CN105543102B - One plant of prevention palm pest coconut knits green muscardine fungus and the application of moth - Google Patents
One plant of prevention palm pest coconut knits green muscardine fungus and the application of moth Download PDFInfo
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- CN105543102B CN105543102B CN201510900251.XA CN201510900251A CN105543102B CN 105543102 B CN105543102 B CN 105543102B CN 201510900251 A CN201510900251 A CN 201510900251A CN 105543102 B CN105543102 B CN 105543102B
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Abstract
The present invention relates to green muscardine fungus and applications that one plant of prevention palm pest coconut knits moth, the Strain Designation is Metarhizium anisopliae (Metarhizum anisopliae) LSY-1, China typical culture collection center (CCTCC), deposit number are preserved on November 9th, 2015：CCTCC M 2015672.The purposes of Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 provided by the present invention is its application in prevention coconut knits moth, by the experiment for directly smearing administration, show that Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 knits moth to coconut and has good insecticidal action, coconut can be efficiently controlled and knit moth population quantity, it is one plant and knits the biocontrol bacterial strain on moth with potential significance in prevention coconut, the development for China's palm industry provides help.
Description
Technical field
The invention belongs to microorganism field, it is related to a kind of green muscardine fungus and its application is specifically related to one plant of prevention palm evil
Worm coconut knits green muscardine fungus and the application of moth.
Background technology
Coconut knits moth Opisina arenosella Walker (also known as coconut wood moth, blackhead crawler belt worm, coconut palm column moth, food leaf
Crawler belt worm), belong to Lepidoptera (Lepidoptera) Zhi E sections (Qecophoridae), English name coconut blackheaded
Caterpillar is distributed mainly on India, Thailand, Indonesia, Malaysia, Sri Lanka, Bangladesh, Burma, Buckie
The ground such as Si Tan are a kind of exotic invasive evils seriously endangering the palm crops such as coconut, betel nut, Chinese fan palm, Middle East nipa palm, big yagua
Worm, in March, 2014 are listed in national forestry danger harmful organism.Coconut knits moth larvae feeding blade, entire tree crown when serious
By invasion gnaw so as to cause plant development slowly, fruit yield be greatly decreased even have no harvest, seriously affect crop quality and
Yield.In recent years palm crops as industrial crops and garden landscape plant the southern large area in China plantation with from foreign countries
A large amount of to introduce, causing coconut to be knitted, the threat that moth is broken out in China is increasing, and Hainan, Guangxi, the wide Three Eastern Provinces have greatly at present
The report of area harm.The prevention of moth is knitted for coconut at present other than using parasitic wasp prevention and control, it is main still with chemical prevention
Based on, although chemical prevention can quickly prevent the worm to a certain extent, residual caused by chemical prevention, resistance with
The problems such as second rice crop season, seriously affects natural environment and the health of the mankind；Although knitting moth using parasitic wasp prevention and control coconut printing
The ground such as degree, Sri Lanka achieve certain control effect, but domestic high-quality parasitic wasp kind introduce with large area raising,
The technologies such as release are not still carried out effectively, therefore it is a kind of more scientific and effective how to combine domestic existing Research foundation to find
The biological control means that control coconut knits moth harm are particularly important.
Metarhizium anisopliae (Metarhizum anisopliae) host range is extensive, can parasitic Orthoptera, Hymenoptera,
7 mesh such as Homoptera, Diptera, Lepidoptera, coleoptera and Semiptera, 42 sections, about more than 200 kind insects, nematode and mite class.
Metarhizium anisopliae is one of the most widely used insect pathogenic fungus in biological control, in forest, crops and public affairs
Altogether all the assessment of effect has been carried out with Metarhizium anisopliae in sanitary insect pest.Using Metarhizium anisopliae biological prevention and control agent come prevention and control Qi
Some pests such as Scarabaeiform, locust, leafhopper, DAmerasan et al. some research it has also been found that, master of the Metarhizium anisopliae to malaria
Propagating insect yellow-fever mosquito has fairly obvious control effect.Metarhizium anisopliae as most important one kind in biocontrol fungi,
It has in use parasitic species it is more, it is adaptable, cause a disease that the period is short, target is strong, will not cause poison to the mankind and livestock
Evil and environmentally friendly feature.Through retrieval, moth is knitted there is not yet relevant report currently with green muscardine fungus control coconut.
Invention content
The object of the present invention is to provide green muscardine fungus and applications that one plant of prevention palm pest coconut knits moth.
One plant of Metarhizium Strains is named as Metarhizium anisopliae (Metarhizum anisopliae) LSY-1, in
On November 9th, 2015 is preserved in China typical culture collection center (CCTCC), address：Wuhan City, Hubei Province Wuchang District Aug. 1st
Road Luo Jia Shan；Deposit number：CCTCC M 2015672.
Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 bacterial strains provided by the present invention, are located away from nature
The coconut of infection knits moth bombys batryticatus, and through morphology, cultural colony, customary physiological is biochemical and DNA sequence dna is identified, which is chafer
The small spore mutation of green muscardine fungus is a new strains for Metarhizium fungi, is named as Metarhizium anisopliae (Metarhizum
anisopliae)LSY-1。
The basic biological characteristics of Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 provided by the present invention
Property is：
Bacterial strain cross in tablet PDA culture medium culture after mycelium be transparent and have diaphragm；In PDA culture medium
Initial stage is white or faint yellow excessive to yellow, and shape is cotton-shaped or villiform protrusion, and the later stage green occurs for basic tone
The color of adjacent chromatography；Conidiophore is in bottleneck shape, has bifurcated, single raw or more lifes to intersect growth, be mutually wound in fence shape.
The each size of conidium is 1.9~2.8 μm of 4.2~8.3 μ m, and oblong is rodlike, and it is most that two terminal circle slips the spore fallen
It is gathered into lumps.
The purposes of Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 provided by the present invention is it anti-
It controls coconut and knits application in moth.
Using Metarhizium anisopliae provided by the present invention (Metarhizum anisopliae) LSY-1 as active constituent
Biological agent also belongs to protection scope of the present invention.When needs, said preparation can include common carrier prepared by biological agent
And auxiliary material.
The present invention is by directly smearing the technique study Metarhizium anisopliae (Metarhizum anisopliae) of administration
LSY-1 knits coconut the insecticidal activity of moth, the experimental results showed that Metarhizium anisopliae (Metarhizum anisopliae) LSY-
1 pair of coconut, which knits moth, good insecticidal action.Show that the bacterial strain is one plant and knits the life with potential significance on moth in prevention coconut
Anti- bacterial strain.
The screening that the present invention knits microorganism in moth bombys batryticatus by the coconut to natural infection detaches, and obtains Metarhizium anisopliae
(Metarhizum anisopliae) LSY-1 shows that the bacterial strain knits moth to coconut and has good desinsection to make by insecticidal test
With can efficiently control coconut and knit moth population quantity, reduce loss, be one plant and knitted in prevention coconut there is potential significance on moth
Biocontrol bacterial strain, have the characteristics that insecticidal toxicity is strong, good disinsection effect, the development for China's Palmae industry provides help.
Description of the drawings
Fig. 1：The bacterial strain spore and hypha form of Metarhizium anisopliae (Metarhizum anisopliae) LSY-1.
Fig. 2：Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 bacterial strains infect coconut and knit moth larvae photo.
A：CK；B：LSY-1 infects coconut and knits moth 2d；C：LSY-1 infects coconut and knits moth 4d；D：LSY-1 infects coconut and knits moth 6d.
Specific implementation mode
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not limited to the scope of the present invention.In the following examples, the experimental methods for specific conditions are not specified, usually
According to normal condition, or according to the normal condition proposed by manufacturer.
The screening and its identification of one bacterial strain of embodiment
1, the screening of bacterial strain
It takes the coconut being collected to knit moth bombys batryticatus sample, first with 70% alcohol disinfecting one minute, is used again after sterile water wash
0.1% mercury chloride carries out surface sterilization 30s, sterile water wash 3 times to susceptible sample, and a little bombys batryticatus tissue of picking is inoculated in choosing
Selecting property SDAY culture mediums (4% glucose, 1% peptone, 1% yeast powder, 2% agar, 100 μ g/ml streptomysins, 100 μ g/ml
Penicillin, 0.8 μ g/ml of carbendazim), it is inverted in 28 ± 1 DEG C of constant temperature and is inverted culture 3-4d, choose monospore and be inoculated in PDA cultures
(peeled potatoes 200g cuts fritter and adds water to 1L boils double gauze filtering after twenty minutes to base, in filtrate plus glucose 20g, fine jade
Fat 20g reheatings are allowed to dissolve, and water is added to supply volume to 1L) it purifies, per 3 points of ware, it is triangularly arranged, after purification through monospore
Obtain with insecticidal action bacterial strain, be named as Metarhizium anisopliae (Metarhizum anisopliae) LSY-1, in
On November 9th, 2015 is preserved in China typical culture collection center (CCTCC), address：Wuhan City, Hubei Province Wuchang District Aug. 1st
Road Luo Jia Shan；Deposit number：CCTCC M 2015672.
2, strain morphology is identified
The bacterial strain isolated and purified is crossed culture in the tablet PDA culture medium of a diameter of 90mm, is put into 29 DEG C of trainings
It supports in case after culture 4-5d, the mycelia and conidiophore of bacterial strain are observed in picking tender white hypha film-making under the microscope
Form；The conidium film-making of picking maturation and the size and shape of paired observation spore again after 10-12d；14d or so is observed
Record the mode of appearance feature of bacterium colony.Bacterial strain is white or faint yellow excessive to yellow before generating spore in PDA culture medium,
Shape is cotton-shaped or villiform protrusion, starts color of the green for the adjacent chromatography of basic tone occur over time.
Mycelium is transparent and has diaphragm.General initial stage covers at white fluff with the dead bombys batryticatus of infection, slowly by depth
The spore of green is substituted；Conidiophore is in bottleneck shape, has bifurcated, single raw or more lifes to intersect growth, be mutually wound in fence
Shape is closely largely gathered into sporodochia.The each size of conidium is 4.2~8.3 μ m, 1.9~2.8 μm of long ellipses
Shape, rodlike, two terminal circle slips the spore multiple aggregation fallen into lumps.
3, ITS sequence amplification, sequencing and the molecular classification of bacterial strain
Constant-temperature table culture 72h uses 65 with gauze collected by suction mycelia under the conditions of 26 DEG C, 230rpm/min respectively
DEG C water-bath preheats 2%CTAB extraction buffers.It scrapes a small amount of green muscardine fungus mycelia (about 1g) to be placed in mortar, be milled to liquid nitrogen white
Color powder；In ground powder, the preheated 2%CTAB extraction buffers of 700ul are added, slowly stir；It will mixing
Liquid is transferred in the sterile centrifugation tube of 2ml, notices that the volume of mixed liquor does not exceed 2/3rds of centrifuge tube；It is positioned over 60 DEG C
Shaking bath in, 100rpm/min shake 1h after take out it is spare；After taking-up after room temperature cooling 2min, chlorine is added into centrifuge tube
Imitative-isoamyl alcohol (24：1) about 0.5ml, slowly shaking up makes the two be uniformly mixed；Supernatant is drawn with liquid-transfering gun, is transferred to containing 600 μ
In the sterile centrifugation tube of l isopropanols, centrifuge tube is slowly shaken into 30sec, can not acutely be shaken, prevention damage DNA make water layer with
Isopropanol is sufficiently mixed, until DNA floccule suspended matters can be seen；After continuing 10000rpm centrifugations 1min, slowly it is sucked out clear
Liquid outwells reservation precipitation, notices that white precipitate is that DNA is sure not to pour out, centrifuge tube handstand is positioned on the paper handkerchief spread out and is drawn
Remaining liq；After 1min, upright centrifuge tube is added the sodium acetate of 80 μ l 5M and 70% ethyl alcohol of 720 μ l is added, gently shakes
It is dynamic, with finger bomb tube bottom, the DNA suspended matters of tube bottom and precipitation is made to float in liquid；30min is placed, makes DNA suspended matters not
Pure object gradually dissolves；After 12000rpm centrifuges 5min again, supernatant is outwelled, 70% ethyl alcohol is added, sediment is washed again
30min；After 12000rpm centrifuges 5min, supernatant is outwelled immediately, spontaneously dries DNA precipitations (super-clean bench blowing)；DNA is precipitated
It is dissolved in 300 μ l sterile waters, RNA enzyme (50 μ g/ml of final concentration) processing solution is added, DNA is made fully to dissolve, -20 DEG C of preservations
It is spare.
Using fungi ribosomes internal gene transcribed spacers (ITS) universal primer ITS4 (5 '-
TCCTCCGCTTATTGATATGC-3 ') ITS5 (5 '-GGAAGTAAAAGTCGTAACAAGG-3 ') amplification pathogenic bacteria ITS1-
5.8s-ITS2rDNA sections, are synthesized by Sangon Biotech (Shanghai) Co., Ltd..PCR instrument：Takara companies.PCR is anti-
Answer system (50 μ L)：50ng DNA profilings, 10 × PCR buffer, 5 μ L, 10pmol/L primer I each 1 μ L of TS4 and ITS5,
1 μ L, 4U/ μ L Taq archaeal dna polymerases of 10mmol/L dNTPs, 1 μ L, add ddH2O to complement to 50 μ L.PCR reaction conditions：94℃
5min, 1 cycle；94 DEG C of 1min, 52 DEG C of 1min, 72 DEG C of 1min, 29 cycles；72 DEG C of extension 10min, 1 cycle；PCR is produced
Object is sequenced by Sangon Biotech (Shanghai) Co., Ltd..It is carried out using the AlignX of 11 softwares of Vector NTI
RDNA-ITS homology analysis, and phylogenetic tree construction development tree.Molecular biology identification result shows the ITS sequences of the bacterial strain
The similitude highest that row belong to Metarhizium anisopliae is 99%.
4, bacterial strain sporulating character
By a concentration of 1 × 108The LSY-1 spore suspensions of cfu/ml are seeded on SDAY plating mediums (100 μ l/
Ware), it is trained under 20 DEG C of different temperatures, 22 DEG C, 24 DEG C, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C, 35 DEG C of condition of culture
Sporulation quantity after supporting 15 days on analytical unit area, it is best to determine that LSY-1 bacterial strains produce spore amount under the conditions of 26 DEG C.
Two bacterial strain of embodiment knits moth indoor insecticidal determination of activity to coconut
Metarhizium anisopliae (Metarhizum anisopliae) the LSY-1 bacterial strains for taking isolation and purification culture good, will be mitogenetic
Conidia powder is fitted into 50ml sterile centrifugation tubes, after 0.1% Tween 80 sterile waters of 10mL are added, vibrates 15min with turbula shaker
Then mixing measures every milliliter of suspension miospore number with blood counting chamber and bacterial strain is configured to spore by suitably adjusting
A concentration of 1.0 × 108A/mL is stored in 10 DEG C of refrigerators and saves backup.By Metarhizium anisopliae (Metarhizum
Anisopliae) spore of LSY-1 bacterial strains is made into the aqua of 5 concentration in the method for gradient dilution, and concentration is 1.0 respectively ×
108、5×107、2.5×107、1.25×107、6.25×106A/ml.Fresh old leaf piece is cut into the blade sections of 8cm or so,
Again blade sections are put into 10ml spore suspensions fully to smear and be infiltrated, is fitted into the test tube after sterilizing for use, often processing inoculation
Test worm 30,3 repetitions are placed in 26 DEG C, raise in 80% growth cabinet of relative humidity.It is observed 1 time per 1d after processing, record
Dead borer population, until 7d after processing, and moisturizing culture is carried out to dead worm, it is determined according to the feature mycelia that worm corpse surface is grown
Whether test worm is that fungal infection is lethal, the results are shown in Table 1.LSY-1 bacterial strains knit moth tool to coconut it can be seen from laboratory experiment result
It is 4.41 × 10 to have lethal effect, half lethal concentration6cfu/ml。
The indoor biological determination of activity of moth larvae is knitted after 1. bacterial strain LSY-1 bacterial strains 5d of table to coconut
Control effect outside three bacterial strain room of embodiment
The aqua of Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 bacterial strains is brought to field, is diluted to and contains
Conidium 1 × 108A/mL, and stir evenly, ultra low volume spray.The similar coconut branch of 3 extents of injury is chosen, with anti-
Worm net surrounds the branch end of the coconut of harm and tightens fly net, and the method by choosing some test worms is controlled to each processing 30
Head coconut knits moth larvae.Start spraying prevention on November 5th, 2015, cloudy turn to overcast for weather at that time, 27 DEG C of temperature, 1 grade of wind-force, empty
Air humidity degree 82%, crosswind angle is 50 ° when spraying, droplet be distributed on blade it is more and uniform, average 40.5 drop of coverage density/
cm3.Every 2 days check once, the results are shown in Table 2.Find out that after processing 4 days, the survival rate of larva starts significantly in trunk by result
Decline, after 14 days the death rate be 96.67%, show Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 have compared with
Strong insecticidal toxicity, and good disinsection effect can efficiently control coconut and knit moth population quantity, reduce loss, be one plant and preventing
It controls coconut and knits the biocontrol bacterial strain with potential significance on moth.
The biological activity determination of moth is knitted to coconut in 2. fields bacterial strain LSY-1 of table
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (3)
1. one plant of Metarhizium Strains is named as Metarhizium anisopliae (Metarhizum anisopliae) LSY-1, in 2015
On November 9, in is preserved in China typical culture collection center, deposit number：CCTCC M2015672.
2. Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 described in claim 1 knits moth in prevention coconut
In application.
3. a kind of prevention coconut knits the biological agent of moth, active constituent is that the deposit number described in claim 1 is CCTCC
Metarhizium anisopliae (Metarhizum anisopliae) LSY-1 of M 2015672.
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| CN201510900251.XA CN105543102B (en) | 2015-12-09 | 2015-12-09 | One plant of prevention palm pest coconut knits green muscardine fungus and the application of moth |
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| CN110669675B (en) * | 2019-10-25 | 2021-07-06 | 云南农业大学 | Metarhizium anisopliae MANGS71814 and application thereof in control of potato tuber moth |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007112257A2 (en) * | 2006-03-24 | 2007-10-04 | The United States Of America, As Represented By The Secretary Of Agriculture | A strain of the fungus metarhizium anisopliae for controlling subterranean termites |
| CN105039181A (en) * | 2015-08-19 | 2015-11-11 | 云南农业大学 | Metarhizium anisopliae MAYX130921 and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007112257A2 (en) * | 2006-03-24 | 2007-10-04 | The United States Of America, As Represented By The Secretary Of Agriculture | A strain of the fungus metarhizium anisopliae for controlling subterranean termites |
| CN105039181A (en) * | 2015-08-19 | 2015-11-11 | 云南农业大学 | Metarhizium anisopliae MAYX130921 and application thereof |
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