CN106520564B - One plant of aspergillus parasiticus and its preparation method and application - Google Patents
One plant of aspergillus parasiticus and its preparation method and application Download PDFInfo
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Abstract
The present invention provides one plant of aspergillus parasiticus, which is aspergillus parasiticus Aspergillus parasiticus RWSF001-02, is preserved in " Guangdong Province's Culture Collection ", and deposit number is GDMCC NO:60080.The aspergillus parasiticus bacterium is filtered out from the aphid because of fungi autoeciousness natural death.The aspergillus parasiticus and conidia powder containing the aspergillus parasiticus spore can be used for preparing insecticide, especially to aphid control rate with higher, and the bacterial strain production traits is good, growth rapidly, sporulation quantity is up to 8 × 10 after cultivating 3~5 days in solid medium8/g。
Description
Technical field
The invention belongs to biological control of insect pests technical fields, are related to one plant of aspergillus parasiticus and preparation method and application.
Background technique
From after invention chemical pesticide, chemical pesticide is increased with annual 5% surprising speed.Although in the past few decades,
Chemical pesticide, as important function, but a large amount of uses of chemical pesticide are played in volume increase and control of insect, is brought in agricultural
The a series of ecological environment problem such as serious drug resistance, pesticide residue.It is long-term largely to lead to drug resistance evil using chemical pesticide
Worm increases, and especially in the past 10 years, the multiple pest such as bollworm, aphid, diamondback moth, prodenia litura is to pyrethroids class, organic phosphorus
The drug resistance of class chemical pesticide increases several hundred or even thousands of times.The application of chemical pesticide simultaneously, makes Residual Pesticides in Farm Produce
Amount increases, and has seriously polluted the environment, jeopardizes mankind Jiankang and life.As the improvement of people's living standards, people are to environment
It is required that it is higher and higher, therefore people is forced to find a kind of control of insect alternative.Increasingly with non-polluted farm product demand
Increase the benign development with market, the cry of microbe insecticide control pest is higher and higher, therefore needs to research and develop
Novel microbial insecticide and its industrialization key technology, especially greenhouse sucking pest such as aphid etc., their reproductive capacity are strong,
Drug resistance is strong, with greater need for biological control.
Summary of the invention
In view of this, the purpose of the present invention is to provide one plant of aspergillus parasiticus bacterium and preparation method thereof, the aspergillus parasiticus
Bacterium can be applied to prepare the lethane of fungus insecticide or the ingredient containing fungi, to prevent eliminate aphis Deng pests.
To achieve the goals above, the technical scheme adopted by the invention is that: one plant of aspergillus parasiticus bacterium, the aspergillus parasiticus
Bacterium is the screening of aspergillus parasiticus bacterial strain Aspergillus parasiticus filtered out from the aphid because of fungi autoeciousness natural death
RWSF001-02 was preserved in " Guangdong Province's Culture Collection " on September 21st, 2016, address: Guangzhou martyr
5 building, the building of compound the 59th of Road 100, Guangdong Microbes Inst, deposit number are GDMCC NO:60080, and the classification of bacterial strain is ordered
Entitled screening of aspergillus parasiticus bacterial strain Aspergillus parasiticus RWSF001-02.
The present invention obtains the step of aspergillus parasiticus bacterium Aspergillus parasiticus RWSF001-02 are as follows: first
The aphid because of fungi autoeciousness natural death is first chosen, is used distilled water flushing 3~5 times after impregnating 30 min using 75% alcohol, it will
The aphid of processing infection is put into the solid plate of PDA culture medium and is 28 DEG C of culture 48h in temperature, then picking single colonie
It is separated, and indoor control aphid effect test is carried out to several plants of isolated bacterial strains, repeated screening, which goes out one plant, has height
Screening of aspergillus parasiticus bacterial strain pesticidal, growth conditions are good.
In order to further increase bacterial strain provided by the present invention growing way and its produce spore character so as to be more suitable for spore batch
Production, by the bacterial strain strain transfer filtered out into PDA liquid medium, is placed on shaking table and cultivates under 28 DEG C of environment
For 24 hours, mycelia liquid seeds are made.It is 1:(1 that the mycelia liquid seeds, which are inoculated into according to 2%~5% inoculum concentration by mass ratio,
~3): in the solid medium that 1 maize flour, wheat bran and dregs of beans forms, keeping air humidity 50%, be 25 DEG C~30 in temperature
Cultivated 3~5 days under the conditions of DEG C, the solid medium carried out after solid culture primary surface is covered with spore it is air-dried, to be made
Content is greater than 8 × 108The conidia powder product of/g.
The obtained bacterial strain of the present invention has following biological characteristics:
Colony morphology characteristic:
It will be inoculated into through PDA liquid medium culture mycelia liquid seeds obtained by maize flour, wheat bran and dregs of beans group
At solid medium in cultivate 3~5 days after, gained bacterium colony is just white, and yellow green then becomes yellowish-brown；Sporulation quantity is up to 8
×108/g；Conidial head is in radiation, and rare loose column, conidiophore is mostly single layer, rare bilayer, conidium
Ellipse.
Growth characteristics:
Growth characteristics research is carried out to the bacterial strain, obtaining its optimum growth temperature is 25 DEG C~30 DEG C, and optimum is empty
Air humidity degree is 50%.
The bacterial strain bacterium colony that will be obtained after cultivating 3~5 days in solid medium extracts its genomic DNA, and using conventional
Aspergillus parasiticus bacterium DNA extension primer pair DNA expanded, and PCR product is examined with agarose gel electrophoresis respectively
It surveys, the PCR product of the ITS DNA sequence dna of the bacterial strain is sequenced in DNA purpose band needed for cutting, and sequencing result is shown in gene sequence
List SEQ.ID No.1.Analysis show that the DNA-ITS sequence of the bacterial strain is shared from the gene order table SEQ.ID No.1
574bp, the bacterial strain belong to the aspergillus parasiticus Aspergillu sparasiticus RWSF001- with aspergillus Aspergillus
02 genetic homology probability is higher.
In conclusion passing through the colonial morphology of the comprehensive bacterial strain, conidium form, condition of culture and molecular biology mirror
The result shows that, which belongs to the aspergillus parasiticus Aspergillu sparasiticus of aspergillus Aspergillus calmly
RWSF001-02。
The screening of aspergillus parasiticus bacterial strain provided by the invention is infected since spore germination to thallus to be generated on polypide again
The process of spore can be divided into 10 stages, i.e., by Spore adhesion in Cuticle, spore germination, penetrate epidermis, mycelia in blood
Intracavity, toxin generate, host is dead, mycelia invades all organs of host, mycelia is pierced by epidermis, generate spore, infect
This ten stages of the diffusion of unit.As long as the bacterial strain can complete the first four stage, it will be able to which host is caused to invade and be infected；
When disease fungus mycelia mass propagation in vivo, so that it may cause host dead.Therefore, provided by the invention described parasitic bent
Mould or conidia powder containing the aspergillus parasiticus bacterium spore can be used to prepare insecticide, in particular by containing described parasitic bent
The conidia powder of mycotic spore is come in the insecticidal fogging aqueous solution for preparing, the aspergillus parasiticus bacterium spore content is 8 × 105~4
×106When to aphid control rate with higher.
To be 1:(1~3 by mass ratio when carrying out anti-eliminate aphis using the aspergillus parasiticus bacterium provided by the invention): 1
Maize flour, wheat bran and dregs of beans composition solid medium made from the Aspergillus strain conidia powder product be diluted with water 500~
It is sprayed after 1000 times, after 3~5 days spraying, when ambient humidity is below 75%, the aphid polypide infected by the strain becomes
It is black, shrivelled, with Stereo microscope it can be found that having mycelia appearance at aphid epidermis stomata；And when ambient humidity is 75% or more
Yellowish-brown and whole body is presented covered with aspergillus parasiticus spore in aphid polypide.The result shows that the Aspergillus strain quilt provided by the invention
The aphid preventive effect of water extension rate 1:500 and 1:750 are up to 92% or more.Therefore, the aspergillus parasiticus bacterium provided by the invention can
For preventing and treating various plant aphids, aphis population quantity can be efficiently controlled.
Detailed description of the invention
The conidiophore shape of Fig. 1, screening of aspergillus parasiticus bacterial strain provided in an embodiment of the present invention under 300,000 times of optical microscopies
State.
The conidium head morphology of Fig. 2, screening of aspergillus parasiticus bacterial strain provided in an embodiment of the present invention under an optical microscope.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be described in further detail.
The screening of aspergillus parasiticus bacterial strain filtered out from the radish aphid because of fungi autoeciousness natural death the present embodiment provides one plant,
Acquisition, culture and the identification method of the bacterial strain the following steps are included:
The separation of bacterial strain: choosing the radish aphid because of fungi autoeciousness natural death first, impregnates 30 using 75% alcohol
After min use distilled water flushing 3~5 times, then by the aphid for handling postoperative infection be put into the solid plate of PDA culture medium and
Temperature is 28 DEG C of culture 48h, and then picking single colonie is separated, and it is raw to carry out indoor aphid to several plants of isolated bacterial strains
Test is tested, and repeated screening, which goes out one plant, has high bacterial strain pesticidal, growth conditions are good.
The identification of bacterial strain: will be inoculated into through the resulting mycelia liquid seeds of PDA liquid medium culture by mass ratio is 1:
Colony morphology characteristic, conidial fructification are observed after cultivating 3~5 days in the solid medium that maize flour, wheat bran and the dregs of beans of 1:1 forms
Referring to Fig. 1 and Fig. 2 with the specific form feature of Spore shapes and size.As can be seen from the figure gained bacterium colony is just white, Huang
Green then becomes yellowish-brown；Conidial head is in radiation, rare loose column, and conidiophore is mostly single layer, rare pair
Layer is oval.
The amplification of DNA ITS sequence, sequencing and the Molecular Identification of bacterial strain: 1 is amplified from the strain gene group DNA
The sequence of about 600bp, PCR product gained sequence after being sequenced are carried out pair by existing nucleic acid sequence in Blast and GenBank
Than, the results showed that the DNA ITS sequence sequencing result of the bacterial strain is shown in SEQ.ID No.1, the sequencing sequence and Aspergillu
The sequence homology of sparasiticus RWSF001-02 is high, so that verifying the bacterial strain is Aspergillus strain Aspergillus
parasiticus RWSF001-02。
In order to further increase strain growth gesture provided by the present invention and its produce spore character, so as to be more suitable for spore batch
Production, by the strain transfer of the bacterial strain into PDA liquid medium, is placed on shaking table and cultivates for 24 hours under 28 DEG C of environment, be made
Mycelia liquid seeds, the mycelia liquid seeds according to 2%~5% inoculum concentration be inoculated by mass ratio be 1:1:1 maize flour,
In the solid medium of wheat bran and dregs of beans composition, air humidity 50% is kept, cultivates 3-5 under the conditions of temperature is 25 DEG C~30 DEG C
It, the solid medium carried out after solid culture primary surface is covered with spore it is air-dried, so that content be made greater than 8 × 108/g
Conidia powder product.
The inhibition aphid effect of aspergillus parasiticus bacterium conidia powder product made from the present embodiment is measured, test effect
Fruit is as follows:
Test one: this experimental plot is located in the green house of vegetables of Zhengzhou City, the garden De Gu, Zhongmou County, is small western calabash for examination crop varieties
Reed aphid.Test sets the aspergillus parasiticus and water thinner ratio according to 1:500,1:750, and 1:1000 times dilutes, and carries out clear water sky
White control.Each test process plot area 15m2, be repeated 3 times test, the random district's groups arrangement of cell is sprayed using back carried hand
Day with fog is spraying, spouting liquid 40kg/667m2, it was sprayed before the dusk and guarantees relative humidity >=85% in canopy.It handles in order to prevent
Between interfere with each other, hidden with plastic foil every neighboring community when application.5 points of samplings in cell, each click 2 plants of plant before processing,
And 3 aptery adult aphid parasitic amounts of calibration are no less than 50 blades on selected plant, investigate insect population, after processing 3d,
5d, 7d count aphid death condition, calculate control efficiency, and the results are shown in Table 1, specific formula for calculation is following,
The preceding insect population of Revision insect recluced rate (%)=(each day remains borer population after insect population-processing before handling)/processing ×
100%
Control efficiency (%)=(treatment region Revision insect recluced rate-check plot Revision insect recluced rate)/(100-check plot insect populations decline
Rate) × 100%
Control efficiency of the aspergillus parasiticus of the different dilution ratios of table 1 to cucurbita pepo aphid
As can be seen from Table 1 after spraying treatment 3d, the parasitic song of extension rate 1:500 and extension rate 1:750
Mould powder product is not significant to the prevention and treatment difference of aphid, and extension rate 1:500 and 1:750 and extension rate 1:1000
There are significant differences for the aspergillus parasiticus bacterium conidia powder product.Meanwhile as a result also showing 3d extension rate 1:500 after spraying treatment
With 1:750 aphid preventive effect 90% or more, the aphid preventive effect of extension rate 1:1000 is also 89% or more.Show aspergillus parasiticus
It is significant to the control efficiency of aphid, illustrate that aphid is substantially all infected after spraying 5d, is also to continue state of a control after 7d, effectively
Solves the short disadvantage of chemical pesticide preventive effect.Therefore, the aspergillus parasiticus bacterium conidia powder product has the prevention and treatment of aphid single-minded
Property is strong, pollution-free, the advantages of persistently preventing and treating.
Test two: this experimental plot is located at Hebi City Shancheng District stone forest town plant area, Ren Yuan biotech development Co., Ltd, supplies
Trying crop varieties is field Li Ke weeds aphid, and test sets aspergillus parasiticus and water ratio according to 1:500,1:750, and 1:1000 times dilute
It releases and blank control totally 4 processing, each processing plot area 1m2, be repeated 3 times, cell random district's groups arrangement.Using backpack
Hand sprayer is spraying, spouting liquid 40kg/667m2, it is spraying before the dusk, prevent outdoor ultraviolet light from killing fungi, while environment
Relative humidity is larger, interferes with each other between handling in order to prevent, is hidden with plastic foil every neighboring community when application.Before processing in cell
3 points of samplings each click 2 plants of plant, and 3 aptery adult aphid parasitic amounts of calibration are no less than 20 blades on selected plant, adjust
Insect population is looked into, aphid death condition is counted in spraying treatment 3d, 5d, 7d, calculates control efficiency, the results are shown in Table 2.
The aphid aspergillus parasiticus of the different dilution ratios of table 2 prevents and treats Li Ke weeds aphid test effect
As can be seen from Table 2 after spraying treatment 3d, extension rate 1:500 and prevention and treatment of the extension rate 1:750 to aphid
Difference is not significant, and there are significant differences with extension rate 1:1000 by extension rate 1:500 and 1:750；As a result also show spraying place
91% or more, the aphid preventive effect of extension rate 1:1000 also exists for 3d extension rate 1:500 and 1:750 aphid preventive effect after reason
86% or more.Show that aspergillus parasiticus is significant to the control efficiency of aphid, illustrates that aphid is substantially all infected after spraying 5d, after 7d
It is to continue state of a control, effective solution chemical pesticide preventive effect short disadvantage.Therefore, the aspergillus parasiticus bacterium conidia powder product
To the prevention and treatment of aphid have the advantages that specificity it is strong, it is pollution-free, persistently prevent and treat.
Test three: this experimental plot is located in ratio Ao Rui Biotechnology Co., Ltd, Beijing green house of vegetables, for studying article
Kind be cowpea, test sets aspergillus parasiticus and water ratio according to 1:500,1:750,1:1000 times of dilution at blank control totally 4
Reason, each processing plot area 15m2, it is repeated 3 times, the random district's groups arrangement of cell.It is spraying using knapsack hand sprayer, spray
Liquid measure 40kg/667m2, it is spraying before the dusk, guarantee relative humidity >=85% in canopy, interfered with each other between handling in order to prevent, is administered
When with plastic foil hide every neighboring community.3 points of samplings in cell, each click 2 plants of plant, and in selected plant subscript before processing
Fixed 3 aptery adult aphid parasitic amounts are no less than 30 blades, investigate insect population, and 3d, 5d, 7d count aphid after spraying treatment
Death condition calculates control efficiency, and the results are shown in Table 3.
The aphid aspergillus parasiticus of the different dilution ratios of table 3 prevents and treats Cowpea Aphid test effect
As can be seen from Table 3 after spraying treatment 3d, extension rate 1:500 and prevention and treatment of the extension rate 1:750 to aphid
Difference is not significant, and there are significant differences with extension rate 1:1000 by extension rate 1:500 and 1:750；As a result also show spraying place
92% or more, the aphid preventive effect of extension rate 1:1000 also exists extension rate 1:500 and 1:750 aphid preventive effect after managing 3d
89% or more.Show that aspergillus parasiticus is significant to the control efficiency of aphid, illustrates that aphid is substantially all infected after spraying 5d, after 7d
It is to continue state of a control, effective solution chemical pesticide preventive effect short disadvantage.Therefore, the aspergillus parasiticus bacterium conidia powder product
To the prevention and treatment of aphid have the advantages that specificity it is strong, it is pollution-free, persistently prevent and treat.
Finally it should be noted that: the above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof；To the greatest extent
The present invention is described in detail with reference to preferred embodiments for pipe, it should be understood by those ordinary skilled in the art that: still
It can modify to a specific embodiment of the invention or some technical features can be equivalently replaced；Without departing from this hair
The spirit of bright technical solution should all cover within the scope of the technical scheme claimed by the invention.
Claims (6)
1. one plant of aspergillus parasiticus, which is characterized in that it is aspergillus parasiticus Aspergillus parasiticus RWSF001-02,
It is preserved in " Guangdong Province's Culture Collection ", deposit number is GDMCC NO:60080.
2. one plant of aspergillus parasiticus according to claim 1, which is characterized in that its DNA-ITS sequence such as SEQ ID NO.1
It is shown.
3. a kind of insecticide, which is characterized in that it includes aspergillus parasiticus of any of claims 1 or 2.
4. a kind of insecticide, which is characterized in that it includes the conidia powder containing aspergillus parasiticus spore, wherein the aspergillus parasiticus
Spore is obtained by aspergillus parasiticus culture of any of claims 1 or 2.
5. insecticide according to claim 4, which is characterized in that the conidia powder containing the aspergillus parasiticus spore prepares step
Suddenly include: that arrive the mycelium inoculation of aspergillus parasiticus of any of claims 1 or 2 by mass ratio be 1:(1~3): 1 maize flour,
In the solid medium of wheat bran and dregs of beans composition, cultivated under the conditions of being placed in 25 DEG C~30 DEG C, to the solid culture primary surface cloth
The solid medium is carried out after full spore to air-dry processing, so that the conidia powder containing the aspergillus parasiticus spore be made.
6. according to the described in any item insecticides of claim 3~5, which is characterized in that it includes for the anti-fungi to eliminate aphis
Insecticide or lethane containing fungi ingredient.
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| CN107058129B (en) * | 2017-03-29 | 2020-05-19 | 四川大学 | Aspergillus awamori strain and its use in field of agricultural pest control |
| CN108477220B (en) * | 2018-02-01 | 2020-05-26 | 鹤壁市人元生物技术发展有限公司 | Aspergillus parasiticus spore composite pesticide and preparation method thereof |
| CN111139185B (en) * | 2018-11-06 | 2023-03-10 | 广州中医药大学(广州中医药研究院) | Aspergillus fungi and application thereof |
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| WO2014117118A1 (en) * | 2013-01-28 | 2014-07-31 | Novozymes Bioag A/S | Compositions and methods for treating pests |
| CN104365679A (en) * | 2014-09-16 | 2015-02-25 | 瑞安市普罗生物科技有限公司 | Anti-aphid reagent and uses thereof |
| CN105705013A (en) * | 2013-11-08 | 2016-06-22 | 诺维信生物农业公司 | Compositions and methods for treating pests |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014117118A1 (en) * | 2013-01-28 | 2014-07-31 | Novozymes Bioag A/S | Compositions and methods for treating pests |
| CN105705013A (en) * | 2013-11-08 | 2016-06-22 | 诺维信生物农业公司 | Compositions and methods for treating pests |
| CN104365679A (en) * | 2014-09-16 | 2015-02-25 | 瑞安市普罗生物科技有限公司 | Anti-aphid reagent and uses thereof |
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