CN105400702B - A kind of friendship branch acremonium bacterial strain and its application - Google Patents
A kind of friendship branch acremonium bacterial strain and its application Download PDFInfo
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Abstract
A kind of friendship branch acremonium bacterial strain（Acremonium implicatum）CQBBW8, deposit number be CGMCC No.11440, the rDNA-ITS sequences of the bacterium andAcremonium implicatumThe sequence homology of isolate LR2 is up to 98%.Present invention friendship branch acremonium bacterial strain CQBBW8 isolated from citrus red mite (crm) body, the production traits is good, growth is rapid, and the colony diameter after cultivating 15 days is up to 5.083cm；The strong, good disinsection effect to the insecticidal activity of various pests simultaneously, 106A/mL kills the 3 instar larvae death rate of papilio xuthus Linnaeus and is up to 100%, kills the aphid death rate and is up to 100%, kills the citrus red mite (crm) death rate and be up to 100%.The disinsection fungal preparation produced using the bacterial strain, has efficiently controlled various pests population quantity.
Description
Technical field
The present invention relates to a kind of microorganism new strains, and in particular to a kind of friendship branch acremonium bacterial strain and its application belong to raw
Object pesticide field.
Technical background
From after invention chemical pesticide, chemical pesticide is increased with annual 5% surprising speed.Although in the past few decades
In, chemical pesticide plays important function, but a large amount of uses of chemical pesticide in agricultural is as volume increase and control of insect, brings
Serious " 3R " problem (i.e. drug resistance Resistance, pesticide residue Residence, then wildness Resurgence), also band
A series of ecological environment problem is carried out.It is long-term largely to cause resistanc pest to increase using chemical pesticide, it is 10 years especially nearly
Come, the multiple pest such as bollworm, aphid, diamondback moth, prodenia litura increases the drug resistance of pyrethroids class, organic phosphates chemical pesticide
Hundreds of or even thousands of times are added.The application of chemical pesticide, makes pesticide residue in agricultural products increase, and has seriously polluted the environment, danger
And mankind Jiankang and life.As the improvement of people's living standards, requirement of the people to environment is higher and higher, therefore force people
Find a kind of control of insect alternative.
With the benign development in the growing and market of non-polluted farm product demand, microbe insecticide control evil
The cry of worm is higher and higher, therefore there is an urgent need for research and development novel microbial insecticide and its industrialization key technologies.Since worm gives birth to
Fungi has economy and ecological significance to the mankind, thus is already concerned by people, especially recent decades, it is ground in the whole nation
The breadth and depth studied carefully all is reinforced.Entomogenous fungi type is more, and metabolic type is complicated, significant popular latent with safe and effective
The advantages that power, easy mass production, increasingly consequence is occupied in the biological control of pest.In the world it is many country
Scientific worker has carried out research extensively and profoundly to them, and up to the present, countries in the world have more than 50 kinds of fungi desinsection
Agent is registered, wherein with the most species of beauveria bassiana.
Invention content
The purpose of the present invention is to provide the good friendship branch acremonium bacterial strain CQBBW8 of a kind of Insecticiding-miticiding, the production traits.
Bacterial strain of the present invention is good by one plant of production traits isolated on citrus red mite (crm) body, has to Insecticiding-miticiding
Friendship branch acremonium (Acremonium implicatum) bacterial strain CQBBW8 of high virulence, on 09 22nd, 2015 in China
Microbiological Culture Collection administration committee common micro-organisms center (CGMCC) preservation, address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica, deposit number：CGMCC No.11440.
The present invention hands over branch acremonium bacterial strain CQBBW8 to be seeded on PDA plate, is shown in bacterium colony in PDA culture medium and slowly gives birth to
It is long, PDA culture medium growth 10d at a temperature of 25 DEG C, colony diameter 40-45mm, aerial hyphae milky, villiform, then
Will present among bacterium colony slowly is orange red, moist, there is pungent smell；The bacterium colony back side is orange-yellow；Conidium length is oval
Shape, fusiform, chain life or fasciation；There is chlamydospore generation.
The technical scheme is that by the classification position of acquisition citrus red mite (crm) → isolate and purify bacterial strain → identification bacterial strain
→ determine that condition of culture → tablet of bacterial strain and solid fermentation → xerospore powder collection → insecticidal effect measure, and sieve repeatedly
Friendship branch acremonium (Acremonium implicatum) bacterial strain that one plant selected has high insecticidal activity, the production traits good
CQBBW8。
In order to further increase the growth potential of bacterial strain of the present invention and its produce spore character, to be more suitable for spore batch production,
It is preferably PDA- sugar culture-mediums (pH 8.0), 121 DEG C of high pressure moist heat sterilization 30min to strain fermentation culture medium of the present invention；It is natural
1: 10 (bacterium solution is pressed after cooling：PDA, V/W) ratio inoculation hand over branch acremonium bacterium CQBBW8 25 DEG C of bacterium solution fermentation tank cultures 3 days after with
Mixing in rice medium (such as in order to reduce cost, low value old rice culture medium may be used), is placed in 25 DEG C of fermenting cellar
Static fermentation collects conidia powder in 10 days, and sporulation quantity is up to 5 × 1010/g.The clear PDA- sugar culture-mediums in this field, in fermentation process
Sugared ingredient is added in PDA culture medium to ferment in mass production more completely.
A kind of sterilization, miticide farm chemical composition, it is characterised in that：It includes a kind of in cuprous oxide, Propineb, Fluoxastrobin
Or a variety of arbitrary combinations, with the conidial powder for handing over branch acremonium (Acremonium implicatum) CQBBW8.
A kind of miticide farm chemical composition, it is characterised in that：It includes clofentezine or/and Envidors, with friendship branch acremonium
The conidial powder of (Acremonium implicatum) CQBBW8.
The advantageous effect that the present invention hands over branch acremonium bacterial strain CQBBW8 specific as follows：
Present invention friendship branch acremonium bacterial strain CQBBW8 isolated from citrus red mite (crm) body, the production traits are good, raw
Long rapid, sporulation quantity is up to 5 × 10 when cultivating 20 days10/g；The strong, good disinsection effect to the insecticidal activity of various pests simultaneously, 106
A/mL spore suspensions kill the 3 instar larvae death rate of papilio xuthus Linnaeus and are up to 100%, kill the aphid death rate and are up to 100%, kill
The citrus red mite (crm) death rate is up to 100%.The disinsection fungal preparation produced using the bacterial strain, has efficiently controlled various pests kind
Group's quantity.
Description of the drawings
Fig. 1：CQBBW8 bacterial strains conidium and conidial fructification form；
Fig. 2：Hand over branch acremonium (Acremonium implicatum) CQBBW8 bacterium colonies front；
Fig. 3：Hand over branch acremonium (Acremonium implicatum) the CQBBW8 bacterium colonies back side；
Fig. 4：Influence of 6 different carbon source of embodiment to handing over branch acremonium CQBBW8 mycelia to grow；
Fig. 5：Influence of 6 different nitrogen sources of embodiment to handing over branch acremonium CQBBW8 mycelia to grow；
Fig. 6：Influence of 6 different temperatures of embodiment to handing over branch acremonium CQBBW8 mycelia to grow；
Fig. 7：Influences of the 6 difference pH of embodiment to handing over branch acremonium CQBBW8 mycelia to grow；
Fig. 8：Embodiment 8 hands over the screening of branch acremonium CQBBW8 growth optimum mediums.
Specific implementation mode
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that following embodiment is only used
In invention is further explained, it should not be understood as limiting the scope of the invention, without departing substantially from spirit of that invention
In the case of essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.If
It does not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The separation of 1 bacterial strain of embodiment
By the 70% alcohol surface sterilization of the citrus red mite (crm) body of collected natural infection, it is put into 25 DEG C of incubators and protects
Wet culture 3-5 days, makes its insect surfaces grow Fresh spores；Aseptically, with the micro Fresh spores of oese picking
In the flat lining out of PDA culture medium containing 100 μ g/ml ampicillins；The culture dish crossed overturning is positioned over 25 DEG C
It is cultivated in insulating box；It selects the typical case grown after 3-4 days and the bacterium colony without miscellaneous bacteria, bacterium is carried with acicula picking is moved in colony edge
One fritter of culture medium of silk, is transferred to test tube slant culture medium central, 25 DEG C of constant temperature incubations 10 days.
The identification of 2 bacterial strain of embodiment
(1) Morphological Identification
By (CQBBW8) inoculation being separated on PDA plate, 25 DEG C of culture observation colony morphology characteristics produce spore knot
Structure and Spore shapes and size.Referring to Fig. 1,2,3, as a result show that bacterium colony is slow-growing, PDA culture medium 10d at a temperature of 25 DEG C
A diameter of 40-45mm, aerial hyphae milky, villiform, then slowly will present among bacterium colony it is orange red, it is moist, have
Pungent smell；The bacterium colony back side is orange-yellow.Conidium oblong, fusiform, chain life or fasciation, there is chlamydospore generation.
Conidiophore is rare, conidium spindle, glossy clear, 6.5-9.8 × 2.0-3.2 μm of size.Multiform is few at pionnote
Number form generates faint rufous at dry short chain, a large amount of spore aggregations.
(2) ITS sequence amplification, sequencing and the Molecular Identification of CQBBW8 bacterial strains
Using ITSl and ITS4 as primer (referring to table 1), the sequence of 1 treaty 600bp is amplified from the strain gene group DNA
Row, PCR product gained sequence after sequencing are compared by Blast and existing nucleic acid sequence in GenBank, as a result table
Bright, the rDNA-ITS sequence results of the bacterial strain are shown in SEQ ID No.1 and Acremonium implicatum isolate
The sequence homology of LR2 is up to 98%.
Table 1 hands over the amplification condition and system of branch acremonium (A.implicatum)
Embodiment 3CQBBW8 is to the 3 active measurement of instar larvae indoor insecticidal of papilio xuthus Linnaeus
Scraping is grown on the CQBBW8 mycelia of 7d on PDA plate in the sterile water of 0.02% tween, and concussion is collected by filtration
Spore suspension is configured to a concentration of 2.47 × 106A/mL spore suspension 10mL are uniformly sprayed at the citrus phoenix of field collection
On 3 instar larvae of butterfly, each processing 30,3 repetitions.25 DEG C of moisturizing cultures of sample, control group spray 10mL and contain 0.02% tween
Sterile water, count the death rate day by day.
Experimental result shows, 3 instar larvae of papilio xuthus Linnaeus is all dead when connecing bacterium 1 day, the death rate 100%.
Embodiment 4CQBBW8 is to the active measurement of aphid indoor insecticidal
Scraping is grown on the CQBBW8 mycelia of 7d on PDA plate in the sterile water of 0.02% tween, and concussion is collected by filtration
Spore suspension is configured to a concentration of 2.05 × 106A/mL spore suspension 20mL, be uniformly sprayed at field acquisition is covered with tangerine
In the citrus tender tip of aphid, 3 branch are each handled, 100 or more citrus aphids on each branch, 25 DEG C of moisturizing cultures of sample, control
Group sprinkling 20mL contains the sterile water of 0.02% tween, counts the death rate day by day.
Experimental result shows, citrus aphid is all dead when connecing bacterium 1 day, the death rate 100%.
Embodiment 5CQBBW8 is to the active measurement of citrus red mite (crm) indoor insecticidal
The CQBBW8 mycelia that scraping is grown on 7d on PDA plate is dissolved in the sterile water of 0.02% tween, and concussion filtering is received
Collect spore suspension, is each configured to 6 spore concentration gradients：1.27×103A/mL, 1.27 × 104A/mL, 1.27 × 105
A/mL, 1.27 × 106A/mL, 1.27 × 107A/mL, 1.27 × 108A/mL.The spore suspension 10mL of each concentration is uniform
It being sprayed on 5 leaves, connects 50 citrus red mite (crm)s on every leaf, control group sprays the sterile water that 10mL contains 0.02% tween,
Day by day the death rate is counted, referring to table 2,3.
Virulence regression equation of the table 2CQBBW8 bacterial strains to citrus red mite (crm)
| Strain | Virulence regression equation | LC50/ (a spore/mL) | Related coefficient (r) |
| Acremonium CQBBW8 | Y=3.3684+0.2680X | 1.2×106 | 0.9291 |
Virulence regression equation of the table 3CQBBW8 bacterial strains to citrus red mite (crm)
| Strain | Virulence regression equation | LT50/(d) | Related coefficient (r) |
| Acremonium CQBBW8 | Y=2.8679+2.9863X | 5.1759 | 0.9893 |
Embodiment 6CQBBW8 bacterial strain sporulating characters
(1) branch acremonium (Acremonium implicatum) is handed over to grow most suitable carbon source screening
On the basis of looking into Bi Shi culture mediums, use respectively the glucose of equivalent, starch, mannitol, inositol, glycerine, lactose,
Maltose is made into the culture medium of different carbon source as carbon source, at 25 DEG C, is cultivated in illumination box under the conditions of L: D=12: 12
15 days.After strain growth 3 days, colony growth diameter is measured daily with crossing method.The shadow that different carbon source grows bacterium colony
It rings result and sees Fig. 4.Acremonium can be grown on various carbon source culture mediums, which can profit to monosaccharide and disaccharide, polysaccharide and alcohol
With wherein sucrose, mannitol grow mycelia the most advantageous, and the influence difference that other carbon sources grow mycelia is little.
(2) the most suitable nitrogen source screening of branch acremonium (Acremonium implicatum) CQBBW8 growths is handed over
On the basis of looking into Bi Shi culture mediums, peptone, potassium nitrate, ammonium chloride, ammonium nitrate, the urea of equivalent is used to make respectively
It is made into the culture medium of different nitrogen sources for nitrogen source, at 25 DEG C, is cultivated 12 days in illumination box under the conditions of L: D=12: 12.Bacterial strain is given birth to
After 3 days long, colony growth diameter is measured daily with crossing method.Fig. 5 is shown in the influence that different nitrogen sources grow mycelia.In nitrogen
In terms of source, nitrate nitrogen, ammonium nitrogen, organic nitrogen can be such that acremonium grows.Wherein, peptone is most beneficial for the growth of mycelia, chlorine
Change growth and production spore that ammonium is most disadvantageous in mycelia.The influence that various nitrogen sources grow mycelia is followed successively by：Peptone > potassium nitrate >
Urea > ammonium nitrate > ammonium chlorides.
(3) branch acremonium (Acremonium implicatum) CQBBW8 growth optimum temperature screenings are handed over
7 are chosen for trying temperature：10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, filter out suitable mycelia growth
Optimum temperature.The CQBBW8 bacterial strains that 7 days are grown in PDA culture medium are transferred to PDA culture medium respectively in a manner of bacteria cake
Center each handles 5 repetitions, is cultivated in the incubator of 7 different temperatures, after 5 days, 10 days, using crossing method
Measure the diameter of bacterium colony.With SPSS software analysis datas, optimum growth temp is obtained.
The experimental results showed that it is 30 DEG C to hand over the optimum growth temp of branch acremonium, the colony diameter after growing 10 days reaches
2.812cm.From 10 DEG C to 30 DEG C, with the rising of temperature, colony diameter gradually increases, and is not grown in 35 DEG C, 40 DEG C.See Fig. 6.
(4) screening of branch acremonium (Acremonium implicatum) CQBBW8 optimum growh pH value is handed over
7 are chosen for trying pH：5,6,7,8,9,10,11, therefrom select the optimum temperature of suitable mycelia growth.It will be in PDA
The PDA culture medium center that 7 days CQBBW8 bacterial strains are transferred to 7 difference pH respectively in a manner of bacteria cake is grown on culture medium, often
5 repetitions of a processing, are cultivated in 25 DEG C of incubators, and after 5 days, 10 days, the diameter of bacterium colony is measured using crossing method.With
SPSS software analysis datas obtain growth optimum growh pH；See Fig. 7.
From figure 7 it can be seen that the colony diameter that CQBBW8 is grown under condition of different pH is without significant difference, so its is right
The variation of pH is insensitive.But when pH value is 8, growth is relatively most fast, and colony diameter reaches 3.02cm after 10 days.
Embodiment 7 hands over branch acremonium (Acremonium implicatum) CQBBW8 compatibilities fungicide, acaricide screening
(1) branch acremonium (Acremonium implicatum) CQBBW8 compatibility Screening of Fungicide is handed over
11 kinds of common fungicide in field are had chosen in total to be used as trying fungicide：86.2% cuprous oxide WP, 40% fluorine
Silicon azoles EC (DuPont Corporation), azoles ether Carbatene, 10% difenoconazole DP (Hainan Boshiway Agricultural Chemical Co., Ltd.),
43% Tebuconazole SC (Baeyer crop science (China) Co., Ltd), 20% thiophanate-methyl SC (the general letter agrochemical shares of Shenzhen promise
Co., Ltd), 50% copper 8-hydroxyquinolinate WP (Zhejiang Hisun Chemical Co., Ltd), 70% Propineb WP (Baeyer crop science (in
State) Co., Ltd), 25% Fluoxastrobin SC (Syngenta Co., Ltd of Britain), carbendazim, pyraclostrobin, be respectively prepared set
The culture medium of fixed pesticide concentration, and utilize the virulence of growth rate method measurement fungicide.According to the property of all fungicide, divide
It does not set for trying concentration.
Culture medium configures：It takes the former pesticides of 1g (1mL) to be configured to the pesticide mother liquor of 100mL with sterile water, takes corresponding concentration institute
Need the pesticide mother liquor (sterile water is as a contrast) of volume and 100mL melted PDA culture medium be uniformly mixed be made it is different dense
The culture medium of degree averagely pours into 3 (i.e. 3 repetitions) a diameter of 9cm and sterile culture dish.With the punching of a diameter of 5mm
Device is beaten on the bacterium colony for handing over branch acremonium takes band bacterium culture medium column (bacteria cake), and sterile or disinfection transfer needle or metal tweezers is used in combination
By culture plate center after bacteria cake access condensation.Inoculated and cultured ware is placed in 28 DEG C of constant incubators and cultivates, and is handed over cross after 7 days
Fork method measures colony diameter.It is for statistical analysis to each pharmacy test result with DPS softwares, obtain poison of each medicament to bacterial strain
Concentration (EC in power regression equation, inhibition50) and related coefficient (r).
As seen from Table 4, the compatibility of pyraclostrobin WP and friendship branch acremonium is worst, is secondly carbendazim, Flusilazole, penta
Azoles alcohol etc..Cuprous oxide and the compatibility of friendship branch acremonium are best, are secondly Propineb, Fluoxastrobin etc..
Virulence regression equation of the 4 11 kinds of fungicide of table to friendship branch acremonium (Acremonium implicatum)
| Medicament | Virulence regression equation | EC50/(mg./mL) | Related coefficient (r) |
| 86.2% cuprous oxide WP | Y=3.7888+0.0436*X | 6.24E+27 | 0.9004 |
| 70% Propineb WP | Y=4.1648+0.3350*X | 311.0357 | 0.9080 |
| 25% Fluoxastrobin SC | Y=4.3151+0.3199*X | 138.3600 | 0.9363 |
| 10% difenoconazole DP | Y=4.0692+0.4746*X | 91.5030 | 0.9014 |
| Azoles ether Carbatene EW | Y=4.3536+0.4073*X | 38.6537 | 0.9141 |
| 20% thiophanate-methyl SC | Y=4.4640+0.4115*X | 20.0683 | 0.9005 |
| 50% copper 8-hydroxyquinolinate WP | Y=3.6342+1.1287*X | 16.2220 | 0.9002 |
| 43% Tebuconazole SC | Y=4.4334+0.5598*X | 10.2828 | 0.9466 |
| 40% Flusilazole EC | Y=4.7628+0.2978*X | 6.2602 | 0.9191 |
| Carbendazim WP | Y=4.8363+0.5073*X | 2.1026 | 0.9009 |
| Pyraclostrobin WP | Y=4.9637+0.2056*X | 1.5021 | 0.9123 |
Remarks：Dosage form code：Wettable powder is WP, water dispersible granules WDG, aqueous emulsion EW, suspending agent SC, breast
Oil is EC, pulvis DP, aqua AS
(2) screening of branch acremonium (Acremonium implicatum) CQBBW8 compatibility acaricides is handed over
5 kinds of common acaricides in field are had chosen in total to be used as trying acaricide：124g/L Envidors SC, 20% clofentezine
SC (Nong Ke limited liability companies of Guangdong CTC (Centell Technology Corporation)), 5% fenpyroximate, 15% pyridaben, 22.4% spiral shell worm ethyl ester.According to acaricidal
Property, each acaricide set 6 for trying concentration.
Culture medium configures：It takes the former pesticides of 1g (1mL) to be configured to the pesticide mother liquor of 100mL with sterile water, takes corresponding concentration institute
Need the pesticide mother liquor (sterile water is as a contrast) of volume and 100mL melted PDA culture medium be uniformly mixed be made it is different dense
The culture medium of degree averagely pours into 3 (i.e. 3 repetitions) a diameter of 9cm and sterile culture dish.With the punching of a diameter of 5mm
Device is beaten on the bacterium colony of the dendritic cladosporium sp of bud takes band bacterium culture medium column (bacteria cake), and sterile or disinfection transfer needle or metal is used in combination
Tweezers are central by culture plate after bacteria cake access condensation.Inoculated and cultured ware is placed in 28 DEG C of constant incubators and cultivates, with ten after 7 days
Word interior extrapolation method measures colony diameter.Inhibiting rate (%)=control bacterium colony of (control colony diameter-processing colony diameter) × 100/ is straight
Diameter
Growth inhibition ratio of the 55 kinds of acaricides of table to friendship branch acremonium
As seen from Table 5, the compatibility of 20 clofentezines and friendship branch acremonium is best, is secondly 124g/L Envidors, other 3
Acaricide inhibiting rate reduces be used cooperatively number as far as possible close to 20%.
Embodiment 8CQBBW8 strain growth optimal mediums screen
5 kinds of culture mediums are selected, including：(1) PDA culture medium；(2) starch agar medium；(3) Bi Shi cultures are looked into
Base；(4) SDAY culture mediums；(5) SEA culture mediums are cultivated under the conditions of temperature is 25 DEG C, and experimental result is shown in Fig. 8.
The experimental results showed that hand over branch acremonium (Acremonium implicatum) CQBBW8 when temperature is 25 DEG C, 5
It can be grown on kind culture medium, according to Fig. 8 it can be seen that different culture media is different to the growth effect of acremonium, wherein in PDA
Growth rate is most fast in culture medium, followed by looks into Bi Shi culture mediums, and bacterium colony growth is most slow on SDAY culture mediums.Hand over branch acremonium
After (Acremonium implicatum) CQBBW8 cultivates 15 days in PDA culture medium, colony diameter is up to 5.083cm, production
Spore amount is up to 108/cm2。
Claims (7)
1. a kind of friendship branch acremonium bacterial strain, it is characterised in that：The bacterial strain is to hand over branch acremonium bacterial strain (Acremonium
Implicatum) CQBBW8, deposit number are CGMCC No.11440.
2. bacterial strain as described in claim 1, it is characterised in that：The TIS1-ITS4 sequences of the bacterial strain are SEQ ID NO.1.
3. bacterial strain as claimed in claim 1 or 2, it is characterised in that：The suitable production spore temperature of the bacterial strain is 30 DEG C, suitable for producing spore
PH is 8.0.
4. bacterial strain as claimed in claim 1 or 2, it is characterised in that：The bacterial strain is PDA culture medium suitable for product spore culture medium.
5. bacterial strain as claimed in claim 1 or 2 is in the fungi preparation for preparing prevention papilio xuthus Linnaeus larva, aphid or citrus red mite (crm)
In application.
6. a kind of sterilization, miticide farm chemical composition, it is characterised in that：It includes a kind of in cuprous oxide, Propineb, Fluoxastrobin or
A variety of combinations, it is described with the conidial powder for handing over branch acremonium (Acremonium implicatum) CQBBW8 bacterial strains
The deposit number of CQBBW8 bacterial strains is CGMCC No.11440.
7. a kind of miticide farm chemical composition, it is characterised in that：It includes clofentezine or/and Envidors, with friendship branch acremonium
The deposit number of the conidial powder of (Acremonium implicatum) CQBBW8 bacterial strains, the CQBBW8 bacterial strains is CGMCC
No.11440。
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