CN115927208A - Fungal virus, attenuated strain, plant disease control agent and application - Google Patents
Fungal virus, attenuated strain, plant disease control agent and application Download PDFInfo
- Publication number
- CN115927208A CN115927208A CN202211008989.1A CN202211008989A CN115927208A CN 115927208 A CN115927208 A CN 115927208A CN 202211008989 A CN202211008989 A CN 202211008989A CN 115927208 A CN115927208 A CN 115927208A
- Authority
- CN
- China
- Prior art keywords
- virus
- fungal
- attenuated strain
- strain
- fungal virus
- 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
- 241000700605 Viruses Species 0.000 title claims abstract description 117
- 230000002538 fungal effect Effects 0.000 title claims abstract description 81
- 230000002238 attenuated effect Effects 0.000 title claims abstract description 66
- 201000010099 disease Diseases 0.000 title claims abstract description 27
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 27
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 31
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims abstract description 28
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
- 241000233866 Fungi Species 0.000 claims abstract description 20
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 10
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 8
- 244000000004 fungal plant pathogen Species 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 18
- 102000039446 nucleic acids Human genes 0.000 claims description 12
- 108020004707 nucleic acids Proteins 0.000 claims description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 12
- 241000193738 Bacillus anthracis Species 0.000 claims description 9
- 230000003032 phytopathogenic effect Effects 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 4
- 241001345406 Colletotrichum alienum Species 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 230000001018 virulence Effects 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 16
- 239000005730 Azoxystrobin Substances 0.000 abstract description 13
- WFDXOXNFNRHQEC-GHRIWEEISA-N azoxystrobin Chemical compound CO\C=C(\C(=O)OC)C1=CC=CC=C1OC1=CC(OC=2C(=CC=CC=2)C#N)=NC=N1 WFDXOXNFNRHQEC-GHRIWEEISA-N 0.000 abstract description 12
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 abstract description 10
- 239000006013 carbendazim Substances 0.000 abstract description 10
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 abstract description 10
- 239000005820 Prochloraz Substances 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 8
- TVLSRXXIMLFWEO-UHFFFAOYSA-N prochloraz Chemical compound C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl TVLSRXXIMLFWEO-UHFFFAOYSA-N 0.000 abstract description 8
- 230000012202 endocytosis Effects 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 description 24
- 239000002245 particle Substances 0.000 description 24
- 210000001938 protoplast Anatomy 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000000975 dye Substances 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 108060004795 Methyltransferase Proteins 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 239000000843 powder Substances 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- 230000007918 pathogenicity Effects 0.000 description 9
- 108090000565 Capsid Proteins Proteins 0.000 description 8
- 102100023321 Ceruloplasmin Human genes 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 241000222199 Colletotrichum Species 0.000 description 7
- 208000031888 Mycoses Diseases 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241001529387 Colletotrichum gloeosporioides Species 0.000 description 4
- 244000070406 Malus silvestris Species 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 241000220324 Pyrus Species 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 244000053095 fungal pathogen Species 0.000 description 4
- WXVXYGWYSRYKKS-UHFFFAOYSA-N methyl n-(1h-benzimidazol-2-yl)carbamate;n-propyl-n-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboxamide Chemical compound C1=CC=C2NC(NC(=O)OC)=NC2=C1.C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl WXVXYGWYSRYKKS-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 210000002845 virion Anatomy 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- 241001639769 Colletotrichum fructicola Species 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 240000001238 Gaultheria procumbens Species 0.000 description 3
- 235000007297 Gaultheria procumbens Nutrition 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000010394 Solidago odora Nutrition 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 239000003899 bactericide agent Substances 0.000 description 3
- 239000013043 chemical agent Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 235000019198 oils Nutrition 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 229910001868 water Inorganic materials 0.000 description 3
- FYGHSUNMUKGBRK-UHFFFAOYSA-N 1,2,3-trimethylbenzene Chemical compound CC1=CC=CC(C)=C1C FYGHSUNMUKGBRK-UHFFFAOYSA-N 0.000 description 2
- QPUYECUOLPXSFR-UHFFFAOYSA-N 1-methylnaphthalene Chemical compound C1=CC=C2C(C)=CC=CC2=C1 QPUYECUOLPXSFR-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 101710134681 40 kDa protein Proteins 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 235000014443 Pyrus communis Nutrition 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 244000269722 Thea sinensis Species 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000021016 apples Nutrition 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000000443 biocontrol Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 235000021017 pears Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- UOHMMEJUHBCKEE-UHFFFAOYSA-N prehnitene Chemical compound CC1=CC=C(C)C(C)=C1C UOHMMEJUHBCKEE-UHFFFAOYSA-N 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000002562 thickening agent Substances 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- ARXJGSRGQADJSQ-UHFFFAOYSA-N 1-methoxypropan-2-ol Chemical compound COCC(C)O ARXJGSRGQADJSQ-UHFFFAOYSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- OAYXUHPQHDHDDZ-UHFFFAOYSA-N 2-(2-butoxyethoxy)ethanol Chemical compound CCCCOCCOCCO OAYXUHPQHDHDDZ-UHFFFAOYSA-N 0.000 description 1
- DGZSVBBLLGZHSF-UHFFFAOYSA-N 4,4-diethylpiperidine Chemical compound CCC1(CC)CCNCC1 DGZSVBBLLGZHSF-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 235000004936 Bromus mango Nutrition 0.000 description 1
- 241000526900 Camellia oleifera Species 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 241000565359 Fraxinus chinensis Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 241001533440 Hypovirus Species 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 240000007228 Mangifera indica Species 0.000 description 1
- 235000014826 Mangifera indica Nutrition 0.000 description 1
- 241000289203 Metarhizium brunneum Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000005561 Musa balbisiana Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 244000025272 Persea americana Species 0.000 description 1
- 235000008673 Persea americana Nutrition 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- HDSBZMRLPLPFLQ-UHFFFAOYSA-N Propylene glycol alginate Chemical compound OC1C(O)C(OC)OC(C(O)=O)C1OC1C(O)C(O)C(C)C(C(=O)OCC(C)O)O1 HDSBZMRLPLPFLQ-UHFFFAOYSA-N 0.000 description 1
- 239000004373 Pullulan Substances 0.000 description 1
- 229920001218 Pullulan Polymers 0.000 description 1
- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 240000003829 Sorghum propinquum Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 235000009184 Spondias indica Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 235000006468 Thea sinensis Nutrition 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 230000000895 acaricidal effect Effects 0.000 description 1
- 239000000642 acaricide Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 235000010407 ammonium alginate Nutrition 0.000 description 1
- 239000000728 ammonium alginate Substances 0.000 description 1
- KPGABFJTMYCRHJ-YZOKENDUSA-N ammonium alginate Chemical compound [NH4+].[NH4+].O1[C@@H](C([O-])=O)[C@@H](OC)[C@H](O)[C@H](O)[C@@H]1O[C@@H]1[C@@H](C([O-])=O)O[C@@H](O)[C@@H](O)[C@H]1O KPGABFJTMYCRHJ-YZOKENDUSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 239000005667 attractant Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 229910052570 clay Inorganic materials 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- HQQADJVZYDDRJT-UHFFFAOYSA-N ethene;prop-1-ene Chemical group C=C.CC=C HQQADJVZYDDRJT-UHFFFAOYSA-N 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- -1 for example Proteins 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008958 fungal pathogenicity Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 230000009643 growth defect Effects 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000028644 hyphal growth Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 239000003350 kerosene Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000005645 nematicide Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000361 pesticidal effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920001495 poly(sodium acrylate) polymer Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000010409 propane-1,2-diol alginate Nutrition 0.000 description 1
- 239000000770 propane-1,2-diol alginate Substances 0.000 description 1
- 235000019423 pullulan Nutrition 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- NNMHYFLPFNGQFZ-UHFFFAOYSA-M sodium polyacrylate Chemical compound [Na+].[O-]C(=O)C=C NNMHYFLPFNGQFZ-UHFFFAOYSA-M 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 239000006097 ultraviolet radiation absorber Substances 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a fungal virus, an attenuated strain, a plant disease control agent and application, and relates to the technical field of biological control, wherein the plant disease control agent has the function of inhibiting plant disease fungi, and has 2 double-stranded RNAs with 1.6-1.8 kb, and the nucleotide sequences of the 2 double-stranded RNAs are shown as SEQ ID NO 1 and SEQ ID NO 2. The fungal virus provided by the invention can influence the growth, spore morphology and endocytosis of host hyphae and the sensitivity of a host to medicaments such as carbendazim, prochloraz and azoxystrobin, so that the aim of efficiently preventing and treating plant diseases is fulfilled.
Description
Technical Field
The invention relates to the technical field of biological control, and particularly relates to a fungal virus, an attenuated strain, a plant disease control agent and application thereof.
Background
Among plant diseases, fungi are one of the most important pathogens, about 80% of the plant diseases are caused by fungi, huge economic losses are caused to agricultural production every year, the prevention and the treatment of the fungal diseases are mainly dependent on chemical agents at present, the continuous application of chemical pesticides is easy to cause the emergence of drug-resistant strains, the drug effect is reduced, the cost is increased, the environment is polluted, and the human health is harmed, so that the search for an efficient and safe prevention and treatment means has important significance for the prevention and the treatment of the fungal diseases.
Fungal viruses (Fungal viruses or mycoviruses) are a class of viruses that infect fungi and are found in a wide variety of fungi including phytopathogenic fungi. Most of the fungal viruses are asymptomatic mixed infection in a host, and some of the fungal viruses with weak toxicity (hypoviruses) can generally cause growth defects, pigment synthesis reduction, spore production reduction, pathogenicity decline and the like of the host, can be used as potential biocontrol factors in biological control of plant fungal diseases, and is used for controlling the plant fungal diseases. Therefore, the excavation of low-toxicity mycovirus with biocontrol potential has great significance for utilizing the mycovirus to carry out plant fungal diseases.
Colletotrichum spp is a very important group of plant pathogenic fungi, which cause diseases of various woody and herbaceous plants. The pathogenic bacteria of the genus can infect plants of up to 30 genera, and cause important loss to fruits with high yield and high economic value, such as mango, strawberry, orange, avocado, banana and the like; the harm to corn, sorghum and other grains and sugarcane is serious, the economic loss caused by the pathogenic fungi is serious, the destructive effect is strong, and the pathogenic fungi are selected as the eighth important plant pathogenic fungi in the world based on the scientific and economic values. At present, the disease is mainly controlled by a chemical method, but a series of related problems such as ecological balance destruction, environmental pollution, pathogenic bacteria resistance and the like caused by long-term use of chemical agents are more and more serious.
Disclosure of Invention
In order to solve the technical problem of the colletotrichum aiming at the diseases caused by woody plants and herbaceous plants, the invention provides the following scheme:
the fungal virus has the function of inhibiting plant disease fungi, and has 2 double-stranded RNAs with the length of 1.6-1.8 kb, and the nucleotide sequences of the 2 double-stranded RNAs are shown as SEQ ID NO. 1 and 2.
Nucleic acid having at least the sequences in SEQ ID NO 1 and 2 or the sequence of a specific part of the sequences having a defined function.
The attenuated strain contains the fungal virus, is classified and named as exotic anthrax (Colletotrichum alienum) YC-31, and has a preservation number of CCTCCM 20221146.
A plant disease control agent comprising at least said fungal virus and/or said attenuated strain.
According to the application of the fungal virus or attenuated strain in inhibiting the pathogenicity of plant pathogenic fungi, the plant pathogenic fungi refer to: colletotrichum pathogenic fungi.
According to the application of the fungal virus in extracellular infection of plant pathogenic fungi, the plant pathogenic fungi refer to: colletotrichum pathogenic fungi.
According to the application of the fungal virus or the attenuated strain in preparing the plant disease control agent caused by the plant pathogenic fungi, the plant pathogenic fungi refer to: colletotrichum pathogenic fungi.
A method for attenuating phytopathogenic fungi by infecting the phytopathogenic fungi in vitro with said fungal virus.
A method for controlling a plant pathogenic fungus, which comprises adding the plant disease controlling agent to a plant.
The invention provides a fungal virus and an attenuated strain with the fungal virus, wherein the fungal virus has low toxicity, and the attenuated strain can inhibit the pathogenicity of plant pathogenic fungi, particularly because the attenuated strain contains the fungal virus, the fungal virus can influence the growth of host hypha, the spore morphology, the endocytosis and the sensitivity of a host to medicaments such as carbendazim, prochloraz and azoxystrobin.
In addition, the fungal virus can be trans-propagated to pathogenic fungi such as fruit anthrax (C.fructicola), ash tree anthrax (C.spaetherianum) and colletotrichum gloeosporioides (C.gloeosporioides) through virus particle transfection, so as to reduce the pathogenicity of the pathogenic fungi.
The attenuated strain is a camellia oleifera fruit which is separated from a permanently defined area in Zhang Jiajiu city in Hunan province, and the attenuated strain is named as alien anthrax bacteria YC-31 (Colletotrichum alienum) according to classification after separation and identification, and is deposited in the China center for type culture collection at 21.07.2022, with the address: the preservation number of the Wuhan university in Wuhan, china is CCTCC NO: M20221146.
Further research shows that the attenuated strain YC-31 contains 2 naturally-occurring mycoviruses with double-stranded RNA of 1.6-1.8 kb, the whole genome sequence of the mycoviruses is obtained by a high-throughput sequencing technology and a ligase-mediated terminal cloning method, the genome sequence is named as Colletotrichum alisimum partivivus 1, called CaPV1 for short, and the gene of the mycoviruses CaPV1 has the nucleotide sequence shown as SEQ ID NO: 1-2 genomic sequences. The fungal virus CaPV1 contains conserved motifs of RdRp (RNA-dependent RNA polymerase) and CP (capsid protein) in the sequence of double-stranded RNA, and the nucleotide sequence encoding RdRp has homology with the known Metarhizium brunneum partitivrus 2, and the nucleotide sequence of CP has homology with the known Colletotrichum partitivrus 1. Therefore, the fungal virus CaPV1 is presumed to be a novel fungal virus based on genome structure analysis, sequence homology comparison and phylogenetic analysis.
The attenuated strain YC-31 is detoxified by a protoplast regeneration technology to obtain the detoxified strains YC-31-P23 and YC-31-P29 of the attenuated strain YC-31, and the pathogenic force of the detoxified germs is recovered through research. The fungal virus CaPV1 provided by the invention can be transmitted to a detoxified strain YC-31-P23 through virus particle transfection, and then an attenuated strain YC-31-P23-T1 is obtained, and the pathogenicity of the obtained attenuated strain is obviously weakened. Therefore, it was concluded that the fungal virus CaPV1 reduces the virulence of the host.
According to observation, the attenuated strain YC-31 provided by the invention has sparse hypha and less branches, while the attenuated strain YC-31-P23 has dense hypha and more branches, so that the fungus attenuated CaPV1 is presumed to influence the growth of host hypha. In addition, in the spore morphology, conidia of the attenuated strain YC-31 are cylindrical, elliptical or long-line shaped, the two ends are blunt or one end is slightly sharp, and abnormal long-line type conidia appear, and the size is 10.1-26.7 × 3.9-8.9 μm (av =15.92+0.65 × 5.81+0.15, n = 50). And the conidiospore of the detoxified strain YC-31-P23 is cylindrical and oval, the two ends are blunt and round or one end is slightly pointed, the size of the conidiospore is 7.5-15.2X 3.5-6.4 mu m (av =12.04+ 0.28X 4.83+0.08, n = 50), so that the influence of the fungal virus CaPV1 on the host conidiospore morphology can be obtained.
The hyphae of the virulent strain YC-31 and the detoxified strain YC-31-P23 are stained by FM4-64 dye, and the endocytosis of the hyphae is observed by a fluorescence microscope. The results show that the dye is absorbed very rapidly in the attenuated strain YC-31-P23, whereas in the attenuated strain YC-31 provided by the invention, the dye is prevented from entering the hyphae obviously, and a small amount of dye is not absorbed by the hyphae until 30min. Thus, it was demonstrated that the fungal virus CaPV1 was able to influence the endocytosis of the host.
The attenuated strain YC-31 and the detoxified strain YC-31-P23 provided by the invention are subjected to medicament sensitivity determination by using carbendazim, prochloraz and azoxystrobin. The sensitivity of the attenuated strain YC-31 provided by the invention to carbendazim and prochloraz is higher than that of the detoxified strain YC-31-P23, and the sensitivity to azoxystrobin is lower than that of the detoxified strain YC-31-P23. It is therefore speculated that the fungal virus CaPV1 influences the host's sensitivity to the agent, resulting in an increased sensitivity to carbendazim and prochloraz and a decreased sensitivity to azoxystrobin.
The fungal virus CaPV1 provided by the invention is trans-propagated to a fruit anthrax (C.fructicola) Cf10-6 strain and a LY5-1 strain, a Fraxinus chinensis (C.spaothenianum) Cs4-1 strain and a Colletotrichum gloeosporioides Cg2-1 strain through virion transfection, and the obtained virus strain also shows obvious reduction of pathogenicity. Therefore, the fungal virus CaPV1 can infect host bacteria through particle transfection, so that the host bacteria contain the fungal virus to reduce the pathogenicity of plant pathogenic fungi and achieve the aim of biologically preventing and treating plant fungal diseases.
The fungal virus provided by the invention can influence the growth, spore morphology and endocytosis of host hyphae and the sensitivity of a host to medicaments such as carbendazim, prochloraz and azoxystrobin, so that the aim of efficiently preventing and treating plant diseases is fulfilled.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings required for the use in the present invention will be briefly described below, it should be understood that the following drawings only show some embodiments of the present invention and therefore should not be considered as limiting the scope, and that for those skilled in the art, other related drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a colony map of attenuated strain YC-31 provided by the present invention;
FIG. 2 is an electrophoretogram of double-stranded RNA in attenuated strain YC-31 provided by the present invention, wherein: m is 5000bp Marker;
FIG. 3 is a schematic structural diagram of the genome of a fungal virus CaPV1 provided by the present invention;
FIG. 4 is a phylogenetic tree constructed by the sequence of the fungal virus CaPV1 RdRp provided by the present invention;
FIG. 5 shows virus particles of the extracted mycovirus CaPV1 from the attenuated strain YC-31 provided by the present invention, wherein: a: protein electrophoresis pattern of virus particles of fungal virus CaPV1, B: electron micrograph of virus particles of fungal virus CaPV1, C: a dsRNA electrophoresis picture after the extraction of the virus particle nucleic acid of the fungal virus CaPV 1;
FIG. 6 is a colony map of the attenuated strain YC-31, the detoxified strains YC-31-P23 and YC-31-P29, and the detoxified recaptured strain YC-31-P23-T1 provided by the present invention on PDA medium;
FIG. 7 is a result chart of the inoculation of pear, camellia sinensis leaves and apple with the strains YC-31, YC-31-P23 and YC-31-P29 of the invention, and the detoxified and detoxified strains YC-31-P23-T1 respectively;
FIG. 8 is a map showing hyphal tips and spore morphology of attenuated strain YC-31 and attenuated strain YC-31-P23 according to the present invention;
FIG. 9 is a photograph of the attenuated strain YC-31 and the attenuated strain YC-31-P23 of the present invention, which were observed under a fluorescence confocal microscope after the hyphae were stained with a plasma membrane FM4-64 dye;
FIG. 10 shows the sensitivity of attenuated strain YC-31 and detoxified strain YC-31-P23 to carbendazim, prochloraz and azoxystrobin;
FIG. 11 shows the study on the control of plant pathogenic fungi by the fungal virus CaPV1 of the present invention, wherein: cf10-6-V, LY5-1-V, cs4-1-V, and Cg2-1-V represent strains infected with the virus.
Detailed Description
The technical solution of the present invention will be described below with reference to the accompanying drawings.
< fungal viruses according to the present invention >
The fungal virus provided by the invention is a fungal virus which is extracted from an attenuated strain YC-31 and has the capability of inhibiting the pathogenicity of plant pathogenic fungi, is named as Colletrichum alienum partitivrus 1 (called CaPV1 for short), and has 2 double-stranded RNAs with 1.6-1.8 kb. Each virus particle of the fungal virus has 2 double-stranded RNAs (double-stranded RNAs) of 1.6-1.8 kb, the 2 double-stranded RNAs are wrapped in one virus particle, and colonies of an attenuated strain YC-31 exist and pass at the same time, and are shown in figure 1.
The fungal virus contains a conserved motif of RdRp (RNA-dependent RNA polymerase) in the sequence of double-stranded RNA, and the nucleotide sequence of the RNA encoding this region is highly homologous to Metharhizium brunneum partiivus 2 (McPV 2) virus and to the region encoding RdRp in the genome of a virus of the genus of the family Bisoviridae. The fungal virus contains a conserved motif of CP (capsid protein) in the sequence of double-stranded RNA, and the nucleotide sequence of RNA encoding the region is highly homologous with Colletrichum partiticurus 1 (CPV 1) virus and has homology with the region encoding CP in the genome of a virus of the genus of the family Bisoviridae.
The invention carries out sequence determination on the fungal virus CaPV1 to obtain a full-length nucleotide sequence. Thus, the present invention encompasses all of the fungal viral genes, nucleic acids having the nucleotide sequences or a portion thereof, proteins encoded by the nucleotide sequences, and the like.
Nucleotide sequences of 2 double-stranded RNAs of the virus are represented by SEQ ID NO:1 to 2. The present invention also encompasses nucleic acids all having all or a part of the above nucleotide sequences. The nucleic acid may be double-stranded or single-stranded, and DNA, cDNA, RNA, and the like are all included. For example, SEQ ID NO:1 to 2, a specific partial sequence having a predetermined function in the sequence, a cDNA having a nucleotide sequence equivalent to these sequences, a recombinant vector (such as a plasmid or a virus) into which a nucleotide sequence equivalent to these sequences has been inserted, and the like are included in the present invention.
The results of the sequence analysis showed that the sequence shown in SEQ ID NO:1, a conserved motif of RdRp (RNA-dependent RNA polymerase), SEQ ID NO:2, there is a conserved motif of CP (capsid protein). Therefore, for example, nucleic acids or recombinant vectors having at least these sequence portions as specific portions having predetermined functions can be prepared and used according to the purpose or use.
The nucleic acid (or gene) of the present invention also includes nucleic acids having homology with the above-mentioned nucleotide sequences, for example, nucleic acids which hybridize under stringent conditions with nucleic acids consisting of nucleotide sequences complementary to the nucleotide sequences and have a fungal pathogenicity-inhibiting effect.
As the host, a known host such as Escherichia coli, yeast, cultured cell, etc. can be used. Considering that fungal viruses infect fungi, yeasts are most suitable as hosts, for example, because they have high proliferation properties and are relatively easy to use. As the recombinant vector, a known vector can be used. Inserted with SEQ ID NO:1 or 2, and co-expressing the two types of genes to reconstitute a fungal virus. In this case, although it is possible to use a known host and a known recombinant vector, yeast may be most suitable from the viewpoint of allowing simultaneous introduction of a plurality of vectors.
< preparation of plant pathogenic fungus Virus >
Since the fungal virus CaPV1 of the present invention is present in a predetermined foreign anthrax YC-31 strain, the fungal virus can be obtained by separating and recovering the virus from the foreign anthrax YC-31 strain infected with the fungal virus CaPV 1.
As a method for separating and recovering a virus, a known method can be used. For example, the cells may be frozen with liquid nitrogen or the like, disrupted, suspended in a predetermined buffer solution, and then virus may be isolated by ultracentrifugation or the like, whereby the virus can be recovered.
However, the fungal virus CaPV1 of the present invention is not limited to only viruses obtained by such production methods. That is, for example, a virus obtained by separating and recovering the anthrax bacteria including the fungal virus CaPV1 is also included in the present invention in a broad sense.
< methods for attenuating phytopathogenic fungi >
The fungal virus CaPV1 of the present invention can be used to infect a specific plant pathogenic fungus or the like, thereby attenuating the fungus.
< Agents for controlling plant diseases >
The plant disease controlling agent of the present invention comprises at least either one of the fungal virus CaPV1 provided by the present invention or the attenuated strain YC-31 provided by the present invention, or may comprise both the fungal virus CaPV1 and the attenuated strain YC-31, or may comprise other components.
The other components include, for example, a predetermined carrier, a binder, a thickener, a fixing agent, an antiseptic and antifungal agent, a solvent, a stabilizer, an antioxidant, an ultraviolet absorber, a crystal precipitation inhibitor, a defoaming agent, a physical property improving agent, a coloring agent, and the like. In addition, other pesticidal ingredients such as acaricides, nematicides, bactericides, antivirals, attractants, herbicides, plant growth regulators, synergists and the like may also be contained.
As the carrier, for example, a solid carrier and/or a liquid carrier can be used. Examples of the solid carrier include animal and plant powders such as starch, activated carbon, soybean powder, wheat flour, wood powder, fish powder and milk powder, and mineral powders such as talc, kaolin, bentonite, zeolite, diatomaceous earth, white carbon, clay, alumina, calcium carbonate, potassium chloride and ammonium sulfate. Examples of the liquid carrier include alcohols such as water, isopropyl alcohol and ethylene glycol, ketones such as cyclohexanone and methyl ethyl ketone, ethers such as propylene glycol monomethyl ether and diethylene glycol mono-N-butyl ether, aliphatic hydrocarbons such as kerosene and light oil, aromatic hydrocarbons such as xylene, trimethylbenzene, tetramethylbenzene, methylnaphthalene and solvent naphtha, amides such as N-methyl-2-pyrrolidone, glycerides of fatty acids, and vegetable oils such as soybean oil and rapeseed oil.
Examples of the binder, thickener, and fixing agent include starch, dextrin, cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, hydroxymethyl starch, pluronic, pullulan, sodium alginate, ammonium alginate, propylene glycol alginate, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene-propylene block polymer, sodium polyacrylate, and polyvinyl pyrrolidone.
The formulation of the control agent is not particularly limited, and may be in the form of, for example, an emulsion, a suspension, a powder, a granule, a tablet, a hydrate, an aqueous solvent, a liquid, a suspension, a granule hydrate, an aerosol, a paste, an oil, an emulsion, or the like.
Examples of the method of applying the controlling agent to the plant include a method of applying the controlling agent to the surface or back surface of the leaf, a method of attaching the controlling agent to the surface or back surface of the leaf using a predetermined carrier or the like, and a method of scattering or supplying the controlling agent to the leaf.
The amount of the control agent to be applied or applied to the surface of the plant is appropriately selected depending on various conditions such as the concentration of the active ingredient, the form of the preparation, the type of the target disease or crop, the degree of damage caused by the disease, the site of application, the method of application, the time of application, the mixing, and the amount and type of the chemical agent or fertilizer to be used.
Example 1:
isolation of attenuated Strain YC-31
Separating the tissues of the boundary of a disease key by adopting a conventional tissue separation method from the field oil tea fruits in the eternal region of Zhang Jiajie city in Hunan province, sterilizing for 1min by 70% alcohol, sterilizing for 1min by mercuric chloride, rinsing with sterile water for three times, then soaking the surface water by using sterilized filter paper, transferring the filter paper to a PDA plate by using sterilized tweezers, and carrying out inverted culture at 28 ℃. The grown colonies were transferred to a new PDA medium, purified by single spore isolation and stored on PDA slants at 4 ℃ for further use.
Example 2:
extracting and purifying double-stranded RNA of attenuated strain YC-31
The attenuated strain YC-31 of the invention is cultured for 1 week in liquid, and then filtered to collect mycelium; grinding mycelium 0.1g into powder in liquid nitrogen, transferring into centrifuge tube, adding 700 μ L STE (1M Tris-HCl, 0.5M EDTA, 5M NaCl, 10% SDS), and shaking with vortex-shaking device for 3min to mix thoroughly; adding 700 μ L RNA extraction phenol, shaking for 3min, and centrifuging at 4 deg.C and 12000rpm for 3min; taking about 650 mu L of supernatant, adding a mixture of equal volume of RNA extraction phenol, chloroform and isoamyl alcohol with the volume ratio of 25; collecting supernatant of about 500 μ L, combining two tubes into 1 tube, adding 0.1g cellulose powder and 200 μ L anhydrous ethanol, shaking for 5min, and standing on ice for 10min. Continuing to oscillate for 3min, and oscillating for 3min at 12000rpm at 4 ℃; discarding supernatant, adding 1020 μ L TE buffer (1M Tris-HCl, 0.5M EDTA, 5M NaCl PH7.0) and 180 μ L anhydrous ethanol, shaking for 1min, and centrifuging at 4 deg.C 12000rpm for 3min; removing supernatant, adding 420 μ L TE, shaking for 3min, and centrifuging at 4 deg.C and 12000rpm for 3min; taking about 400 mu L of supernatant, putting into a 1.5mL new centrifuge tube, adding equal volume of isopropanol, precipitating at-20 ℃ for 2h, centrifuging at 12000rpm at 4 ℃ for 15min, and removing the supernatant; adding 1mL of 70% ethanol, centrifuging at 12000rpm for 10min, discarding the supernatant, washing once again, completely removing the residual ethanol, and adding 30 mu Ldd H2O to dissolve the precipitate; mu.L of DNaseI (RNase-free), 1. Mu.L of S1 Nuclear and 1/5 volume of DNA buffer were added thereto, and the mixture was reacted at 37 ℃ for 40 minutes to obtain the objective double-stranded RNA.
Example 3:
sequencing the whole genome of the attenuated strain YC-31 double-stranded RNA
And adding a 5' phosphorylated and 3' aminated adaptor Primer A to the 3' end of the double-stranded RNA, and purifying and recovering the double-stranded RNA after the end of the adaptor is filled. Then, cDNA was synthesized by reverse transcription using a random Primer and a Primer B Primer (reverse complement Primer of Primer A). cDNA was synthesized as a template and then sequencing of the clone of the terminal sequence was performed. And designing gene specific primers and Primer B primers by using fragments obtained by high-throughput sequencing to perform PCR, purifying and recovering the obtained strips, connecting the strips to a T vector, and converting escherichia coli for screening to obtain positive clones. Each positive clone was then sequenced. Obtaining the full-length sequence of the genome information of the fungal virus CaPV 1. The fungal virus CaPV1 is composed of 2 double-stranded RNAs (see figure 3), and the nucleotide sequence of each double-stranded RNA is shown in SEQ ID NO:1 to 2. In the sequence listing, "uracil" is replaced with "thymine" in order to perform sequence determination after replacing double-stranded RNA with cDNA.
The evolutionary tree constructed from the RdRp sequence of the fungal virus CaPV1 is shown in FIG. 4.
Example 4:
analysis of the Biochemical characteristics of the viral particles of the fungal Virus CaPV1
Taking a plurality of bottles of attenuated strain YC-31 which is subjected to amplification culture in PDA, filtering, drying by using sterile filter paper, and grinding into powder in liquid nitrogen; adding 0.1M phosphate buffer (pH7.0) into the mycelium at a ratio of 1g mycelium dry powder to 4mL phosphate buffer, shaking for 5min, and centrifuging at 4 deg.C and 10000rpm for 15min; the supernatant was aspirated, 10% by volume of n-butanol was added: chloroform (1); the supernatant was aspirated, 50% PEG6000 and 5M NaCl were added to give final concentrations of 6% and 0.1M, respectively, and the mixture was left to stand at 4 ℃ for overnight precipitation. Centrifugation was carried out at 12000rpm at 4 ℃ for 30min, the supernatant was discarded, and the pellet was suspended in 0.01M phosphate buffer (about 30ml, which was sufficient to dissolve the virus particles in the phosphate buffer), and centrifuged at 12000rpm at 4 ℃ for 30min, leaving the supernatant (about 30 ml). Then, the supernatant was put into an ultra-high speed centrifuge tube, 20% sucrose was added to the bottom, 100000g was centrifuged for 4 hours, and the obtained precipitate was dissolved in a small amount of 0.01M phosphate buffer (pH 7.0), which was a crude extract of virions.
A part of the crude extract of the virus particles was subjected to SDS-PAGE (8% gel, tris-glycine buffer (pH 8.8), 20mA, 90 minutes) electrophoresis, and stained with CBB (Coomassie Brilliant blue) to analyze the molecular weight of the major component (coat protein) of the virus protein. The results of protein electrophoresis are shown in FIG. 5, and a band of about 40kDa protein derived from viral particles is detected. From the fraction in which the 40kDa protein was detected, nucleic acid extraction was performed by the SDS-phenol method, and as a result, 2 double-stranded RNAs of 1.6 to 1.8kbp were detected (see FIG. 5). Therefore, the presence of the viral particles of the fungal virus of the present invention can be confirmed by this example.
Example 5:
identification of the presence of virus particles outside the cell by electron microscopy
This strain YC-31 was cultured in PDA medium, and virus particles were isolated from the culture supernatant. The culture supernatant was centrifuged (10000 g, 5 min) and then ultracentrifuged (100000 g, 30 min) to obtain a precipitate containing virus particles. This precipitate was dissolved in 0.05M phosphate buffer (pH 7.0, negatively stained with phosphotungstic acid or uranium acetate, and observed with an electron microscope (magnification:. Times.20000 to 40000). As a result, the virus particle of the fungal virus CaPV1 of the present invention was identified by the electron microscope, and the virus particle was in a hexagonal shape of about 30 to 40nm and enclosed in an envelope-like structure (see B in FIG. 5). Therefore, it was confirmed by this example that the virus particle of the fungal virus of the present invention was present in the liquid medium of the strain containing the fungal virus CaPV 1.
Example 6:
the attenuated strain YC-31 is subjected to protoplast elimination to obtain the attenuated strains YC-31-P23 and YC-31-P29.
Activating the attenuated strain YC-31 provided by the invention on a PDA flat plate, picking up agar blocks containing hyphae to transfer to a PD culture medium after young hyphae grow out, and continuously culturing for 12-24 h at 28 ℃ and 180 rpm; filtering and collecting hyphae by using gauze, washing the hyphae for 2-3 times by using sterile deionized water, continuously washing the hyphae for 2-3 times by using 0.7M NaCl, selecting a proper amount of hyphae to 5mL of protoplast preparation solution, placing the solution in 30 ℃, shaking and culturing for 1-3 h at 80rpm, and performing microscopic examination until the protoplasts are released in large quantity. Filtering with three layers of lens wiping paper to collect protoplast, centrifuging at 4 deg.C for 8min at 6000g, and collecting protoplast. Adding an appropriate amount of STC solution to suspend the protoplast, centrifuging for 8min at 4 ℃, adding a small amount of STC to re-suspend the protoplast, and observing the enrichment condition of the protoplast by microscopic examination. Protoplast suspensions were diluted with STC in different gradients and plated onto regeneration medium plates. After 1-2 days, the grown fresh single colony is picked up and cultured on a PDA plate, and verified by dsRNA extraction and RT-PCR.
Example 7:
PEG-mediated protoplast transformation is carried out on virus particles of a fungal virus CaPV1, and the protoplast transformation is successfully introduced into a detoxified strain YC-31-P23 to obtain a detoxified and recovered strain YC-31-P23-T1.
After extracting the viral particles of CaPV1 by example 4, protoplasts in the detoxified strain YC-31-P23 were prepared as in example 6, 10. Mu.L of the viral particle extract was added to 100. Mu.L of the protoplast suspension, incubated on ice for 30min, added with 1mL of PTC, and left to stand at room temperature for 30min. Dripping the mixed solution into regeneration culture medium, picking mycelium block to PDA plate after mycelium grows over the plate, culturing for 2-3d, performing dsRNA extraction and RT-PCR verification, and making corresponding colony map as shown in FIG. 6.
Example 8:
the needling inoculation method is used for researching whether the fungal virus CaPV1 is effective in preventing and controlling plant pathogenic fungi.
Sterilizing pear, mountain tea tree leaf and apple with 75% ethanol, and air drying; forming wounds on the surfaces of pears, mountain tea leaves and apples by using sterilized pins; beating 5mm fungus cakes on the edges of a fresh attenuated strain YC-31, a detoxified strain YC-31-P23 and a detoxified strain YC-31-P29 and a detoxified and obtained strain YC-31-P23-T1 PDA plate of the invention cultured for 5 days to be inoculated on pears, mountain tea leaves and apples; placing the inoculum in a clean transparent polystyrene box, moistening with cotton wetted with sterile water, culturing at constant temperature of 28 deg.C, repeating the treatment for three times, and inoculating for 14d to count the morbidity.
The results are shown in FIG. 7, which shows that the lesion spots formed by the strain YC-31 and the detoxified and detoxified strain YC-31-P23-T1 of the invention are smaller than the lesion spots formed by the detoxified strains YC-31-P23 and YC-31-P29. It is demonstrated that the fungal virus CaPV1 of the present invention is effective in the control of phytopathogenic fungi.
Example 9:
the hyphal tips and spore morphology of the attenuated strain YC-31 and the attenuated strain YC-31-P23 of the present invention were observed by electron microscopy.
Fresh mycelia of the attenuated strain YC-31 and the attenuated strain YC-31-P23 were inoculated into the center of PDA medium by punching with a punch having a diameter of 5 mm. Sterile cover slips were inserted at an angle of 45 ° to approximately 3cm from the hyphal mass, incubated in a constant temperature incubator at 28 ℃ and repeated three times for each treatment. When the hyphae grow on the cover glass, the cover glass is picked and covered on the glass slide, and the hyphae tip shape is observed and recorded by an optical microscope.
Selecting fresh attenuated strain YC-31 and detoxified strain YC-31-P23, placing fresh hypha blocks in 100mL PD culture medium, shaking at 28 deg.C and 180rpm, filtering to remove hypha, placing 10 μ L spore liquid on glass slide, observing spore morphology with optical microscope, and recording.
The attenuated strain YC-31 was found to have sparse hyphae and less branching (see first panel of FIG. 8), whereas the attenuated strain YC-31-P23 had dense hyphae and more branching (see second panel of FIG. 8), and thus the fungal virus CaPV1 was presumed to affect host hyphal growth. The strain of the attenuated strain YC-31 has abnormal long-line conidia, the conidia are cylindrical, elliptical or long-line, both ends are blunt round or one end is slightly sharp (see the third picture of figure 8), and the size is 10.1-26.7X 3.9-8.9 μm (av = 15.92)+0.65×5.81+0.15,n = 50), whereas the conidia of the detoxified strain YC-31-P23 were cylindrical, oval, blunt-rounded at both ends or slightly pointed at one end, and had a spore size of 7.5-15.2 × 3.5-6.4 μm (av = 12.04)+0.28×4.83+0.08,n = 50), and thus CaPV1 affected host conidiospore morphology (see fig. 8, fourth panel).
Example 10:
the hyphae of the virulent strain YC-31 and the detoxified strain YC-31-P23 are stained by FM4-64 dye, and the endocytosis of the hyphae is observed by a fluorescence microscope.
Mycelia cultured in MM liquid medium for 48h with shaking were collected and placed on a slide glass, and the mycelia were stained with lipid-compatible FM4-64 dye by dropping on the mycelia for various lengths of time. After dyeing, immediately washing with clear water for 3 times to elute redundant dyes, and placing under a fluorescence microscope to observe the dye absorption condition of the mycelium.
The results are shown in FIG. 9, which shows that the dye is absorbed very rapidly in the detoxified strain YC-31-P23, whereas in the attenuated strain YC-31 of the present invention the entry of the dye into the hyphae is significantly hindered, and only a small amount of the dye is absorbed by the hyphae up to 30min. Thus, the fungal virus CaPV1 influences the endocytosis of the host.
Example 11:
the sensitivity of the attenuated strain YC-31 and the detoxified strain YC-31-P23 of the invention to carbendazim, prochloraz and azoxystrobin is tested.
By adopting a hypha growth rate method, 97.17 percent of carbendazim is configured into PDA culture medium plates of 0.01, 0.02, 0.04, 0.08 and 0.16mg/L, 97 percent of prochloraz is configured into PDA culture medium plates of 0.0125, 0.025, 0.05, 0.1 and 0.2mg/L, and 98 percent of azoxystrobin is configured into PDA culture medium plates of 2, 4, 8, 16 and 32 mg/L. PDA medium without added bactericide was inoculated as a blank. Repeating each concentration for 3 times, and adding bactericide with different concentrations into PDA culture medium at 50 deg.C to obtain culture medium containing medicine with different final concentrations and making into PDA plate. The bacterial cake was punched out from the edge of newly cultured 5d colony by a 5mm diameter punch and inoculated in the center of the drug-containing and control PDA plates of different drugs and concentrations. After inoculation, the inoculated strain is placed in a constant temperature incubator at 28 ℃ for 5d, the diameter of each treated colony is measured by a cross method, and the hypha growth inhibition rate is calculated. Recording data and analyzing by SPSS20.0 software, preparing toxicity curve by using inhibition rate as y axis and logarithm value of concentration as x axis, and calculating toxicity regression equation, correlation coefficient (r) and lethal middle concentration (EC) 50 )。
The results are shown in FIG. 10, and the attenuated strain YC-31 of the present invention was used for carbendazim EC 50 0.037mg/L, and the detoxified strain YC-31-P23 vs carbendazim EC 50 0.068mg/L; the attenuated strain YC-31 of the invention is to prochloraz EC 50 0.031mg/L, and the detoxified strain YC-31-P23 vs. carbendazim EC 50 Is 0.121mg/L; attenuated bacteria of the present inventionStrain YC-31 p-azoxystrobin EC 50 25.990mg/L, and the detoxified strain YC-31-P23 azoxystrobin EC 50 It was 16.929mg/L. This indicates that the attenuated strain YC-31 of the present invention has a higher sensitivity to carbendazim and prochloraz than the detoxified strain YC-31-P23, and a lower sensitivity to azoxystrobin than the detoxified strain YC-31-P23. It is therefore speculated that CaPV1 affects host sensitivity to the agent, resulting in increased sensitivity to carbendazim and prochloraz, and decreased sensitivity to azoxystrobin.
Example 12:
research on prevention and treatment of plant pathogenic fungi by using CaPV1 virus
After extraction of virions of the fungal virus CaPV1 by example 4, the virions were introduced by PEG-mediated protoplast transformation into other non-toxic wild-type anthracnose bacteria Cf10-6 and LY5-1 (C.fructicola), cs4-1 (C.spaeatianum) and Cg2-1 (C.gloeosporioides). Cf10-6-V, LY5-1-V, cs4-1-V, and Cg2-1-V represent strains infected with the virus. Therefore, the fungal virus CaPV1 can infect host bacteria through particle transfection, so that the host bacteria contain the fungal virus, thereby reducing the pathogenicity of plant pathogenic fungi and achieving the aim of biologically controlling plant fungal diseases, as shown in figure 11.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. The fungal virus has the function of inhibiting plant disease fungi, and has 2 double-stranded RNAs with the length of 1.6-1.8 kb, and the nucleotide sequences of the 2 double-stranded RNAs are shown as SEQ ID NO. 1 and 2.
2. The fungal virus of claim 1, which is capable of inhibiting plant disease fungi on woody and herbaceous plants.
3. Nucleic acid having at least the sequences in SEQ ID NO 1 and 2 or the sequence of a specific part of the sequences having a defined function.
4. An attenuated strain comprising the fungal virus of claim 1, which is classified and designated as alien anthrax (Colletotrichum alienum) YC-31, with a collection number of CCTCCM 20221146.
5. A plant disease control agent comprising at least the fungal virus of claim 1 and/or the attenuated strain of claim 4.
6. Use of a fungal virus according to claim 1 or an attenuated strain according to claim 4 for inhibiting the virulence of a phytopathogenic fungus.
7. Use of a fungal virus according to claim 1 for the extracellular infection of a plant pathogenic fungus.
8. Use of the fungal virus according to claim 1 or the attenuated strain according to claim 4 for the preparation of an agent for controlling a plant disease caused by a plant pathogenic fungus.
9. A method for attenuating a plant pathogenic fungus by infecting the plant pathogenic fungus in vitro with the fungal virus of claim 1.
10. A method for controlling phytopathogenic fungi, which comprises adding the plant disease-controlling agent according to claim 5 to plants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211008989.1A CN115927208A (en) | 2022-08-22 | 2022-08-22 | Fungal virus, attenuated strain, plant disease control agent and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211008989.1A CN115927208A (en) | 2022-08-22 | 2022-08-22 | Fungal virus, attenuated strain, plant disease control agent and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115927208A true CN115927208A (en) | 2023-04-07 |
Family
ID=86696512
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211008989.1A Pending CN115927208A (en) | 2022-08-22 | 2022-08-22 | Fungal virus, attenuated strain, plant disease control agent and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115927208A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102083973A (en) * | 2008-01-21 | 2011-06-01 | 国立大学法人东京农工大学 | Novel mycovirus, attenuated strain of phytopathogenic fungus, plant disease controlling agent, method of producing mycovirus, method of attenuating phytopathogenic fungus and method of controlling plant disease |
| WO2021178496A1 (en) * | 2020-03-04 | 2021-09-10 | Virginia Tech Intellectual Properties Inc. | Boxwood endophyte burkholderia sp ssg as potential biocontrol agent against a wide range of pathogens |
| KR20220068663A (en) * | 2020-11-19 | 2022-05-26 | 충북대학교 산학협력단 | Novel Metarhizium anisopliae SDTu1 strain with simultaneous control activities to insect and plant pathogens and uses thereof |
-
2022
- 2022-08-22 CN CN202211008989.1A patent/CN115927208A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102083973A (en) * | 2008-01-21 | 2011-06-01 | 国立大学法人东京农工大学 | Novel mycovirus, attenuated strain of phytopathogenic fungus, plant disease controlling agent, method of producing mycovirus, method of attenuating phytopathogenic fungus and method of controlling plant disease |
| WO2021178496A1 (en) * | 2020-03-04 | 2021-09-10 | Virginia Tech Intellectual Properties Inc. | Boxwood endophyte burkholderia sp ssg as potential biocontrol agent against a wide range of pathogens |
| KR20220068663A (en) * | 2020-11-19 | 2022-05-26 | 충북대학교 산학협력단 | Novel Metarhizium anisopliae SDTu1 strain with simultaneous control activities to insect and plant pathogens and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| JUN ZI ZHU等: "A novel partitivirus conferring hypovirulence by affecting vesicle transport in the fungus Colletotrichum", MBIO, vol. 15, no. 2, 14 February 2024 (2024-02-14), pages 0253023 * |
| 朱俊子等: "植物炭疽病菌真菌病毒生防因子筛选及致病相关基因挖掘", 博士电子期刊, no. 4, 15 April 2024 (2024-04-15), pages 1 - 154 * |
| 李春霞等: "刺盘孢菌真菌病毒研究进展", 南方农业学报, vol. 1, 15 January 2020 (2020-01-15), pages 123 - 132 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107034146B (en) | Antagonistic trichoderma for promoting crop growth and application thereof | |
| CN110564651A (en) | bacillus siamensis and application thereof | |
| CN111778216A (en) | Xanthomonas carpet grass phage, and composition, kit and application thereof | |
| CN108865900B (en) | Isaria javanicus strain and application thereof | |
| CN106146630A (en) | A kind of mould albumen exciton EPTP and the application in improving disease resistance of plant thereof | |
| CN112280751B (en) | Mycovirus SlMV1, attenuated strain and application | |
| CN102994401A (en) | Method for preparing apple tree valsa ceratosperma transformant and GFP (Green Fluorescent Protein) labelled strain | |
| CN112126630A (en) | Virus particle for preventing and treating wheat stem basal rot and method for preventing and treating wheat stem basal rot | |
| CN114657109B (en) | Pseudomonas putida and application thereof | |
| CN111534525A (en) | Fusarium graminearum FgARL1 gene and application thereof | |
| CN109294961B (en) | Biocontrol strain PNC25 for preventing and treating litchi downy blight and application thereof | |
| CN110423762A (en) | A kind of construction method of PVMV full length infectious cDNA | |
| CN109666655B (en) | Fusarium graminearum single-stranded circular DNA virus FgGMTV1/HB58 and application thereof | |
| CN103387942B (en) | Metarrhizum anisopliae for preventing and treating palm insect rhynchophorus ferrugineus and application thereof | |
| CN112029665A (en) | Rubber tree colletotrichum resistant strain HcgHNQZ1736 and application thereof in drug resistance research | |
| CN115927208A (en) | Fungal virus, attenuated strain, plant disease control agent and application | |
| CN114921421B (en) | Fungus virus VpFV1, weak virulence strain and application thereof | |
| CN114196551B (en) | Isaria javanica strain for preventing and controlling citrus pests and application thereof | |
| CN109735457A (en) | One plant of mutagenesis Infected barnyardgrass and its application in prevention and treatment barnyard grass | |
| CN116240126A (en) | Multifunctional bacillus belgium SB10 and application thereof | |
| CN113416679A (en) | Bacillus methylotrophicus, microbial inoculum comprising bacillus methylotrophicus and application of bacillus methylotrophicus | |
| CN113046249A (en) | Verticillium lecanii LL-01 and biocontrol application thereof | |
| Soliman | Xanthomonas euvesicatoria associated with bacterial spot on pepper fruits in Egypt | |
| CN109810997B (en) | Construction method of fusarium graminearum single-stranded circular DNA virus FgGMTV1/HB58 infectious clone | |
| CN105543102B (en) | One plant of prevention palm pest coconut knits green muscardine fungus and the application of moth |
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 |