CN117187078A - Metarhizium anisopliae strain and application thereof in preventing and treating tea lissajous - Google Patents
Metarhizium anisopliae strain and application thereof in preventing and treating tea lissajous Download PDFInfo
- Publication number
- CN117187078A CN117187078A CN202311126848.4A CN202311126848A CN117187078A CN 117187078 A CN117187078 A CN 117187078A CN 202311126848 A CN202311126848 A CN 202311126848A CN 117187078 A CN117187078 A CN 117187078A
- Authority
- CN
- China
- Prior art keywords
- tea
- metarhizium anisopliae
- lissajous
- larvae
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000223250 Metarhizium anisopliae Species 0.000 title claims abstract description 65
- 241001122767 Theaceae Species 0.000 title 1
- 239000002689 soil Substances 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 238000007598 dipping method Methods 0.000 claims abstract description 6
- 239000000725 suspension Substances 0.000 claims description 16
- 241000262154 Metarhizium pinghaense Species 0.000 claims description 6
- 241000219748 Cyamopsis Species 0.000 claims description 5
- 238000005507 spraying Methods 0.000 claims description 2
- 241000208367 Euonymus Species 0.000 claims 1
- 241000743698 Pinicola Species 0.000 claims 1
- 241001646398 Pseudomonas chlororaphis Species 0.000 claims 1
- 244000269722 Thea sinensis Species 0.000 abstract description 42
- 238000000034 method Methods 0.000 abstract description 12
- 230000007918 pathogenicity Effects 0.000 abstract description 11
- 206010024642 Listless Diseases 0.000 abstract description 7
- 235000009024 Ceanothus sanguineus Nutrition 0.000 abstract description 5
- 240000003553 Leptospermum scoparium Species 0.000 abstract description 5
- 235000015459 Lycium barbarum Nutrition 0.000 abstract description 5
- 206010059866 Drug resistance Diseases 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 4
- 230000018109 developmental process Effects 0.000 abstract description 4
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052782 aluminium Inorganic materials 0.000 abstract description 2
- 230000019617 pupation Effects 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract 1
- 235000013616 tea Nutrition 0.000 description 35
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 21
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 21
- 102000004243 Tubulin Human genes 0.000 description 14
- 108090000704 Tubulin Proteins 0.000 description 14
- 208000036815 beta tubulin Diseases 0.000 description 14
- 241000254173 Coleoptera Species 0.000 description 9
- 241000254171 Curculionidae Species 0.000 description 9
- 241000238631 Hexapoda Species 0.000 description 9
- 241000233866 Fungi Species 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 230000006378 damage Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 4
- 229920000053 polysorbate 80 Polymers 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 description 3
- 241000382353 Pupa Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 235000020567 organic tea Nutrition 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000751139 Beauveria bassiana Species 0.000 description 2
- 240000006491 Ehretia microphylla Species 0.000 description 2
- 235000001911 Ehretia microphylla Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241000218652 Larix Species 0.000 description 2
- 235000005590 Larix decidua Nutrition 0.000 description 2
- 206010040925 Skin striae Diseases 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000443 biocontrol Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000032669 eclosion Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 240000005369 Alstonia scholaris Species 0.000 description 1
- 241001124134 Chrysomelidae Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000110847 Kochia Species 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 235000006468 Thea sinensis Nutrition 0.000 description 1
- 241000223258 Thermomyces lanuginosus Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000000853 biopesticidal effect Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 230000001055 chewing effect Effects 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000000967 entomopathogenic effect Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000009569 green tea Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 238000011392 neighbor-joining method Methods 0.000 description 1
- 235000020333 oolong tea Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses metarhizium anisopliae and application thereof in controlling tea listless amabilis. The metarhizium anisopliae Ma0628 is a strain separated from the larvae of the tea lissajous which endangers tea trees for the first time, is suitable for the environment condition of acid soil rich in aluminum of tea gardens, is simple to culture and rapid to grow, and has strong pathogenicity to the tea lissajous. Experimental results of the dipping method and the soil mixing method show that the metarhizium anisopliae Ma0628 conidium has strong pathogenicity to larvae under the pupation temperature condition of the larvae of the tea-leaf scholaris, and the application of the metarhizium anisopliae has strong pathogenicity to adults of the tea-leaf scholaris by the dipping method, and is environment-friendly, pollution-free and difficult to generate drug resistance. Therefore, the metarhizium anisopliae Ma0628 can be applied to the control of the larvae of the tea lissajous in the soil-out period, can also be used for the control of the adults of the tea lissajous, and has good development potential and application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to metarhizium anisopliae and application thereof in controlling tea listless amabilis.
Background
The tea-line weevil (Myllocerinus aurolineatus Voss) belongs to Coleoptera (Coleoptera) and is mainly parasitic to tea trees and seriously damages spring tea in oolong tea areas and summer tea in green tea areas in China.
The current field control of the tea-beautiful vein beetles is mainly controlled by chemical pesticides, and is mainly controlled by aiming at the adult period. However, chemical control methods are easy to cause pesticide residues and the like, which is unfavorable for the sustainable development of the health of the tea industry. In addition, the chemical control mode also has non-target biological potential safety hazards and the risk of affecting the ecological environment.
Tea tree is mainly endangered by tea tree by chewing leaves with adults, larvae and pupae live in soil, and before pupation and eclosion, the larvae need to migrate to surface soil. When the temperature of the soil surface layer reaches about 20 ℃, the larvae centralize and emerge. If the characteristic of the tea leaf weevil can be utilized to prevent and treat the tea leaf weevil in the larva or pupa stage, the damage of the tea leaf weevil adults can be relieved from the source, and a plurality of defects of chemical pesticide prevention and treatment can be avoided. The biocontrol fungi have the advantages of short pathogenic period, strong specificity, environmental friendliness and the like, and the development of the biocontrol fungi for effectively preventing and controlling the cyamopsis grossedentata has important application value.
Disclosure of Invention
The invention aims to provide metarhizium anisopliae strain and application thereof in controlling tea listless amabilis.
The invention acquires natural sick beauveria bassiana larva from a horizontal town flat yang village organic tea garden in a Shaoxing city of Zhejiang province, separates from the beauveria bassiana larva body and cultures on a potato glucose PDA culture medium, and transfers hyphae to a new culture medium for purification to obtain the green muscardine fungus (Metarhizium pingshaense) Ma0628.
Morphological identification of Metarhizium anisopliae Ma 0628:
on PDA culture medium, bacterial colony is milky white in initial stage, white in center, fine and fluffy in aerial hyphae, and spread from center to periphery. As the culture time is prolonged, light green spores begin to appear at the colony center, the colony is gradually changed from milky white to light gray, and the hypha is gradually changed from smooth plush to Mao Xuzhuang with obvious granular feel. The colony back is light gray, and the center is light yellow. The mycelium is colorless and slender, has branches and smooth surfaces, and is about 1.6-2.8 mu m; the conidiophores are straight and are on the vegetative hypha, are multiple and single, and the upper ends of the conidiophores are provided with single or multiple closely arranged sporulation peduncles; conidium is planted on the top of small spore stem, and is colorless, round at both ends, short bar-shaped, single, with size of (2.39-3.66) × (5.73-7.67) μm, and the spores are adhered together by mucus and long chain.
Molecular biological identification of metarhizium anisopliae Ma 0628:
the 5 'and 3' ends of EF-1 alpha and beta-tubulin sequences of the strain Metarhizium anisopliae Ma0628 are amplified by PCR and sequenced, and the results show that the fragment sizes are 1228bp, 1154bp and 1316bp respectively. The size of the sequence fragment obtained after sequence splicing of EF-1 alpha sequence is 1734bp. The comparison result of EF-1 alpha sequences of the metarhizium anisopliae Ma0628 in the nucleic acid database of NCBI shows that the similarity of the EF-1 alpha sequences of the strain Ma0628 and the model metarhizium anisopliae strain CBS257.90 and ARSEF7929 is the highest and is 99.94%, and secondly, the similarity of the EF-1 alpha sequences of the strain and other 11 metarhizium anisopliae strains is also above 99.85%. Similarly, the comparison of the β -tubulin sequence of strain Ma0628 in the nucleic acid database of NCBI shows that the similarity of the β -tubulin sequences of strain Ma0628 and the model metarhizium anisopliae strains ARSEF7929 and ARSEF 3210 are both 100%. Phylogenetic tree analysis shows that the EF-1 alpha and beta-tubulin sequences of strain Ma0628 are clustered with known Metarrhizium anisopliae strains with a confidence of 99% and 98%, respectively.
Accordingly, a first object of the present invention is to provide a strain of Metarrhizium anisopliae (Metarhizium pingshaense) Ma0628 with deposit number GDMCC No.63616.
The invention also provides application of the metarhizium anisopliae Ma0628 in controlling tea listless amabilis.
Preferably, the tea lissajous is tea lissajous larva, pupa and adult.
Preferably, the application comprises an application step of contacting the live cells of the metarhizium anisopliae Ma0628 or a culture containing the live cells of metarhizium anisopliae Ma0628 with the tea listless beetle bodies.
Preferably, the culture containing the live metarhizium anisopliae Ma0628 is spore suspension of metarhizium anisopliae Ma0628. Such as 1.0X10 5 Individual spores/mL, 1.0X10 6 Individual spores/mL, 1.0X10 7 Individual spores/mL, 1.0X10 8 The spore suspension with the concentration of individual spores/mL can have better control effect on the tea stria.
Preferably, the concentration of the spore suspension of the metarhizium anisopliae Ma0628 is 1.0X10 7 Individual spores/mL and above.
Preferably, the application mode is a soil mixing method, an immersion method, a spraying method or the like.
The invention also provides a biological control preparation for controlling the tea-leaf vein beetles, which contains the live bacteria of the metarhizium anisopliae Ma0628 and/or a culture containing the live bacteria of the metarhizium anisopliae Ma0628.
The metarhizium anisopliae Ma0628 provided by the invention and the application thereof in the aspect of controlling tea listless amabilis have the following advantages:
1. the separated Metarhizium anisopliae Ma0628 strain is a fungus strain derived from naturally morbid tea lissajous larvae, has strong pathogenicity to tea lissajous, and has little biological harm to other soil in tea gardens, especially organic tea gardens.
2. The metarhizium anisopliae Ma0628 strain adapts to the environment of the aluminum-rich acidic tea garden soil, and has higher pathogenicity to the tea-beautiful scholartree larvae under the environment condition.
3. The metarhizium anisopliae Ma0628 strain has stronger pathogenicity to the tea striae weevil under the conditions of lower culture temperature (22 ℃) and 24-28 ℃.
4. The conidium of the metarhizium anisopliae Ma0628 strain has strong pathogenicity to the tea lissajous larvae under the condition of soil mixing application, is environment-friendly and pollution-free, is not easy to generate drug resistance, can be widely used for preventing and controlling the tea lissajous larvae in the soil emergence period, can reduce the harm of the tea lissajous adults to tea trees from the source, and has good development potential and application prospect.
Preservation description:
metarhizium pingshaense Ma0628 (Metarhizium anisopliae Ma 0628) of the present invention was deposited at the microorganism strain collection (GDMCC) of Guangdong province at the year 07 and 03, with the deposit number of GDMCC No.63616, and the deposit address of building 5, 100 th university of Mitsui, guangzhou, national institute of microbiology, guangdong province.
Drawings
FIG. 1 is a graph showing colony morphology and mycelium, conidiophore and spore morphology of Metarhizium anisopliae Ma0628 on PDA medium containing 0.1g/L chloramphenicol; wherein, FIG. a-b are front side of spore-free and spore-containing colonies, FIG. c is back side of spore-containing colonies, FIG. d is mycelium, FIG. e-f is spore-producing structure, FIG. g is spore-producing structure and spores, and FIG. h is spore chain.
FIG. 2 is an EF-1. Alpha. Phylogenetic tree of Metarrhizium anisopliae Ma0628.
FIG. 3 is a beta-tubulin phylogenetic tree of Metarhizium anisopliae Ma0628.
FIG. 4 is mortality of larvae of Rhizoma theali infected with Metarhizium anisopliae Ma0628; wherein, the graph A shows the death rate of the tea vein beetle larvae caused by inoculating the spore suspension with different concentrations by an immersion method, and the graph B shows the death rate of the tea vein beetle larvae caused by inoculating the spore suspension with different concentrations by a soil mixing method, and the numerical value is the average value plus or minus standard deviation.
FIG. 5 is mortality of adult tea-line weevils infected with Metarhizium anisopliae Ma0628 by infusion; values are mean ± standard deviation.
FIG. 6 is a phenotype map of uninfected tea-leaf beetle larvae and dead larvae and adults infected with Metarhizium anisopliae Ma0628; wherein, figure a is uninfected tea-like striated beetle larvae, figure b is dead larvae infected with metarhizium anisopliae Ma0628, and figures c and d are dead adults infected with metarhizium anisopliae Ma0628.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Entomopathogenic fungi are a class of fungi that infect insects through their body surfaces and propagate on their body surfaces and in vivo, resulting in the death of the insects when they are ill. Because of being environment-friendly, the novel pesticide has continuous control capability on pests, is not easy to generate drug resistance, and is considered to be the next generation novel biopesticide most likely to replace chemical pesticides. The inventor collects natural sick tea-like striated larva in the organic tea garden of the horizontal village of the Kochia scoparia in Shaoxing city of Zhejiang at 4 months 12 days 2021. Through separation, purification and toxicity measurement, the strain is identified to be the Metarhizium anisopliae M.pingshaense strain with the number of Ma0628, and has strong pathogenicity to the tea-beautiful scholaris larva under the condition of the soil temperature (22 ℃) when the tea-beautiful scholaris larva intensively emerges from the soil to form pupa for eclosion. In addition, the strain has higher pathogenicity to the adult tea striae at room temperature (24-28 ℃). The result shows that the metarhizium anisopliae Ma0628 strain has good application prospect in controlling the tea listless amabilis.
Example 1: separation and identification of Metarhizium anisopliae Ma0628
1.1 isolation of strains
Sterilizing naturally ill Metarhizium anisopliae with 75% alcohol surface in super clean bench for 5min, cleaning with sterile water for 3 times to remove adhered soil, and cutting with high temperature sterilized scissors into small pieces. A group of every 3 cadavers was then inoculated in an equilateral triangle shape onto potato dextrose PDA medium containing 0.1g/L chloramphenicol. Culturing in a 27 deg.C incubator for 7d, and inoculating the mycelia on PDA culture medium; after bacterial colonies with different forms, colors and sizes are picked up after bacterial colonies grow out, and the bacterial colonies are transferred to a new PDA culture medium by a plate streaking method. The above procedure was repeated until pure colonies were obtained, and stored in a refrigerator at 4℃for further use.
1.2 identification of strains
1.2.1 morphological identification
The isolated and purified strain was inoculated onto sterilized PDA medium containing 0.1g/L chloramphenicol by streaking using a sterile inoculating loop in an ultra clean bench. 2-3 sterile coverslips were inserted obliquely along the inoculation streaking site, respectively, and the PDA medium plates were placed in an incubator at 27℃for cultivation. And taking out the cover slips with hyphae at 5-10 d after inoculation, and observing the microstructures of bacterial strain hyphae, spore-forming structures and conidium forms by using an optical microscope (Kernel, VHX-7000).
On a PDA culture medium, bacterial colony is milky white at the initial stage, white at the center, fine and fluffy aerial hyphae and spread from the center to the periphery; as the culture time is prolonged, light green spores begin to appear at the colony center, the colony is gradually changed from milky white to light gray, and the hypha is gradually changed from smooth plush to Mao Xuzhuang with obvious granular feel. The colony was light gray in back and pale yellow in center (FIGS. 1 a-c). The mycelium is colorless and slender, has branches, has a septum and a smooth surface, and is about 1.6-2.8 μm (FIG. 1 d); the conidiophores are straight and grow on the vegetative hypha, are multiple single, and have single or multiple small sporophores closely arranged at the upper end (fig. 1 e-f); conidium is adhered to the top of small spore stem, is colorless, has rounded ends, is in short stick shape, is single, has size of (2.39-3.66) x (5.73-7.67) μm, and has long chain shape (figure 1 g-h).
1.2.2 molecular biological identification
Inoculating the isolated pathogenic strain into chloramphenicol containing 0.1g/LAfter 5d incubation on PDA medium, spores and hyphae were scraped off and genomic DNA was extracted using a fungal genomic DNA extraction kit (Shanghai Biotechnology, cat. No. B518259). The 5 'and 3' ends of EF-1. Alpha. Of this strain were PCR amplified and the beta-tubulin sequence was sequenced. Wherein the amplification primer of the EF-1 alpha 5' end about 1200bp sequence is EF1T (5 ' -ATGGGTAAGGARGACAAGAC-3 ')/1567R (5 ' -ACHGTRCCRATACCACCSATCTT-3 '). The amplification primer of the EF-1 alpha 3' end about 1100bp sequence is tef1fw (5'-GTGAGCGTGGTATCACCA-3')/1750-R (5'-GACGCATGTCACGGACGGC-3'). The beta-tubulin sequence amplification primers were Pseta-F (5'-ACCCTCCATTGTCTAGGACC-3')/Pseta-R (5'-CACATCATTGACGGGACTTAC-3'), respectively. The PCR reaction system is as follows: 2 XTaq Master Mix 25. Mu.L, genomic DNA template 30-60ng, 10. Mu. Mol/L upstream and downstream primers 1. Mu.L each, ddH 2 O was made up to 50. Mu.L. The PCR reaction program of the EF-1 alpha sequence 5 'and 3' end sequences and beta-tubulin is as follows: pre-denaturation at 95℃for 3min; pre-denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 20s for 30 cycles; extending at 72℃for 5min. The PCR product was then ligated intoThe Zero vector (Beijing full gold, cat No. CB 501-01) was transformed into Trans 1T 1 competent cells (Beijing full gold, cat No. CD 501-02) and positive clones were selected and sequenced. After sequencing, the 5 'end sequence and the 3' end sequence of EF-1 alpha are spliced by using Seqman software to obtain the full-length sequence. The sequencing of the resulting EF-1. Alpha. And beta. -tubulin sequence fragments was performed at NCBI for Blast analysis of their homology. Downloading EF-1 alpha and beta-tubulin sequences of a kindred species, performing multi-sequence alignment on the EF-1 alpha and beta-tubulin sequences of the isolated strain and the kindred species sequences by using ClustalW of MEGA 5.0 software, and then constructing a molecular phylogenetic tree by using a neighbor-joining method and checking the molecular phylogenetic tree, wherein a Bootstrap value is 1000.
The 5 'and 3' ends of EF-1 alpha and beta-tubulin sequences of the strain Metarhizium anisopliae Ma0628 are amplified by PCR and sequenced, and the results show that the fragment sizes are 1228bp, 1154bp and 1316bp respectively. The size of a sequence fragment obtained by sequence splicing of the EF-1 alpha sequence is 1734bp, and the nucleotide sequence of the sequence fragment is shown as SEQ ID NO. 1. The comparison result of the Metarhizium anisopliae Ma0628 EF-1 alpha sequence in the nucleic acid database of NCBI shows that the similarity of the EF-1 alpha sequences of the strain Ma0628 and the model Metarhizium anisopliae strain CBS257.90 and ARSEF7929 is the highest and is 99.94%, and secondly, the similarity of the EF-1 alpha sequences of the strain and other 11 Metarhizium anisopliae strains is also above 99.85%. Similarly, the comparison of the β -tubulin sequence of strain Ma0628 (the nucleotide sequence of which is shown in SEQ ID No. 2) in the NCBI nucleic acid database shows that the similarity of the β -tubulin sequences of strain Ma0628 and the metarhizium anisopliae strains ARSEF7929 and ARSEF 3210 is 100%. Analysis of the phylogenetic tree of FIGS. 2 and 3 showed that the EF-1α and β -tubulin sequences of strain Ma0628 clustered together with known Metarrhizium anisopliae strains with 99% and 98% confidence, respectively.
Morphological and molecular biological identification results show that the obtained strain is metarhizium anisopliae and is named metarhizium anisopliae (Metarhizium pingshaense) Ma0628.
Example 2: determination of pathogenicity of Metarhizium anisopliae Ma0628 on larvae of Pharhizoma tea
1.1 preparation of spore suspension
Adding distilled water (2000 times of Tween-80 diluent) containing 0.5 per mill (v/v) Tween-80 into a super clean bench to wash out spores in the dish, transferring into a sterilized triangular flask, stirring at medium speed for 20min with a magnetic stirrer, and dispersing to obtain mother liquor. The stock solution was then diluted by 10-fold dilution and counted under a microscope with a blood cell counting plate, 3 plate replicates. Dilution of spore suspension to 1X 10 8 Per mL, 1X 10 7 Per mL, 1X 10 6 Per mL, 1X 10 5 And (3) one/mL for later use.
1.2 inoculation of test insects
Dipping method: placing the mature larva of the healthy tea lissajous with concentration of 1.0X10 5 ～1.0×10 8 Each/mL of the spore suspension was immersed for 20s and then transferred to a small culture cup (height and width: 4 cm. Times.4.5 cm) containing 20g of soil. Immersing the adults of the Philippine tea in 1.0X10 by the same method 8 The individual/mL spore suspension was then placed in an insect cage (length. Times. Width. Times. Height: 20 cm. Times.18 cm)X 38 cm) were fed with fresh tea shoots.
And (3) a soil mixing method: will be mixed with 2mL of 1.0X10-concentration respectively 5 ～1.0×10 8 20g of soil of each/mL spore suspension was transferred to a small culture cup, and then the healthy aged larvae of Thermomyces lanuginosus were transferred to the culture cup (height and width: 4 cm. Times.4.5 cm).
Each concentration gradient was repeated 3 times, and 10 replicates of the control group of the insect-dipping method and the soil-mixing method were treated with a sterile aqueous solution containing 0.5%o (v/v) tween-80. All larvae were incubated in an incubator at 22℃with a photoperiod 16L:8D and a Relative Humidity (RH) of 70%. The adults are kept under room temperature (24-28 ℃ and RH 70% + -5%, 16L: 8D) for feeding. Starting on day 3 after treatment, observations were made 1 time per day, and the death number of the test insects was recorded. The growth of hyphae (the hyphae growing on the insect corpse can be seen with naked eyes) is observed by the wet-keeping culture of dead insects, and the insect state of the infected stiff insects is recorded by photographing.
The statistics of mortality from infection with the larvae of the tea-liriope are shown in figure 4. As can be seen from FIG. 4, under the culture condition of 22℃1X 10 was used respectively 5 、1×10 6 、1×10 7 And 1X 10 8 After inoculating the larva of the tea-line weevil with the spore suspension of each/mL by the dipping method for 11 days, the mortality rate of the larva of the tea-line weevil reaches 60.00%, 63.33%, 73.33% and 76.67%, respectively (fig. 4A); after 11 days of inoculation with the larch larvae by the soil mixing method, the mortality rates of the larch larvae reached 60.00%, 76.67%, 80% and 76.67%, respectively (fig. 4B). The statistics of mortality from adult tea-liriope infection are shown in figure 5. As can be seen from FIG. 5, the adults of the Philippine tea had been exposed to 1X 10 under room temperature culture conditions 8 After each/mL of metarhizium anisopliae spore suspension, the mortality rate was 83.33% on day 14, whereas the mortality rate of 0.05%o (v/v) Tween-80 treated control group adult tea weevil was only 3.33%. In addition, the white hyphae and olive green spores were evenly distributed on the surfaces of the green muscardine fungus spore suspension-treated tea leaf beetle larvae and adults (fig. 6 b-c), while the control group did not have symptoms of infection (fig. 6 a).
In conclusion, the metarhizium anisopliae Ma0628 provided by the invention is a biological fungus for preventing and treating the tea lism, has the advantages of strong pathogenicity to tea lism larva and adults, environmental protection, no pollution and difficult generation of drug resistance, and can be widely used for preventing and treating tea lism in the peak period of soil emergence.
Claims (9)
1. A strain of metarhizium anisopliae (Metarhizium pingshaense) Ma0628, characterized by the deposit number GDMCC No.63616.
2. The use of metarhizium anisopliae Ma0628 according to claim 1 for controlling cyamopsis grossedentata.
3. The use according to claim 2, wherein the cyamopsis grossedentata is cyamopsis grossedentata larvae, pupae and adults.
4. The use according to claim 2, characterized in that it comprises an administration step of contacting the living cells of metarhizium anisopliae Ma0628 or a culture containing the living cells of metarhizium anisopliae Ma0628 with the bodies of the species euonymus theani.
5. The use according to claim 4, wherein the culture containing live cells of metarhizium anisopliae Ma0628 is a spore suspension of metarhizium anisopliae Ma0628.
6. The use according to claim 5, wherein the spore suspension of Metarhizium anisopliae Ma0628 has a concentration of 1.0X10 5 Individual spores/mL and above.
7. The use according to claim 6, wherein the spore suspension of Metarhizium anisopliae Ma0628 has a concentration of 1.0X10 7 Individual spores/mL and above.
8. The use according to claim 4, wherein the application is by soil mixing, dipping or spraying.
9. A biological control agent for controlling cyamopsis pinicola, characterized by comprising the living cells of metarhizium anisopliae Ma0628 according to claim 1 and/or a culture comprising the living cells of metarhizium anisopliae Ma0628 according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311126848.4A CN117187078A (en) | 2023-09-04 | 2023-09-04 | Metarhizium anisopliae strain and application thereof in preventing and treating tea lissajous |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311126848.4A CN117187078A (en) | 2023-09-04 | 2023-09-04 | Metarhizium anisopliae strain and application thereof in preventing and treating tea lissajous |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117187078A true CN117187078A (en) | 2023-12-08 |
Family
ID=88986263
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311126848.4A Pending CN117187078A (en) | 2023-09-04 | 2023-09-04 | Metarhizium anisopliae strain and application thereof in preventing and treating tea lissajous |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117187078A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117701395A (en) * | 2022-09-15 | 2024-03-15 | 慕恩（广州）生物科技有限公司 | Metarhizium anisopliae and application thereof |
-
2023
- 2023-09-04 CN CN202311126848.4A patent/CN117187078A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117701395A (en) * | 2022-09-15 | 2024-03-15 | 慕恩（广州）生物科技有限公司 | Metarhizium anisopliae and application thereof |
| CN117701395B (en) * | 2022-09-15 | 2024-06-04 | 湖南慕恩生物科技有限公司 | Metarhizium anisopliae and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102776130B (en) | Metarhizium anisopliae and application thereof | |
| CN102363750B (en) | Insecticidal fungus and application thereof | |
| KR101785098B1 (en) | Composition for simultaneous control of both aphid and Pythium ultimum using Isaria javanica Pf185 and its culture media | |
| Kawuri et al. | Destructive leaf rot disease caused by Fusarium oxysporum on Aloe barbadensis Miller in Bali | |
| CN112143658B (en) | Beauveria bassiana strain MQ-08 and application and microbial preparation thereof | |
| CN106479943A (en) | The Java Isaria BE01 of one plant height effect preventing and treating fall webworms and its application | |
| CN111925963A (en) | Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease | |
| CN117187078A (en) | Metarhizium anisopliae strain and application thereof in preventing and treating tea lissajous | |
| Arzanlou et al. | Fruit rot of olive (Olea europaea) caused by Truncatella angustata | |
| CN113308385B (en) | Isaria javanicus strain DMC01 and application thereof in prevention and treatment of locusta migratoria in east Asia | |
| CN112553086B (en) | Violet purpurea bacterial strain and application thereof in preventing and treating phyllotreta striolata | |
| CN111808888B (en) | Chinese fir endophytic fungi fermentation filtrate and extract thereof, and preparation method and application of Chinese fir endophytic fungi fermentation filtrate and extract | |
| CN113061559A (en) | Composite biological agent and application thereof in preventing and treating rice blast and promoting rice growth | |
| CN114806898B (en) | Beauveria bassiana BbKMND202111 strain and application thereof | |
| CN105112315B (en) | A kind of tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion endophyte Alcaligenes faecalis bacterial strain | |
| CN114606140B (en) | Isaria fumosorosea and application thereof in controlling diaphorina citri | |
| CN113373065B (en) | Isaria javanicus strain DMC01 and application thereof in prevention and treatment of pine caterpillars | |
| Smith et al. | Pathogenicity of the fungus, Colletotrichum gloeosporioides (Penz.) Sacc., to Eurasian watermilfoil (Myriophyllum spicatum L.) | |
| CN106591153B (en) | One plant of Metarhizium Strains and its application to carpocapsa pononella highly pathogenicity | |
| CN115161240A (en) | Bacillus velezensis strain and application thereof | |
| CN105274007B (en) | One plant of Metarhizium anisopliae var. Anisopliae MaTS02 and its application in terms of preventing and treating Bemisia tabaci | |
| CN114958622B (en) | Metarhizium anisopliae and application thereof in preventing and treating tea lissajous | |
| CN112760237A (en) | Cladosporium cladosporioides strain with strong pathogenicity to diaphorina citri and application thereof | |
| CN112961782A (en) | Pediococcus kaoliang, microbial inoculum and herbicide containing same and application thereof | |
| KR101785099B1 (en) | Composition for simultaneous control of both aphid and Sclerotinia sclerotiorum using Beauveria bassiana Bb18 and its culture media |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |