CN106479943A - The Java Isaria BE01 of one plant height effect preventing and treating fall webworms and its application - Google Patents
The Java Isaria BE01 of one plant height effect preventing and treating fall webworms and its application Download PDFInfo
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Abstract
The invention discloses the Java Isaria of one plant of preventing and treating fall webworms, its Classification And Nomenclature is Java Isaria（Isaria javanica）BE01, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.13192, and preservation date is on November 28th, 2016, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.The Java Isaria BE01 bacterial strain of the present invention is upper isolated from fall webworms larva bombys batryticatus, is confirmed by test, 1.0 × 104Under the spore concentration of individual/more than mL, the fall webworms to different worm ages are respectively provided with preferable virulence, infection rate more than 53.33%, 1.0 × 108Under the spore concentration of individual/mL, infection rate can efficiently prevent and treat fall webworms more than 73.33%.
Description
Technical field
The invention belongs to microbial technology field, and in particular to Java Isaria bacterial strain and its answering in terms of pest control
With.
Background technology
Fall webworms (Hyphandria cunea) also known as autumn curtain caterpillar, Qiu Mue, category Lepidoptera (Lepidor tera),
Arctiidae (Arctiidae), the high subfamily of lamp (Arctiinae), are all a kind of important insects in a lot of countries.The worm originates in
North America, late 1940s, travels to Europe and Asia drop by mankind's activity and delivery vehicle.Liaoning Province of China in 1979
Operation in Dandong Area finds fall webworms first, and which causes serious harm, travels to Beijing, Tianjin, Hebei, Shandong and Shanxi etc. again afterwards
Provinces and cities.At present, due to the harm of this invasive lepidoptera pest, huge in forestry and fruit tree or even agriculturally causing
Big loss.The host of this insect almost contain all of forest, fruit tree, ornamental plant, ornamental flower, vegetables, crops,
Herbal medicine and shrub.While the U.S. is high small in vain but heavy, and persistently takes food and cause harm, being therefore otherwise known as, " forest is smokeless
Fire ".
In recent years, the prevention and controls of fall webworms are mainly chemical prevention.Chemical prevention is mainly used in fall webworms children
Worm, and have preferable effect to the larva before 4 ages, the fall webworms decreased effectiveness to diffusion of scoring a goal.And Long-Time Service chemistry is killed
Worm agent, can bring serious environmental pollution, or even the impact mankind's is healthy.
The Natural Enemies of Insects of fall webworms is nibbled with Zhou Shi based on chalcid fly (Chouioia cunea Yang), infects fall webworms
Fungi only have beauveria bassiana (Beauveria bassiana), powder Isaria (Isaria farinose) to appear in the newspapers.But
Have no the report that fall webworms are prevented and treated using Java Isaria (Isaria javanica).
Content of the invention
Goal of the invention：For the deficiencies in the prior art, it is an object of the invention to provide a kind of efficient Java cluster
Spore bacterial strain, meets use demand.It is a further object of the present invention to provide the efficient Java Isaria bacterial strain prevents and treats fall webworms
Application.
Technical scheme：In order to realize foregoing invention purpose, the technical solution used in the present invention is：
The Java Isaria of one plant of preventing and treating fall webworms, its Classification And Nomenclature are Java Isaria Isaria javanica, protect
China Committee for Culture Collection of Microorganisms's common micro-organisms center is hidden in, deposit number is CGMCC No.13192, preservation
Date is on November 28th, 2016, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Application of the described Java Isaria BE01 in preventing and treating fall webworms.
Described application, is sprayed directly on fall webworms larva using the spore liquid of Java Isaria BE01 and is prevented
Control.
Described spore liquid and preparation method thereof is：From isolating and purifying in the Java Isaria BE01 bacterial strain that obtains, picking mycelia
It is inoculated into and is covered with the SDAY flat board of glassine paper, cultivate 20 days at 25 DEG C, scrapes the mycelia on glassine paper and spore is put into 0.1%
The Tween-80 aqueous solution in, be sufficiently stirred for concussion, filter, obtain spore mother liquor, spore concentration be adjusted to 1.0 × 104Individual/
ML～1.0 × 108Individual/mL.It is preferred that spore liquid concentration is 1.0 × 106Individual/mL～1.0 × 108Individual/mL.
The Java Isaria BE01 adopts Czapek's medium culture, cultivates 13 days under the conditions of 25 DEG C.
The present invention uses Natural Selection method, by gather natural susceptible insect, isolate and purify out bacterial strain, Fungal identification,
The steps such as Pathogenicity obtain one plant of Java Isaria Isaria javanica BE01 efficient to fall webworms.Java rod
The morphological feature of bundle spore BE01：As shown in Figure 1-Figure 3, on Czapek's medium flat board, bacterium colony irregular shape, white mycelium,
Spore layer color purplish grey, back are faint yellow, the visible coremium of Extending culture.Under microscope, mycelia is transparent, colourless, width 0.3-
1.2 μm, transparent smooth.On conidiophore raw 2-4 bottle obstruct the verticil to be formed, bottle metulae portion ellipse expands, upwards
It is tapered；Conidium is transparent smooth, and oblong, two ends are sharper.Molecular Identification carried out afterwards, phylogenetic tree construction,
Finally determine the bacterial strain for Java Isaria (Isaria javanica) BE01.
Beneficial effect：Compared with prior art, the Java Isaria BE01 bacterial strain of the present invention is from fall webworms larva bombys batryticatus
Upper isolated, confirmed by test, 1.0 × 104Fall webworms under the spore concentration of individual/more than mL, to different worm ages
Be respectively provided with preferable virulence, infection rate more than 53.33%, 1.0 × 108Under the spore concentration of individual/mL, infection rate exceedes
73.33%, can efficiently prevent and treat fall webworms.
Description of the drawings
Fig. 1 is the aspect graph that BE01 bacterial strain grows 13 days on Czapek's medium；
Fig. 2 is the conidial fructification figure of BE01 bacterial strain；
Fig. 3 is the conidium form figure of BE01 bacterial strain.
Specific embodiment
The present invention is further elaborated on reference to specific embodiment.Following embodiments are preferably implemented for the present invention
Mode, the present invention mainly elaborate the invention thought of the bacterial strain, and embodiment is not related one by one in instances, but not therefore
Limit the present invention, other any Spirit Essences away from the present invention and the change that is made under principle, modification, replacement, combine, simple
Change, equivalent substitute mode should be considered, be included in the present invention.
If the experimental technique used in following embodiments is conventional method nothing specified otherwise.Material used, reagent
Deng if nothing specified otherwise, all commercially obtaining：
For trying insect：Fall webworms (Hyphandria cunea) larva bombys batryticatus：Xiuzhou City, Anhui Province gathers；Fall webworms
(Hyphandria cunea) larva：Jiangsu Province Huai'an gathers.
SDAY culture medium prescription is as follows：1% yeast extract powder, 4% glucose, 1% tryptone, 2% agar, plus aseptic
Water is quantitative to 1L, and 121 DEG C sterilize 20 minutes.
Czapek's medium：0.2% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% 7 hydration sulphur
Sour magnesium, 0.001% green vitriol, 3% sucrose, 2% agar, plus sterilized water are quantitative to 1L, and 121 DEG C sterilize 20 minutes.
1 Java Isaria BE01 of embodiment is separated and is identified
Java Isaria BE01, is that the fall webworms larva bombys batryticatus collected from Anhui Suzhou is upper isolated.Method
As follows：From the upper picking mycelia of bombys batryticatus polypide, it is inoculated on the SD AY culture medium added with ampicillin and streptomycin sulphate and trains
Support, cultivate 5 days under the conditions of 25 DEG C.In the bacterium colony center picking mycelia for being formed, it is inoculated into and has added ampicillin and sulfuric acid strepto-
Purifying culture is carried out on the SDAY culture medium of element, and purifying culture is repeated 3 times.The fungal strain numbering that screening is obtained is BE01.
1) Morphological Identification：As Figure 1-3, by inoculation after purification to Czapek's medium, bacterium colony is irregular
Shape, white mycelium, spore layer color purplish grey, back are faint yellow, the visible coremium of Extending culture.In the micro- sem observation of Zeiss
Under, mycelia is transparent, colourless, 0.3-1.2 μm of width, transparent smooth.On conidiophore raw 2-4 bottle obstruct to be formed verticillate
Body, bottle metulae portion ellipse expand, and are tapered upwards；Conidium is transparent smooth, and oblong, two ends are sharper.
2) molecular biology identification
The extraction side of Java Isaria BE01 STb gene adopts CTAB method, obtains the DNA sample for extracting；With the bacterial strain for extracting
STb gene as gene magnification masterplate, using fungi EF1- α primer 983F (5 '-GCYCCYGGHCAYCGTGAYTTYAT-3 ') and
2218R (5 '-ATGACACCRACRGCRACRGTYTG-3 '), enters performing PCR amplification, and PCR primer recovery purifying, connection conversion are gone forward side by side
After row DNA sequencing, the sequence is as shown in SEQ ID NO.1.All Eukaryotic EF1- α sequences for having determined with GenBank
Row compare homology, build BE01 bacterial strain phylogenetic tree, as a result show BE01 bacterial strain and 1 (Isaria of KT225600.
Javanica) gather for a class, and its morphological feature meets the feature of Java Isaria (Isaria javanica), so can
To identify that BE01 bacterial strain, as Isaria javanica, is named as Java Isaria BE01.
2 Java Isaria BE01 preventing and treating fall webworms of embodiment
The preparation of spore suspension：From isolating and purifying in the Java Isaria BE01 bacterial strain that obtains, picking mycelium inoculation is arrived
It is covered with the SDAY flat board of glassine paper, cultivates 20 days at 25 DEG C, scrapes the mycelia on glassine paper and spore is put into 0.1% tell
In warm -80 aqueous solution, concussion is sufficiently stirred for, filters, obtain spore mother liquor.Spore concentration is measured with blood counting chamber, by spore
Concentration is adjusted to 1.0 × 104Individual/mL, 1.0 × 105Individual/mL, 1.0 × 106Individual/mL, 1.0 × 107Individual/mL, 1.0 × 108Individual/
mL.
Using spraying, the spore liquid of each concentration for preparing is sprayed onto with 3-5 instar larvae, control sprinkling 0.1%
Tween-80 solution, each concentration in each worm age are inoculated with 10, and each processes 3 repetitions, and postvaccinal larva is placed in 25 DEG C of trainings
Brightness 12 in foster case:12, raised, regularly observe, record the concrete death condition of each process daily, and dead larvae is put
At 25 DEG C with moisturizing cotton balls moisturizing culture in the culture dish for being placed with oneself the wet filter paper of sterilizing, the head number of bombys batryticatus, result of calculation is recorded
As table 1.
Infection rate formula：Infection rate (%)=bombys batryticatus number/supply test worm number
Infection ability of the 1 Java Isaria BE01 of table to 3,4,5 instar larvae of fall webworms
As a result show, Java Isaria BE01 fall webworms are had pathogenic, 1.0 × 104The spore concentration of individual/mL
Under, the death rate is more than 53.33%.The bacterial strain has preferable effect to fall webworms 3,4 instar larvaes.
SEQUENCE LISTING
<110>Anhui Academy of Forestry of Nanjing Forestry University
<120>The Java Isaria BE01 of one plant height effect preventing and treating fall webworms and its application
<130> 100
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 643
<212> DNA
<213> Isaria javanica
<400> 1
ggagaccaag gccggcaagt ccaccggcaa gaccctcctc gaggctattg acgccatcga 60
gccccccaag cgtcccctcg acaagcccct ccgtcttcct cttcaggatg tctacaagat 120
cggtggtatc ggaacggtgc ccgtcggtcg tgtcgagact ggtatcatca agcccggcat 180
ggtcgtcacc tttgctcctt ccaacgtcac cactgaagtc aagtccgtcg agatgcacca 240
cgagcagctg cccgagggtg ttcccggtga caacgtcggc ttcaacgtga agaacgtttc 300
cgtcaaggaa atccgtcgtg gtaacgtcgc tggtgactcc aagaacgacc ctcccaacgg 360
tgctgcctcc ttcaacgccc aggtcattgt catcaaccac cctggccaga tcggtaacgg 420
ttacgcccct gttcttgact gccacactgc ccacattgct tgcaagttct ccgagctcct 480
cgagaagatc gaccgccgta ctggtaagtc ggttgagaac aaccccaagt tcatcaagtc 540
tggtgactcc gccatcgtca agatggttct ctccaagccc atgtgcgttg aggctttcac 600
cgactacccc cctctgggcc gtttcgccgt ccgtgacatg cgt 643
<210> 2
<211> 23
<212> DNA
<213> Artificial
<220>
<223>Primer 983F sequence
<400> 2
gcyccygghc aycgtgaytt yat 23
<210> 3
<211> 23
<212> DNA
<213> Artificial
<220>
<223>Primer 2 218R sequence
<400> 3
atgacaccra crgcracrgt ytg 23
Claims (7)
1. one plant preventing and treating fall webworms Java Isaria, its Classification And Nomenclature be Java Isaria（Isaria javanica）
BE01, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC
No.13192, preservation date are on November 28th, 2016, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
2. application of the Java Isaria BE01 described in claim 1 in preventing and treating fall webworms.
3. application according to claim 2, it is characterised in that：It is sprayed directly on using the spore liquid of Java Isaria BE01
Prevented and treated on fall webworms larva.
4. application according to claim 3, it is characterised in that：Described spore liquid and preparation method thereof is：From isolating and purifying
To Java Isaria BE01 bacterial strain in, picking mycelium inoculation is cultivated 20 days at 25 DEG C to being covered with the SDAY flat board of glassine paper,
Mycelia and spore on scraping glassine paper is put in the 0.1% Tween-80 aqueous solution, is sufficiently stirred for concussion, is filtered, obtain spore
Mother liquor, spore concentration is adjusted to 1.0 × 104Individual/mL ~ 1.0 × 108Individual/mL.
5. application according to claim 3, it is characterised in that：Described spore liquid concentration is 1.0 × 106Individual/mL ~ 1.0 ×
108Individual/mL.
6. according to claim 1 preventing and treating fall webworms Java Isaria, it is characterised in that：The Java Isaria
BE01 adopts Czapek's medium culture.
7. according to claim 6 preventing and treating fall webworms Java Isaria, it is characterised in that：The Java Isaria
BE01 adopts Czapek's medium culture, cultivates 13 days under the conditions of 25 DEG C.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108102929A (en) * | 2017-12-26 | 2018-06-01 | 华南农业大学 | A kind of Java Isaria of anti-pymetrozine and its application |
| CN109479903A (en) * | 2018-11-28 | 2019-03-19 | 华南农业大学 | A kind of Java Isaria bacterium conidium oil-suspending agent and its application |
| CN110720471A (en) * | 2019-11-15 | 2020-01-24 | 韶关市华实现代农业创新研究院 | Compound pesticide of Isaria javanicus mutagenic strain IJUV-6 and thiamethoxam and application thereof |
| CN111378582A (en) * | 2020-03-17 | 2020-07-07 | 南京林业大学 | Culture medium, Isaria javanicus, wettable powder containing Isaria javanicus and application of Isaria javanicus |
| CN113373065A (en) * | 2021-07-16 | 2021-09-10 | 广西大学 | Isaria javanicus strain DMC01 and application thereof in preventing and treating pine moth |
| WO2021211677A1 (en) * | 2020-04-16 | 2021-10-21 | The United States Of America, As Represented By The Secretary Of Agriculture | Compositions and methods to reduce insect pests |
| CN114134046A (en) * | 2021-11-12 | 2022-03-04 | 广东省科学院动物研究所 | Isaria farinosa GIZ _ PH5-1 and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108102929A (en) * | 2017-12-26 | 2018-06-01 | 华南农业大学 | A kind of Java Isaria of anti-pymetrozine and its application |
| CN109479903A (en) * | 2018-11-28 | 2019-03-19 | 华南农业大学 | A kind of Java Isaria bacterium conidium oil-suspending agent and its application |
| CN109479903B (en) * | 2018-11-28 | 2021-03-19 | 华南农业大学 | Isaria javanicus conidium oil suspending agent and application thereof |
| CN110720471A (en) * | 2019-11-15 | 2020-01-24 | 韶关市华实现代农业创新研究院 | Compound pesticide of Isaria javanicus mutagenic strain IJUV-6 and thiamethoxam and application thereof |
| CN111378582A (en) * | 2020-03-17 | 2020-07-07 | 南京林业大学 | Culture medium, Isaria javanicus, wettable powder containing Isaria javanicus and application of Isaria javanicus |
| WO2021211677A1 (en) * | 2020-04-16 | 2021-10-21 | The United States Of America, As Represented By The Secretary Of Agriculture | Compositions and methods to reduce insect pests |
| CN113373065A (en) * | 2021-07-16 | 2021-09-10 | 广西大学 | Isaria javanicus strain DMC01 and application thereof in preventing and treating pine moth |
| CN113373065B (en) * | 2021-07-16 | 2022-05-20 | 广西大学 | Isaria javanicus strain DMC01 and application thereof in prevention and treatment of pine caterpillars |
| CN114134046A (en) * | 2021-11-12 | 2022-03-04 | 广东省科学院动物研究所 | Isaria farinosa GIZ _ PH5-1 and application thereof |
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