mannitol, glycerol and starch are drawn, after preparing plate, streak inoculation HJG-5 bacterial strain, after 28 DEG C of constant temperature incubation 7d,
Observe strain growth situation.
2.2.5 melanin generates
By HJG-5 strain inoculated in tyrosine culture medium, 28 DEG C of constant temperature incubations, the 7d and 14d of difference after inoculation,
Whether there is or not melanin generations for observation.
2.2.6 hydrogen sulfide generates
HJG-5 bacterial strain is accessed in Chai Sina culture medium, after 28 DEG C are cultivated 7 days, has seen whether that black or brown occur.
2.2.7 nitrate reduction test
By HJG-5 strain inoculated in nitrate fluid nutrient medium, 28 DEG C, culture 7d-10d after, take a culture solution to add
It reddens color after Griess reagent and aniline reagents or pink colour is the positive, then take a part culture solution, be added after aniline reagents if indigo plant
Color is then negative, is the positive if non-discolouring.
2.2.8 gelatin liquefaction
HJG-5 bacterial strain is accessed into gelatin culture medium, 28 DEG C, culture 7d-14d observe cooling 30min in preceding 4 DEG C of refrigerators, such as
Periphery of bacterial colonies gelatin is flowable, then is the positive, is otherwise feminine gender.
2.2.9 milk is solidified or is peptonized
HJG-5 bacterial strain is accessed in milk-based liq culture medium, 28 DEG C of cultures, grumeleuse such as occur in 5d, 10d, 15d observation
Become liquid again afterwards, is then the positive.
2.2.10 Starch Hydrolysis
By the streak inoculation of HJG-5 bacterial strain on starch agar medium plate, iodine solution, observation is added dropwise in periphery of bacterial colonies after 7d
There is situation in transparent circle, has then for the positive.
2.2.11 cellulose utilization
By HJG-5 strain inoculated in immerse culture medium filter paper item on, 28 DEG C of culture 7d, if bacterial strain can give birth on filter paper item
It is long, and it is threadiness that filter paper, which is decomposed thinning, for the positive.
2.2.12 molecular biology identification
The DNA of HJG-5 bacterial strain is extracted using CTAB method, expands 16S rDNA gene.Primer is 27f:5 '-
AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACGGYTACCTTGTTACGACTT-3 '.Amplification system and procedure reference
The method of Coombs, amplified production send to Jin Wei intelligence Biotechnology Co., Ltd and are sequenced.By the 16SrDNA sequence of acquisition in NCBI
It after upper BLast, compares, chooses and the higher strain gene sequence of its homology, utilization through row with the sequence in Genbank
MEGA5.1 software is analyzed, and constructs HJG-5 bacterial strain phylogenetic tree using Neighbor-Joining method.
2.3 endogeny rayungus HJG-5 fermented and cultureds
2.3.1 the preparation of seed liquor
The HJG-5 bacterial strain of preservation is activated on PDA plate, beats and takes two Ф 5mm bacteria cakes, be seeded to 100mL/250mL
In the triangular flask of PD culture medium, 28 DEG C, 160rmin-1, seed culture fluid is obtained after shaking flask culture 48h.
2.3.2 the preparation of fermentation liquid
By the seed liquor of HJG-5 bacterial strain by 10% (V/V) amount access PD fermentation medium, 28 DEG C, 160rmin-1, shake
After bottle culture 7d, the fermentation liquid of bacterial strain, 12000rmin are obtained-1Centrifugation, 0.22 μm of filtering with microporous membrane to get HJG-5 hair
Ferment filtrate, 4 DEG C save backup.
2.3.3 fermentation liquid bacteriostatic activity detects
It is CK with blank PD culture solution using A.solani as indicator bacteria using aforementioned growth performance rate method.
2.3.4 the accumulation of thalli growth and Substance
Seed culture fluid is accessed into PD fermentation medium (250mL/500mL) with 10% (V/V), 28 DEG C, 160rmin-1,
It cultivates different time (1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 9d), takes fermentation liquid 100mL, 12000rmin daily-1Centrifugation,
Supernatant with growth rate method, surveys its bacteriostatic activity to A.solani, precipitating thallus dries after 0.22 μm of filtering with microporous membrane
Dry weighing.
2.3.5 culture medium and condition of culture screening
By the seed fermentation liquid of HJG-5 bacterial strain by the inoculum concentration of 10% (V/V) be inoculated in each sterilizing basic culture solution (A:
Gao Shi No. 1 fluid nutrient medium, B: millet fluid nutrient medium, C: soybean powder fluid nutrient medium, D: soybean, corn powder liquid culture medium,
E:PD culture medium) in, 28 DEG C, 160rmin-1, after 7d, measure HJG-5 strain fermentation under the conditions of different fermentations basal medium
Filtrate determines best fermentation basal medium to the bacteriostasis rate of A.solani.
Basis is replaced respectively with the soluble starch of equivalent (20g/L), maltose, sucrose, lactose, mannitol, corn flour
Glucose in culture medium, other compositions are constant, with the basal medium that ferments for CK, when measuring the different sources C, and HJG-5 bacterial strain hair
Ferment filtrate determines optimum carbon source to the bacteriostatic activity of A.solani.
Using the above-mentioned best source C filtered out as the source C, equivalent (10g/L) soybean powder, peptone, tryptose are added respectively
Peptone, yeast powder, beef extract, urea, (NH4)2SO4、KNO3, under the conditions of measuring the different sources N, HJG-5 strain fermentation filtrate pair
The bacteriostatic activity of A.solani, determines optimum nitrogen source, makees CK with original PD culture medium.
Further to study influence of the fermentation condition to HJG-5 bacterial strain Substance yield, using orthogonal test L16
(45), referring to table 1, revolving speed, PH, liquid amount, inoculum concentration, fermentation time are optimized, so that it is best to filter out bacterial strain HJG-5
Fermentation condition.
1 fermentation condition Orthogonal Experiment and Design of table
2.4 HJG-5 bacterial strain fermentation liquor Stability Determinations
2.4.1 thermal stability determination
Take six sterile test tubes, every dress ferment filtrate 10mL, respectively at 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C, 121 DEG C of temperature
30min is handled under the conditions of degree, its bacteriostatic activity is measured after being cooled to room temperature, and is CK with untreated ferment filtrate.
2.4.2 ultraviolet radiation stability measures
Take 10mL ferment filtrate in sterilizes culture dish respectively, irradiated respectively at ultraviolet lamp (15cm) 2h, 4h, 6h, 8h,
10h, 12h, for 24 hours after, survey its bacteriostatic activity, be CK with the ferment filtrate without ultraviolet irradiation.
2.4.3 ph stability measures
It takes 10mL ferment filtrate in sterilizing test tubes respectively, is distinguished its pH with 1moL/L HCl and 1moL/LNaOH solution
It is adjusted to: 2.0,3.0,4.0,5.0,7.0,8.0,9.0,10,11.0,12.0, original pH 6.0 is recalled to after placing 2h at room temperature,
Its bacteriostatic activity is measured, is control with the ferment filtrate (PH6.0) handled without soda acid.
2.4.4 storage stability measures
Take ferment filtrate, be stored in sterile test tube, 4 DEG C of refrigerator dark save, respectively at 0d, 15d, 30d, 45d, 60d,
Its bacteriostatic activity of 75d, 90d, 105d, 120d sample detection.
The detection of the above bacteriostatic activity is all made of growth rate method, using A.solani as target bacterium.
The Primary Study of 2.5 HJG-5 strain fermentation filtrate bacteriostasis
2.5.1 to the growth of germ mycelia and the influence of form
Repressed A.solani mycelia is on glass slide in picking Antibacterial Activity, microexamination hypha form, with
The A.solani mycelia of normal growth is CK, records simultaneously microphotograph.
2.5.2 to the influence of germ cell membrane permeability
The A.solani of volume production spore big after culture is connected in PD culture solution, 28 DEG C, 160rmin-1, after 5-6d,
7500r·min-1It is collected by centrifugation mycelium, after sterile water wash 3 times, weighs 5 parts, every part of 5g is placed in 100mL triangular flask, is added
Enter 50mL sterile water, then be separately added into ferment filtrate, making final concentration is respectively 5%, 10%, 13%, and 20%, CK adds isometric
PD culture solution, 28 DEG C, 160rmin-1, respectively in 0,0.5h, 1h, 1.5h, 2h, 4h, 6h, 20h, for 24 hours, after taking 5mL to be centrifuged, survey
Determine supernatant conductivity value.Influence of the Substance to A.solani cell leakage is indicated with conductivity value variation.
2.6 HJG-5 bacterial strain fermentation liquor growth-promotings and Biocontrol Effect measure
2.6.1 to the influence of Tomato Seeds Germination
Choose full tomato seeds uniform in size, after surface sterilization, respectively fermentation liquid stoste, 5x, 10x, 20x,
It soaks seed in 50x, 80x, 100x dilution for 24 hours, using the processing of blank run liquid as CK, 30, every ware, which is placed in, is covered with two layers of sterilizing
In the culture dish of filter paper, 27 DEG C of dark culturings, timing is unified plus sterile water keeps wet, and 3 repetitions of every processing, test repeats 2
It is secondary, it observes daily and measures the germination index respectively handled in 7 days.
Germination index (GI)=∑ Gt/Dt
In formula: Gt: the germinative number in t days；Dt: germination number of days.
2.6.2 to the influence of tomato seedling growth
Tomato seeds are in the above way chosen, are sowed after surface sterilization, when seedling grows the 4th true leaf, choose growing way
Seedling of the same size stays seedling, and respectively with fermentation liquid stoste, the diluted fermentation liquid of 5X, 10X, 20X, 50X, 80X and 100X into
Row pouring root, every plant of 20mL, pouring root again after five days are CK, incubated at room temperature, timely and appropriate discovery watering, after sowing 70d with PD culture solution
Measure plant height, fresh weight, dry weight and root long.
2.6.3 excised leaf control effect testing
Close leaf position on tomato plant, the consistent healthy leaves of growth are chosen, after surface treatment disinfection, using stab inoculation
Method sprays fermentation liquid stoste and 5X, 10X, 20X dilution respectively on blade, to spray blank PD culture solution for CK, is followed by for 24 hours
Kind of tomato early blight bacterium bacteria cake, by treated, blade is placed in bottom is covered in the culture dish of sterile wet filter paper, every processing six
Blade is tested in triplicate, 27 DEG C of moisturizing dark alternate cultures, is observed blade incidence after 4d, is calculated bacteriostasis rate.
* 100/ control lesion area of inhibiting rate/%=(control lesion area-processing lesion area)
2.6.4 in vitro fruit control effect testing
Healthy tamato fruit is chosen, 75% alcohol disinfecting is spare after aseptic water washing is clean, using stab inoculation,
Middle part of fruit draws the wound that Φ is 5mm with sterile needle, sprays fermentation liquid stoste, 5X, 10X, 20X dilution, to spray blank respectively
PD is CK, is inoculated with A.solani bacteria cake afterwards for 24 hours, is placed in bottom and is covered in the sterile tray of wet filter paper, preservative film environmental sealing.
5 fruits of every processing, test are repeated 3 times, 27 DEG C of cultures, observe fruit incidence after 4d.
The initial gross separation of 2.7 Substances and biological activity determination
2.7.1 the screening of best extractant
By bacterial strain HJG-5 ferment filtrate loaded on (200mL/500mL) in five triangular flasks, it is separately added into isometric petroleum
Ether, ethyl acetate, chloroform, methylene chloride, n-butanol, 28 DEG C, 160rmin-1, after 4h in separatory funnel, 4-6h is stood, point
Not Shou Ji organic phase and water phase, after multiple extraction merging Organic phase, revolving uses first to dry under appropriate temperature conditions
Alcohol dissolves and is settled to 10mL, its bacteriostatic activity to A.solani is surveyed with growth rate method, with the optimal organic extraction of determination
Solvent.
2.7.2 silica gel column chromatography
2.7.2.1 the preparation of Substance crude extract
HJG-5 bacterial strain fermentation liquor 3L is prepared, through 12000rmin-1After centrifugation, 0.22 μm of filtering with microporous membrane, it must ferment
Filtrate, with n-butanol equal-volume extraction three times after, merge organic phase, rotate at 55 DEG C, dissolve crude extract, 0.45 μ with 5mL methanol
After the organic filtering with microporous membrane of m, 4 DEG C of refrigerator storages.
2.7.2.2 silica gel column chromatography
Dress column: silica gel (200-300 mesh) is adjusted to paste with petroleum ether, is poured into chromatographic column (30 × 450mm), side edged
Stirring, gently taps cylinder with rubber hammer, bubble in column is discharged, gently twists cock, settles silica gel slowly, be filled to from nozzle 5-
7cm drenches column with appropriate petroleum ether, until cylinder stops decline, while in column bed surface one layer of quartz sand of uniform fold, with
Guard column bed surface it is smooth.
Sample-adding: when liquid surface in column is away from Silica Surface 1cm or so, with suction pipe by the crude extract (2mL) of preparation along column
Inner wall is slow, is homogeneously added into, and when sample is moved to Silica Surface or less, successively carries out ladder with the ascending reagent of polarity
Degree elution.
Elution and sample collection: eluent is followed successively by petroleum ether: chloroform (1:1), chloroform: methanol (2:1), chloroform: methanol
(1:1), chloroform: methanol (1:2), methanol, each gradient eluent 200mL.Coutroi velocity is 3~4 drops/S, every pipe 20mL.
2.7.2.3 silica gel thin-layer chromatography detects
Collected flow point is through TLC detection (solvent is chloroform: methanol 4:1, and iodine distillation is used as color developing agent), root in test tube
Merge identical flow point according to Rf value.
2.7.2.4 each component biological activity determination
Suppression after group lease making after merging is concentrated under reduced pressure, with the inhibition zone method measurement separated substance of each component to A.solani
Bacterium rate.
3. result and analysis
The separation screening and Antibacterial Activity of raw Antagonistic Actinomycetes in 3.1 Chinese prickly ashes
3.1.1 Chinese prickly ash endogeny rayungus isolates and purifies
According to bacterium colony appearance features, size, whether there is or not equal to the actinomyces grown on isolation medium progress for soluble pigment
Classification purifying, is obtained 11 plants of endogeny rayungus, wherein 8 plants from root, 3 plants come from stem.Bacterium is had no on CK culture medium
It falls, it was demonstrated that surface sterilization is thorough, and obtained strains are Chinese prickly ash endophyte.
3.1.2 the screening of Antagonistic Actinomycetes is given birth in Chinese prickly ash
Using B.cinerea and A.solani as indicator bacteria, using 4 tablet face-off methods to raw in isolated 11 plants
Actinomyces carry out primary dcreening operation, wherein 6 plants can be observed bacteriostasis (table 2), to the bacteriostasis rate of A.solani 44.4%~
Between 73.59%, to the inhibiting rate of B.cinerea between 29.25%~60.10%, wherein HJG-5 bacterial strain is to two kinds of germs
Bacteriostasis rate be best.
It with growth rate method to 6 plants of Antagonistic Fungi secondary screenings, can be obtained by table 2, inhibition of six plants of bacterium metabolites to A.solani
Rate is between 47.18%~81.92%, and wherein the bacteriostasis rate of HJG-5 strain fermentation filtrate is highest 81.92%.It is real herein
On the basis of testing, optimal bacterial strain HJG-5 is selected to carry out subsequent research.
The screening of raw Antagonistic Actinomycetes in 2 Chinese prickly ash of table
Note: table Chinese and English lowercase indicates newly the significance of difference of the range test in the level of P < 0.05 again.
3.1.3 HJG-5 bacterial strain antimicrobial spectrum measures
3.1.3.1 HJG-5 bacterial strain Antibacterial Activity
Using 12 kinds of pathogens as indicator bacteria measure HJG-5 bacterial strain antimicrobial spectrum, table 3 it is found that HJG-5 bacterial strain to for try cause of disease
Bacterium has inhibiting effect, inhibiting rate 42.87%-76.55%；It is best to the inhibiting rate of M.laxa and A.solani, be respectively
77.42%, 76.55%；Suppression for V.mali, S.sclerotiorum (lib.) de Bary and C.lindemuthianum
Bacterium effect is poor, respectively 45.98%, 49.84% and 42.87%；For other pathogens bacteriostasis rate in 51.91%-
Between 70.54%, effect is shown in Fig. 1.
Inhibiting effect of the 3 HJG-5 bacterial strain of table to 12 kinds of pathogens
3.1.3.2 HJG-5 strain fermentation filtrate antimicrobial spectrum measures
The measurement of antimicrobial spectrum is carried out, to the ferment filtrate of HJG-5 bacterial strain to ensure its research and development value.As shown in Table 4,
There are significant differences for inhibiting effect of the strain fermentation filtrate to 12 kinds of germs for examination, to the inhibiting rate of M.laxa and A.solani
Most preferably, respectively 80.47%, 82.39%, to the inhibiting rate of F.graminearum, L.libanotis, M.fructicola
Preferably, respectively 73.88%, 75.26%, 70.32%.And to R.solani, V.mali, S.sClerotiorum (lib.)
The inhibiting rate of deBary is poor, is lower than 40%, effect is shown in Fig. 2.
Inhibiting effect of the 4 HJG-5 strain fermentation filtrate of table to 12 kinds of pathogens
3.2 HJG-5 strain classification status
3.2.1 morphological feature
HJG-5 bacterial strain cultivates 2-3d on oatmeal agar medium, and bacterium colony surface is smooth white, after culture to 6d
The aerial hyphae for beginning with white grows from colony edge, starts to gradually become light grey after 8d.Colonial morphology in incubation
For cone protuberance, the concentric wheel stripe that colony edge has 4-6 to enclose is with dizzy shape earth side outside.Pass through microexamination, aerial hyphae hair
Reach, branch it is more, for substrate mycelium without tabula, not broken, effect is shown in Fig. 3.
3.2.2 cultural characteristic
Referring to table 5, HJG-5 bacterial strain is more excellent in ISP2, PDA and grown on nutrient agar, and bacterium colony surface elevation is in densification
Suede powdery, aerial hyphae is flourishing, for white or canescence, substrate mycelium brown color or yellowish-brown.Medium, gas is grown on ISP5
Raw mycelia is light gray, and substrate mycelium is light light yellow.In ISP4, poor, gas life is grown on Cha Shi and Gause I culture medium
Mycelia is thin, is in very light lime color more, and no substrate mycelium hardly grows on ISP3.On 8 kinds of culture mediums for examination
Do not generate soluble pigment.
Cultural characteristic of the 5 endogeny rayungus HJG-5 of table in different culture medium
Note: it "-": does not grow；"+": growth；" ++ ": well-grown；" +++ ": growth is vigorous
3.2.3 physiological and biochemical test
Referring to table 6, Physiology and biochemistry testing result shows that interior raw Antagonistic Actinomycetes HJG-5 can produce melanin, and part can be made bright
Dispergation, nitrate reduction are the positive, and Starch Hydrolysis, milk is solidified and peptonized, and do not generate H2S is not grown on cellulose.Bacterial strain
Starch, xylose, arabinose and fructose cannot be utilized, to glucose, maltose, sucrose, lactose, mannitol and glycerol these types
Carbon source has relatively good producing level.
6 HJG-5 bacterial strain physiological and biochemical property of table and utilization of carbon source
Note: "-": negative；"+": positive
3.2.4 molecular biology
Using primer 2 7F/1492R, using HJG-5 bacterial strain DNA as template, PCR amplification, agarose gel electrophoresis detection are carried out
PCR product is as shown in figure 4, band is clear, single.After sequencing, sequence length 1361bp.By 16SrDNA sequence and Genbank
In listed allied species 16SrDNA sequence be compared, choose with the gene order of the higher bacterial strain of its homology, utilize
MEGA5.1 software is analyzed, and constructs HJG-5 bacterial strain phylogenetic tree, final HJG-5 using Neighbor-Joining method
Bacterial strain and streptomyces cinnamocastaneus, AB184588 gather in same branch, and referring to Fig. 5, homology reaches
99%, affiliation is nearest, therefore, bacterial strain HJG-5 is classified as cortex cinnamomi maroon streptomycete
streptomycescinnamocastaneus。
3.3 fermentation condition optimization
3.3.1 thalli growth curve
The growth curve of HJG-5 bacterial strain and the change of production of Substance are as shown in fig. 6, bacterial strain is quick in 0-7d
Growth, with the extension of fermentation time, dry mycelial weight increases, and peak is reached in 7d, and 8-9d is stationary phase, and fermentation 10d is opened
There is decline trend in beginning.
By the bacteriostasis rate curve of HJG-5 bacterial strain it is found that the yield of Substance gradually increases with the extension of fermentation time
Add, start fungistatic effect occur after the 1d that ferments, reach maximum value in 7d, be 85.89%, when 8-9d, bacteriostasis rate compared with
Stablize, with peak without significant difference, 10d starts downward trend occur, bacterial strain itself life known to ligative hyphae dry weight curve
The long yield with Substance is consistent substantially.
3.3.2 culture medium and condition of culture screening
3.3.1 basal medium screens
Using five kinds of basal mediums of A~E, A: Gause I fluid nutrient medium, B: millet fluid nutrient medium, C: soybean powder
Fluid nutrient medium, D: soybean, corn powder liquid culture medium, E:PD culture medium carry out fermented and cultured, use growth rate method after 7d
Its ferment filtrate is surveyed to the bacteriostasis rate of A.solani.As shown in Figure 7, the bacteriostatic activity of bacterial strain has significance difference in each fermentation medium
It is different, wherein the fungistatic effect of the ferment filtrate of E culture medium is best, is 84.63%, secondly trains for 76.27%, A of D culture medium
The fungistatic effect for supporting base is worst, and only 4.52%, therefore, E culture medium i.e. PD is selected to carry out the excellent of next step for basic culture medium
Change test.
3.3.2 carbon source is screened
It is basic culture medium with PD, under the conditions of adding the different sources C, the ferment filtrate of HJG-5 bacterial strain is antibacterial to A.solani
Activity is there are significant difference, such as Fig. 8, CK PD, and when with maltose being the source C, it is 86.29% that bacteriostatic activity is best, secondly grape
Sugared CK is 84.28%, and mannitol, starch, lactose and corn flour take second place, and when using sucrose as carbon source, fungistatic effect is worst, are
31.28%.Therefore with maltose for the best source C.3.3.3 nitrogen source is screened
The different sources N are to difference such as Fig. 9 of HJG-5 bacterial strain bacteriostatic activity, in 8 kinds of sources N of examination, tryptone nutritional condition
The bacteriostatic activity of lower bacterial strain is best, is 87.60%, compared with the CK for not adding nitrogen source that bacteriostasis rate is 84.32%, significant difference,
It secondly is yeast powder and (NH4)2SO4, respectively 83.64%, 83.52%, beef extract, peptone, KNO3Take second place with soybean powder,
And bacteriostatic activity is respectively less than CK, when addition urea is nitrogen source, worst fungistatic effect is 19.32%, therefore later period test selection pancreas
Peptone is as the source N.
3.3.4 fermentation condition orthogonal optimization
On the above-mentioned optimal medium component base filtered out, revolving speed, pH, liquid amount, inoculum concentration, fermentation time are selected
Five factors further optimize fermentation condition, are as a result wherein the 5th group of fungistatic effect is best such as table 7
91.30%, corresponding horizontal combination is A2B1C2D3E4, is intuitively analyzed from the tendency of Figure 10, it is known that optimal horizontal group
It is combined into A4B3C4D3E4, this group test is not comprised in positive quadraturing design test table, therefore under additional primary level
Test, to increase the accuracy of test result, the bacteriostasis rate of supplementary test is 91.78%, the fungistatic effect higher than the 5th group.From
Range analysis it is found that five factors to HJG-5 strain fermentation filtrate to the bacteriostatic activity influence degree size of A.solani successively
It is therefore to turn by HJG-5 strain fermentation condition after optimization of orthogonal test for revolving speed > liquid amount > fermentation time > PH > inoculum concentration
Fast 180rmin-1, PH 8, liquid amount 125mL/250mL, inoculum concentration 8%, fermentation time 9d.
7 HJG-5 strain fermentation condition orthogonal test analysis table of table
3.3.5 the bioactivity detection after optimizing
According to the fermentation of culture medium prescription and condition of culture progress HJG-5 bacterial strain after optimization, growth rate method measurement hair
Ferment filtrate to the bacteriostatic activity of A.solani, the experimental results showed that, the ferment filtrate bacteriostatic activity after optimization is significantly improved,
It is increased to 91.78% by 83.70% before optimizing, referring to Figure 11.
3.4 HJG-5 strain fermentation filtrate Stability Determinations
3.4.1 thermal stability
HJG-5 bacterial strain fermentation liquor bacteriostatic activity such as Figure 12 after treatment of different temperature 30min, as the temperature rises, suppression
Bacterium activity gradually has reduction, but in addition to bacteriostatic activity is down to 22.54% after 121 DEG C of processing, Substance is more steady within 80 DEG C
Fixed, bacteriostasis rate maintains 80% or so, 100 DEG C treated fermentation liquid has A.solani 63.63% bacteriostasis rate, i.e., still have
Stronger bacteriostatic activity, the test result show HJG-5 Metabolite better heat stability.
3.4.2 ph stability
Fermentation liquid is through soda acid treated bacteriostasis rate variation such as Figure 13.Bacteriostatic activity is not best under pH7-pH9 environment, and not
Through processing group CK compared to bacteriostatic activity without significant difference, with the increase of acid-base property, bacteriostasis rate gradually has reduction but is also maintained at
80% or so.It can to sum up obtain, which has stronger pH stability.
3.4.3 ultraviolet radiation stability
The bacteriostatic activity that ferment filtrate obtains after different time ultraviolet light is as shown in figure 14, with irradiation time
Cumulative, fermentation liquid is in a slight decrease to the inhibiting rate of A.solani, but bacteriostasis rate is overall 80% or so, after irradiation 24 hours
Bacteriostasis rate is 79.51%, and the CK group bacteriostasis rate compared to not ultraviolet processing only has dropped 6.5%, it can thus be concluded that ultraviolet irradiation out
Under, Substance is more stable, and degradation will not occur and change.
3.4.4 storage stability
At 4 DEG C, ferment filtrate has preferable storage stability as shown in figure 15, in 30 days, bacteriostatic activity 80% with
On, when 40d-80d, there was no significant difference for bacteriostasis rate, 77% or so, when storing 90d, still has to the inhibiting rate of A.solani
74.03%, it is the 85.15% of original fermentation liquid bacteriostatic activity.
3.5 HJG-5 strain fermentation filtrate bacteriostasis
3.5.1 to the influence of A.solani hypha form
Influence of the strain fermentation filtrate to A.solani hypha form such as Figure 16, A are normal mycelia；B, C is bacterial strain HJG-5
The mycelia of processing；Compare that the growth and development of A.solani mycelia is normal, and mycelia is smooth and even thickness, interval is clear, and mycelium is thin
Born of the same parents' Dissolve things inside is evenly dispersed, structural integrity.After HJG-5 fermentation liquor treatment, mycelia growth failure, lopsided swelling, thickness is uneven, out
Phenomena such as existing cytoplasm aggregation, extravasation.
3.5.2 to the influence of A.solani cell leakage
When mycelial cell membrane permeability changes, and intracellular organic matter exosmoses, the variation of conductivity value will cause.Therefore often pass through
Conductivity value is measured, to reflect influence of the antibacterial substance to thallus membrane permeability.Referring to Figure 17, with the extension of time, each processing
Conductivity value there is the trend increased, and ascensional range and be added ferment filtrate amount it is proportional, and in the conductivity of CK
Increasing degree degree very little, curve are gentle.In the same time, conductivity value is positively correlated with Filtrate.Therefore, it can obtain
Know, Metabolite is affected to tomato early blight bacterium cell leakage, increase and time with ferment filtrate concentration
Extension, the aggravation of this extent of the destruction.
3.6 HJG-5 strain fermentation filtrate growth-promotings and Biocontrol Effect measure
3.6.1 to the influence of Tomato Seeds Germination
The GI of tomato seeds is in the situation fallen after rising with the increase of ferment filtrate concentration, referring to Figure 18, wherein 20X,
The diluted fermentation liquid of 50X and 80X remarkably promotes effect to seed sprouting, sends out after the diluted fermentation liquor treatment tomato seeds of 20X
Bud index is 49.07, compared to the 42.03 of control, improves 16.75%, when extension rate is gradually increased, facilitation subtracts
Weak, 100 times of fermentation liquid dilution treated GI and CK are without significant difference, and the sprouting after the seed soaking of the fermentation liquid of high concentration to seed
Inhibition trend is presented.
3.6.2 to the influence of Growth of Tomato Seedling
Referring to Figure 19, after various concentration fermentation liquid root irrigation, the growing state of tomato seedling, when fermentation liquid concentration exists
When 100X-20X dilutes, tomato seedling plant height, root long, fresh weight, dry weight increase with the increase of fermentation liquid concentration, especially with 20X
The effect of dilution is best, has been respectively increased 29.0%, 20.4%, 24.9%, 35.9% compared with CK group, and with fermentation liquid concentration
Increase, growth-promoting functions weaken, and inhibiting effect gradually has enhancing, and fermenation raw liquid has the indices of plant aobvious relative to CK
Land inhibiting effect, it follows that bacterial strain fermentation liquor has certain facilitation in low concentration to plant development, and highly concentrated
When spending, then the development of plant is restricted, referring to fig. 20.
3.6.3 in vitro preventive effect
3.6.3.1 preventive effect of the HJG-5 strain fermentation filtrate to excised leaf A.solani
HJG-5 bacterial strain fermentation liquor on excised leaf to the preventive effect of A.solani, referring to table 8, the fermentation liquid of various concentration
Start to fall ill after thering is A.solani on excised leaf different degrees of preventive effect, the tomato leaf of CK to be inoculated with pathogen 1 day, 4d
Lesion area is gradually expanded to 485.10mm afterwards3, the blade control efficiency for spraying fermentation liquid stoste is best, and lesion area is only
122.33mm3, inhibiting rate 80.60%, when increasing fermentation liquid extension rate, preventive effect is gradually decreased, anti-with fermenation raw liquid
Imitate significant difference, referring to fig. 21.
Control efficiency of the 8 HJG-5 strain fermentation filtrate of table to excised leaf A.solani
3.6.3.2 preventive effect of the HJG-5 strain fermentation filtrate in vitro fruit A.solani
Various concentration ferment filtrate prevents and treats the inhibitory effect significant difference of A.solani referring to table 9 in vitro fruit
Effect is minimum for the diluted fermentation liquid of 20X, and the bacteriostasis rate to A.solani is 44.34%, and the fermentation liquid of high concentration, from
There are apparent preventive effect, the optimal fermentation liquid stoste of control efficiency to A.solani on body tomato fruit, bacteriostasis rate reaches 82.96%.
Control efficiency of the 9 HJG-5 strain fermentation filtrate of table in vitro fruit A.solani
The initial gross separation of 3.7 Substances and biological activity determination
3.7.1 the screening of best extractant
Substance in HJG-5 strain fermentation filtrate is slightly mentioned using the organic solvent of 5 kinds of opposed polarities, is revolved
Each organic phase and water phase are measured after inspissation contracting to the bacteriostatic activity of A.solani, referring to table 10, the organic phase and water of five processing
Xiang Junyou different degrees of bacteriostasis, wherein the fungistatic effect difference of organic phase and water phase is most when using n-butanol as extractant
Greatly, organic phase bacteriostasis rate is 93.12%, water phase 43.39%.With the polar decrease of organic solvent, each extractant organic phase
Bacteriostatic activity gradually decrease, the bacteriostatic activity of petroleum ether extraction liquid is only 30.03%, show the active material be polarity it is larger
Liposoluble substance therefore select n-butanol for optimal extractant.
The bacteriostatic activity of 10 bacterial strain HJG-5 different organic solvents extract of table
3.7.2 silica gel column chromatography separates Substance
Fermentation liquid obtains active material crude extract after extracting n-butyl alcohol, through petroleum ether, chloroform, methanol after wet process loading
After the gradient elution of different ratio, every pipe 20mL is collected into 60 pipes altogether, after TlC is detected, merges Rf (Rf value) identical stream
It there are 10 components after point, after each component is concentrated under reduced pressure, its bacteriostatic activity to A.solani measured using inhibition zone method, join
See Figure 22.The fungistatic effect of component 8 is best, antibacterial circle diameter 18.63mm, and 4,5 and 10 couples of A.solani of component almost without
Bacteriostatic activity.
4 conclusions
The separation screening of raw Antagonistic Actinomycetes and identification in 4.1 Chinese prickly ashes
The present invention isolates to obtain 11 plants of endogeny rayungus from each tissue site of Chinese prickly ash plant using tissue homogenate method, leads to
It crosses tablet face-off method and the screening of fermentation liquid growth rate method obtains 1 plant of HJG-5 bacterial strain with fine bacteriostatic activity, pass through measurement
Its antimicrobial spectrum can especially inhibit the growth of M.laxa and A.solani, viable bacteria pair it is found that the bacterial strain antimicrobial spectrum is wider well
For its inhibiting rate 70% or more, fermentation liquid then has 80% or more bacteriostasis rate to it, and the bacteriostatic activity of generally metabolite is wanted
Higher than viable bacteria thallus, therefore also it is inferred that HJG-5 bacterial strain generates Substance by metabolism to inhibit the life of pathogen
It is long, and be the bacterial strain with good Biocontrol Potential.
By strain morphology feature, cultural characteristic, Physiology and biochemistry identification and bacterial strain 16SrDNA sequencing analysis, by bacterial strain
HJG-5 is accredited as cortex cinnamomi maroon streptomycete, but the bacterial strain can use lactose, mannitol, glycerol, synthesizes No. 1 in Gao Shi and looks into
The upper growing pullets of family name are weak, and can then grow well on PDA, and observe not on 8 kinds of cultural characteristics used observation culture medium
To soluble pigment, this is retouched with the cortex cinnamomi maroon streptomycete streptomyces cinnamocastaneus's of Shen Mei hair tonic table
Difference is stated, on the other hand, HJG-5 strain isolation is from the root of Chinese prickly ash plant, and the streptomyces of Shen Meisheng
Cinnamocastaneus bacterial strain is then located away from soil, therefore speculates that endogeny rayungus strain HJG-5 is initially soil actinomycete
A part, by long-term evolution, into the root of plant, because of the change of living environment, itself physicochemical property changes.
The research of 4.2 HJG-5 strain fermentation conditions
The present invention filters out optimal basal medium PD from for 5 kinds of culture mediums of examination, and is filtered out most with single_factor method
The good source C maltose and optimum N source tryptone；The condition of culture of optimum bacterial strain is filtered out using Orthogonal Optimization are as follows: turn
Fast 180rmin-1, pH8, liquid amount 125mL/250mL, inoculum concentration 8%, fermentation time 9d.Culture medium after optimization is matched
Side and fermentation condition carry out biological activity determination, it is found that it significantly improves the bacteriostatic activity of A.soLani.
4.3 HJG-5 Metabolite physicochemical properties and antimicrobial mechanism
For the physicochemical property for exploring Substance, various stability tests are carried out to bacterial strain fermentation liquor, the results showed that,
HJG-5 Metabolite has preferable thermal stability, and 80 DEG C or less bacteriostasis rates maintain 80% or so, in neutral and alkalescent
It is highly stable under environment；Antibacterial substance does not change bacteriostatic activity through ultraviolet irradiation；Under the conditions of 4 DEG C, storage stability is good, 90d
When, the 85.15% of original fermentation liquid bacteriostatic activity can be reached to the inhibiting rate of A.solani.Know the antibacterial work that the bacterial strain generates
Property substance stable in physicochemical property.
The shadow that the present invention grows A.solani mycelia using optical microscope inspection HJG-5 bacterial strain Substance
It rings, while determining its influence to hyphal cell membrane permeability.The result shows that after the processing of HJG-5 bacterial strain fermentation liquor, A.solani
There is phenomena such as bending fold, lopsided swelling, cytoplasm aggregation, extravasation in the Hyphal form of mycelia and normal development ratio.Pass through measurement
The conductivity value of pathogen cultural hypha liquid is it is found that the addition of fermentation liquid increases conductivity value, with fermentation liquid concentration and place
The reason time is positively correlated, i.e. hyphal cell film is destroyed, intracellular organic matter extravasation, this result kissing also arrived with mycelia microexamination
It closes.
4.4 HJG-5 bacterial strain fermentation liquor growth-promotings and Biocontrol Effect
The present invention passes through the measurement that Tomato Seeds Germination, growth of seedling influences to various concentration HJG-5 bacterial strain fermentation liquor
It is found that fermentation liquid can promote seed sprouting and plant strain growth in a certain concentration, effect is best when being diluted with 20X, processing
Seed GI improves 16.75% relative to CK afterwards, and tomato seedling plant height, root long, fresh weight, dry weight also have significant growth compared with CK group.
The present invention using stabs inocalation method measurement HJG-5 bacterial strain fermentation liquor in vitro preventive effect, find its ferment filtrate kind
Fine to the fungistatic effect of A.solani on eggplant excised leaf and fruit, fermenation raw liquid can achieve 80% or more bacteriostasis rate.
By the growth-promoting ability and biological and ecological methods to prevent plant disease, pests, and erosion ability of HJG-5 bacterial strain, it is known that it has good Biocontrol Potential, has in control of plant disease very well
Application prospect.
The initial gross separation of 4.5 HJG-5 bacterial strain Substances and biological activity determination
The present invention carries out Substance in HJG-5 strain fermentation filtrate using the organic solvent of 5 kinds of opposed polarities
Slightly mention, the results showed that, n-butanol can extract Substance to greatest extent, i.e., the active material is the biggish liposoluble of polarity
Property substance.10 groups are obtained after the separation of silicagel column gradient elution, TLC detection in the active material crude extract being obtained by extraction
Point, its bacteriostatic activity to A.solani is measured using inhibition zone method, wherein the bacteriostatic activity of component 8 is best, antibacterial circle diameter
For 18.63mm.The bacteriostatic activity for the high-efficiency broad spectrum that Chinese prickly ash endogeny rayungus HJG-5 is shown shows that it is latent with stronger biological and ecological methods to prevent plant disease, pests, and erosion
Power.
It should be appreciated that specific embodiment described above is only used for explaining invention, it is not intended to limit the present invention.By sending out
The obvious changes or variations that bright spirit is extended out are still in the protection scope of this invention.
Claims (10)
1. a kind of Chinese prickly ash endogeny rayungus HJG-5, which is characterized in that the HJG-5 bacterial strain is preserved in Chinese microorganism strain guarantor
Hide administration committee's common micro-organisms center, preservation day: 2017.11.30, preservation registration number 14969.
2. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 1, which is characterized in that the HJG-5 bacterial strain is classified as
Cortex cinnamomi maroon streptomycete.
3. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 1, which is characterized in that the HJG-5 bacterial strain is in swallow
2-3d is cultivated on flour agar medium, bacterium colony surface is smooth white, and the aerial hyphae of white is begun with after cultivating to 6d
It is grown from colony edge, starts to gradually become light grey after 8d；Colonial morphology is cone protuberance, colony edge in incubation
The concentric wheel stripe for having 4-6 to enclose, is with dizzy shape earth side outside；By microexamination, aerial hyphae is flourishing, branch is more, substrate mycelium
Without tabula, not broken.
4. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 1 or 2, which is characterized in that the HJG-5 bacterial strain
Cultural characteristic be, in ISP2, for bacterium colony surface elevation in fine and close suede powdery, aerial hyphae is flourishing, is on PDA and nutrient agar
White or canescence, substrate mycelium brown color or yellowish-brown；Aerial hyphae is light gray on ISP5, and substrate mycelium is light light yellow
Color；In ISP4, raw mycelia is thin on Cha Shi and Gause I culture medium, be in very light lime color, no substrate mycelium more；In ISP3
On hardly grow.
5. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 3, which is characterized in that the HJG-5 bacterial strain can produce
Melanin can make part of gelatin liquefy, and nitrate reduction is the positive, and Starch Hydrolysis, milk is solidified and peptonized, and not generate H2S, it is fine
It is not grown on dimension element；Cannot utilize starch, xylose, arabinose and fructose, can utilize glucose, maltose, sucrose, lactose,
Mannitol and glycerol.
6. a kind of Chinese prickly ash endogeny rayungus HJG-5 described in claim 1-5 is living in production growth promoting activity substance, broad-spectrum antibacterial
Purposes in terms of property substance.
7. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 6 is in production growth promoting activity substance, broad-spectrum antibacterial
Purposes in terms of active material, which is characterized in that the growth promoting activity substance, broad-spectrum antibacterial active material are from described
Fermentation liquid after HJG-5 strain fermentation culture.
8. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 7 is in production growth promoting activity substance, broad-spectrum antibacterial
Purposes in terms of active material, which is characterized in that the condition of culture of the HJG-5 strain fermentation culture is revolving speed 180rmin-1, pH=8, liquid amount 125mL/250mL, inoculum concentration 8%, cultivation temperature is 28 DEG C, incubation time 9d；Fermentation medium makes
Carbon source is maltose, nitrogen source is tryptone, and maltose: the proportion of tryptone is 2:1.
9. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 8 is in production growth promoting activity substance, broad-spectrum antibacterial
Purposes in terms of active material, which is characterized in that the fermentation liquid has preferable thermal stability, and 80 DEG C or less bacteriostasis rates maintain
It is highly stable under neutral and weakly alkaline environment 80% or so；Do not change bacteriostatic activity through ultraviolet irradiation；Under the conditions of 4 DEG C, storage
It deposits and has good stability, after placing 90d, the 85.15% of original fermentation liquid bacteriostatic activity can be reached to the inhibiting rate of A.solani.
10. a kind of Chinese prickly ash endogeny rayungus HJG-5 according to claim 9 is in production growth promoting activity substance, wide spectrum suppression
Purposes in terms of bacterium active material, which is characterized in that described 20 times of fermentation liquid dilution preferable compared with fruit, at 20 times of diluted fermentation liquids
Germination index improves 16.75% after reason tomato seeds；Tomato seedling plant height, root long, fresh weight, dry weight are respectively increased
29.0%, 20.4%, 24.9%, 35.9%.
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Application publication date: 20181207 |