CN118667663B - A strain of Trichoderma and its microcapsule preparation method and application - Google Patents
A strain of Trichoderma and its microcapsule preparation method and application Download PDFInfo
- Publication number
- CN118667663B CN118667663B CN202410973026.8A CN202410973026A CN118667663B CN 118667663 B CN118667663 B CN 118667663B CN 202410973026 A CN202410973026 A CN 202410973026A CN 118667663 B CN118667663 B CN 118667663B
- Authority
- CN
- China
- Prior art keywords
- trichoderma
- pepper
- microcapsules
- montmorillonite
- spores
- 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.)
- Active
Links
- 239000003094 microcapsule Substances 0.000 title claims abstract description 104
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 241000223259 Trichoderma Species 0.000 title abstract description 12
- 235000002566 Capsicum Nutrition 0.000 claims abstract description 50
- 239000006002 Pepper Substances 0.000 claims abstract description 37
- 235000016761 Piper aduncum Nutrition 0.000 claims abstract description 37
- 235000017804 Piper guineense Nutrition 0.000 claims abstract description 37
- 235000008184 Piper nigrum Nutrition 0.000 claims abstract description 37
- 239000000661 sodium alginate Substances 0.000 claims abstract description 29
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 29
- 241000196324 Embryophyta Species 0.000 claims abstract description 26
- 241000894006 Bacteria Species 0.000 claims abstract description 24
- 201000010099 disease Diseases 0.000 claims abstract description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims abstract description 17
- GUJOJGAPFQRJSV-UHFFFAOYSA-N dialuminum;dioxosilane;oxygen(2-);hydrate Chemical compound O.[O-2].[O-2].[O-2].[Al+3].[Al+3].O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O GUJOJGAPFQRJSV-UHFFFAOYSA-N 0.000 claims abstract description 13
- 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 claims abstract description 8
- 244000061176 Nicotiana tabacum Species 0.000 claims abstract description 8
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 8
- 241000675573 Trichoderma afroharzianum Species 0.000 claims abstract description 8
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 8
- 241000722363 Piper Species 0.000 claims abstract 11
- 241000223260 Trichoderma harzianum Species 0.000 claims description 59
- 238000011282 treatment Methods 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 239000000725 suspension Substances 0.000 claims description 22
- 238000000855 fermentation Methods 0.000 claims description 17
- 230000004151 fermentation Effects 0.000 claims description 17
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 230000000813 microbial effect Effects 0.000 claims description 12
- 229910052901 montmorillonite Inorganic materials 0.000 claims description 10
- 241000123650 Botrytis cinerea Species 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 9
- 230000012010 growth Effects 0.000 claims description 8
- 241001530056 Athelia rolfsii Species 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 244000052769 pathogen Species 0.000 claims 5
- 230000001717 pathogenic effect Effects 0.000 claims 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims 2
- 239000001110 calcium chloride Substances 0.000 claims 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims 2
- 235000011148 calcium chloride Nutrition 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 22
- 230000000443 biocontrol Effects 0.000 abstract description 9
- 239000000463 material Substances 0.000 abstract description 9
- 238000004321 preservation Methods 0.000 abstract description 7
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 6
- 230000003042 antagnostic effect Effects 0.000 abstract description 5
- 244000000004 fungal plant pathogen Species 0.000 abstract description 3
- 238000001125 extrusion Methods 0.000 abstract description 2
- 240000003889 Piper guineense Species 0.000 description 44
- 239000000243 solution Substances 0.000 description 22
- 240000008574 Capsicum frutescens Species 0.000 description 13
- 239000001390 capsicum minimum Substances 0.000 description 12
- 241000813090 Rhizoctonia solani Species 0.000 description 11
- 239000002689 soil Substances 0.000 description 11
- 244000052616 bacterial pathogen Species 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 210000001339 epidermal cell Anatomy 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 241000233616 Phytophthora capsici Species 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 230000002262 irrigation Effects 0.000 description 5
- 238000003973 irrigation Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 241001001916 Colletotrichum scovillei Species 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 241000233614 Phytophthora Species 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000012681 biocontrol agent Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 241001477352 Bengalia Species 0.000 description 3
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 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 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000005538 encapsulation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 2
- 101100313266 Mus musculus Tead1 gene Proteins 0.000 description 2
- 241001361634 Rhizoctonia Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 235000010633 broth Nutrition 0.000 description 2
- 239000000648 calcium alginate Substances 0.000 description 2
- 229960002681 calcium alginate Drugs 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000012054 meals Nutrition 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 238000004382 potting Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- WOSNCVAPUOFXEH-UHFFFAOYSA-N thifluzamide Chemical compound S1C(C)=NC(C(F)(F)F)=C1C(=O)NC1=C(Br)C=C(OC(F)(F)F)C=C1Br WOSNCVAPUOFXEH-UHFFFAOYSA-N 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241001290235 Ceratobasidium cereale Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000026010 Dendrobium candidum Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 101001091269 Escherichia coli Hygromycin-B 4-O-kinase Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 108050002220 Green fluorescent protein, GFP Proteins 0.000 description 1
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 1
- 241000710921 Infectious pancreatic necrosis virus Species 0.000 description 1
- 244000199866 Lactobacillus casei Species 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- 238000007476 Maximum Likelihood Methods 0.000 description 1
- 208000012868 Overgrowth Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000984202 Peperomia obtusifolia Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000233647 Phytophthora nicotianae var. parasitica Species 0.000 description 1
- 241000758706 Piperaceae Species 0.000 description 1
- 241001646398 Pseudomonas chlororaphis Species 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000221662 Sclerotinia Species 0.000 description 1
- 241000970978 Streptomyces griseoaurantiacus Species 0.000 description 1
- 101001091268 Streptomyces hygroscopicus Hygromycin-B 7''-O-kinase Proteins 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
- 241000499912 Trichoderma reesei Species 0.000 description 1
- 238000010162 Tukey test Methods 0.000 description 1
- 230000006750 UV protection Effects 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- AEMOLEFTQBMNLQ-ORELYVPDSA-N alpha-L-glucopyranuronic acid Chemical compound O[C@@H]1O[C@@H](C(O)=O)[C@H](O)[C@@H](O)[C@@H]1O AEMOLEFTQBMNLQ-ORELYVPDSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 210000004027 cell Anatomy 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
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 1
- 229940097277 hygromycin b Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229940017800 lactobacillus casei Drugs 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 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
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940126578 oral vaccine Drugs 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 239000011253 protective coating Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000004804 winding Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 239000001052 yellow pigment Substances 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/38—Trichoderma
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pest Control & Pesticides (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Agronomy & Crop Science (AREA)
- General Chemical & Material Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明公开了一株木霉菌及其微胶囊制备方法和应用，属于植物病害生防菌领域，该非洲哈茨木霉菌(Trichoderma afroharzianum)YZ‑1已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 20241084。本发明提供的非洲哈茨木霉菌YZ‑1对于辣椒白绢病具有优异的防效，防效可达97.57%；另外还对辣椒和烟草上的其他5种植物病原真菌均具有拮抗活性，抑菌率在51.12±1.20%到81.09±1.28%之间。同时，本发明以海藻酸钠和蒙脱石K‑10为壁材，通过挤压法制备了非洲哈茨木霉菌YZ‑1的微胶囊，其盆栽防效为74.13%。本发明提供的非洲哈茨木霉菌及其微胶囊有较大的潜力开发成商业化的活菌生防制剂。
The present invention discloses a strain of Trichoderma and a method for preparing microcapsules thereof and an application thereof, and belongs to the field of plant disease biocontrol bacteria. The African Trichoderma ( Trichoderma afroharzianum ) YZ-1 has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20241084. The African Trichoderma YZ-1 provided by the present invention has excellent control effect on pepper white rot, and the control effect can reach 97.57%; in addition, it also has antagonistic activity against other 5 plant pathogenic fungi on pepper and tobacco, and the antibacterial rate is between 51.12±1.20% and 81.09±1.28%. At the same time, the present invention uses sodium alginate and montmorillonite K-10 as wall materials, and prepares microcapsules of African Trichoderma YZ-1 by extrusion, and the potted plant control effect is 74.13%. The African Trichoderma and its microcapsules provided by the present invention have great potential to be developed into commercial live bacterial biocontrol preparations.
Description
Technical Field
The invention relates to the field of biocontrol bacteria for plant diseases, in particular to trichoderma as well as a microcapsule preparation method and application thereof.
Background
The biocontrol agent refers to a biological product processed by microorganisms (such as bacteria, fungi, viruses and the like) and metabolites thereof and the like, and is used for controlling plant diseases. The research and application of the biocontrol agent can play a good role in relieving the problems of environmental pollution, disease resistance enhancement, human health threat and the like caused by applying chemical pesticides, and is necessary for sustainable development of agriculture.
However, the instability of the application of biological agents in the soil due to temperature, humidity, uv radiation, etc., is currently a major challenge in the field of agriculture. Microencapsulation is an innovative and effective technique that refers to the process of encapsulating an active ingredient within a protective coating or matrix. The microbial agent can be used as a barrier to protect microorganisms from being stressed by living or non-living things, so that the stability of the microbial agent is improved, the limitation of other preparations such as solid or liquid formulations in storage and application can be overcome, the service life of the microorganisms is prolonged by controlling the release of the microorganisms, and the use efficiency of the microorganisms is improved.
The microcapsule properties are determined by the nature of the carrier or encapsulating material, which plays a critical role in the success and efficiency of the microencapsulation of the biocontrol agent. Sodium alginate is a material commonly used as a carrier and is a polysaccharide consisting of alpha-l-glucuronic acid and beta-mannic acid units. The material is nontoxic, biodegradable, and easy to gel with various crosslinking agents such as calcium ions, so that the material becomes a preferred carrier material for delivering and releasing encapsulated cells, medicines and other bioactive molecules. However, alginate has limitations such as high mechanical rigidity, varying size, poor physical properties, etc., which make it unsuitable for providing long-term stability of the encapsulated microorganism. Therefore, in order to overcome these limitations, suitable fillers, one of which is montmorillonite, are generally added to the formulation. It has large surface area, excellent expansion capability and naturally exists in soil, and is a proper packing.
At present, few biocontrol agents are found in the aspect of preventing and treating pepper southern blight. For example, CN 104988079B discloses a Trichoderma harzianum JSNL1404 which has strong antibacterial ability on dendrobium candidum southern blight, and CN 117327593A discloses a Trichoderma harzianum ZM1 which has prevention effect on peanut southern blight. The biocontrol microbial agents for preventing and treating the pepper southern blight are relatively few, and the report on the trichoderma microcapsule is more recently reported. Therefore, there is a need in the art to screen a trichoderma strain having a high biocontrol effect on pepper southern blight and to realize its application by microencapsulation.
Disclosure of Invention
The invention aims to provide a trichoderma reesei and a microcapsule preparation method and application thereof, so as to solve the problems in the prior art, and the trichoderma harzianum YZ-1 and the microcapsule thereof provided by the invention have excellent prevention effect on pepper southern blight and have great potential for developing a commercial living bacteria biocontrol preparation.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides trichoderma harzianum (Trichoderma afroharzianum) YZ-1, wherein the preservation number of the trichoderma harzianum YZ-1 is CCTCC NO: M20241084.
The invention also provides a trichoderma harzianum YZ-1 microcapsule, which comprises the trichoderma harzianum YZ-1.
The invention also provides a preparation method of the trichoderma harzianum YZ-1 microcapsule, which comprises the following steps:
dissolving 1-4 g of montmorillonite K-10 in 100-500 mL of deionized water, adding 6-9 g of sodium alginate, and then adding glycerol for fully and uniformly mixing to obtain a montmorillonite-sodium alginate solution;
Taking Trichoderma harzianum YZ-1 spore liquid, centrifuging to obtain spores, adding the montmorillonite-sodium alginate solution to suspend spores, and obtaining montmorillonite-sodium alginate spore suspension with the spore concentration of 1X 10 7～1×109 spores/mL;
extruding the montmorillonite-sodium alginate spore suspension into 2% CaC1 2 solution, and eluting with sterile water to obtain Trichoderma harzianum YZ-1 microcapsule.
Further, the preparation method comprises the following steps:
Dissolving 2.5 g montmorillonite in 300 mL deionized water, adding 7.5 g sodium alginate, adding glycerol, and mixing to obtain montmorillonite-sodium alginate solution;
Taking Trichoderma harzianum YZ-1 spore liquid, centrifuging to obtain spores, adding the montmorillonite-sodium alginate solution to suspend spores, and obtaining montmorillonite-sodium alginate spore suspension with the spore concentration of 1X 10 8 spores/mL;
extruding the montmorillonite-sodium alginate spore suspension into 2% CaC1 2 solution, and eluting with sterile water to obtain Trichoderma harzianum YZ-1 microcapsule.
The invention also provides a microbial agent containing the trichoderma harzianum YZ-1.
Further, the viable bacteria concentration of the microbial agent is 1×10 8 spores/mL.
The invention also provides an application of the trichoderma harzianum YZ-1, the trichoderma harzianum YZ-1 microcapsule or the microbial agent in inhibiting the growth of pathogenic bacteria, wherein the pathogenic bacteria comprise southern blight germ of capsicum, botrytis cinerea, phytophthora capsici, tobacco target spot germ, rhizoctonia solani and anthracnose germ of capsicum.
The invention also provides an application of the trichoderma harzianum YZ-1, the trichoderma harzianum YZ-1 microcapsule or the microbial agent in preventing and treating plant diseases, wherein the plant diseases are pepper southern blight caused by pepper southern blight.
The invention also provides a method for preventing and controlling the capsicum southern blight caused by capsicum southern blight, which comprises the following steps:
treating a pepper plant by adopting the trichoderma harzianum YZ-1 or fermentation liquor or bacterial suspension thereof or the trichoderma harzianum YZ-1 microcapsule;
The concentration of viable bacteria in the fermentation liquor or the bacterial suspension is 1 multiplied by 10 8 spores/mL.
Further, the treatment is that fermentation liquor or bacterial suspension of the trichoderma harzianum YZ-1 with the concentration of 1X 10 8 spores/mL or the trichoderma harzianum YZ-1 microcapsule is subjected to root irrigation treatment on pepper plants.
The invention discloses the following technical effects:
The invention separates and screens to obtain the Trichoderma harzianum (Trichoderma afroharzianum) YZ-1, and experiments prove that the Trichoderma harzianum YZ-1 provided by the invention has excellent control effect on pepper southern blight caused by pepper southern blight, the control effect can reach 97.57%, and in addition, the Trichoderma harzianum YZ-1 has antagonistic activity on other 5 plant pathogenic fungi on peppers and tobacco, and the bacteriostasis rate is between 51.12+/-1.20% and 81.09 +/-1.28%. Meanwhile, the microcapsule of trichoderma harzianum YZ-1 is prepared by an extrusion method by taking sodium alginate and montmorillonite K-10 as wall materials, and the potting prevention effect is 74.13%. The trichoderma harzianum and the microcapsules thereof provided by the invention have great potential to be developed into commercial living bacteria biocontrol preparations.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the effect of Trichoderma harzianum YZ-1 on inhibiting pepper southern blight, wherein the left side is a control, and the right side is a culture of YZ-1 against pepper southern blight;
FIG. 2 shows the colony morphology of Trichoderma harzianum YZ-1 on different media, a-b colony morphology on the front and back sides of PDA, c-d colony morphology on the front and back sides of CMD. e-f colony morphology of the front and back sides of SNA;
FIG. 3 is a phylogenetic tree of multiple genes constructed based on ITS, tef-1 gene sequences;
FIG. 4 shows antagonistic activity of Trichoderma harzianum YZ-1 on different pathogenic bacteria, wherein the first row sequentially comprises tobacco target spot bacteria (Rhizoctonia solani), pepper gray mold bacteria (Botrytis cinerea), pepper anthracnose bacteria (Colletotrichum scovillei), pepper phytophthora capsici (Phytophthora capsica) and pepper rhizoctonia rot bacteria (Rhizoctonia solani), and the second row sequentially comprises tobacco target spot bacteria (Rhizoctonia solani), pepper gray mold bacteria (Botrytis cinerea), pepper anthracnose bacteria (Colletotrichum scovillei), pepper phytophthora capsici (Phytophthora capsica) and pepper rhizoctonia rot bacteria (Rhizoctonia solani) added with Trichoderma harzianum YZ-1;
FIG. 5 shows the Control effect of different treatment groups on pepper southern blight, wherein A is a pepper plant in a pot, and II is a pepper plant after soil removal, B is a relative Control bar graph of different treatments, control is a blank Control, and 0.4% Tiofuramide is a positive Control;
FIG. 6 shows the effect of different treatment groups on the growth of pepper plants, A. Pepper plants in pot culture, B. Pepper plants after soil removal;
FIG. 7 shows the form of mycelia after the re-hosting of Trichoderma harzianum YZ-1, african trichoderma harzianum, a, attachment, B, winding, c, digestion, B, overgrowth, and a;
FIG. 8 shows the verification of YZ-1-GFP strain transformant, A. Molecular verification, B. Fluorescence verification;
FIG. 9 shows the observation of the colonization of YZ-1-GFP1 in pepper plants, the attachment of spores to lateral root tips (c) observed 1 day after inoculation, the aggregation of spores in epidermal cells on root surface (d) on day 2, the colonization of main roots (a) 3 days after inoculation, fluorescence (g) detected in epidermal cells at the root-stem junction on day 5, the penetration of mycelia into epidermal cells of stems (f) 7 days after inoculation, no fluorescence observed in leaves (b), the observation of cross-section of the stem base on day 6, and the observation of root tips on day 4;
FIG. 10 shows the release of YZ-1 microcapsules in physiological saline;
FIG. 11 is a chart showing the statistics of the particle size of YZ-1 microcapsules, wherein A and B are the particle sizes of empty microcapsules and YZ-1 microcapsules in wet state, respectively, and C and D are the particle sizes of empty microcapsules and YZ-1 microcapsules in dry state, respectively;
FIG. 12 shows the morphology of YZ-1 microcapsules observed by a scanning electron microscope, wherein A and B are the morphologies of empty microcapsules at 200X and 50000X, respectively, and C and D are the morphologies of YZ-1 microcapsules at 200X and 50000X, respectively;
FIG. 13 is a FTIR spectrum of YZ-1 microcapsules, wherein the left side is empty microcapsules and the right side is YZ-1 microcapsules;
FIG. 14 shows the results of UV stability measurements of YZ-1 microcapsules, A dry YZ-1 microcapsules, B wet YZ-1 microcapsules, C free YZ-1 spores;
FIG. 15 shows the results of temperature stability measurements of YZ-1 microcapsules, wherein a, b, and C are the survival rates of the dried YZ-1 microcapsules at 7 ℃, 27 ℃, and 37 ℃ and d, e, and f are the survival rates of the wet YZ-1 microcapsules at 7 ℃, 27 ℃, and 37 ℃ in order;
FIG. 16 shows the results of bacteriostasis of YZ-1 microcapsules on a plate, YZ-1-GFP1: YZ-1-GFP1 strain, YZ-1-GFP1+SA+K-10: YZ-1-GFP1 microcapsules, THE SAME TIME: YZ-1-GFP1 microcapsules inoculated simultaneously with pathogenic bacteria XTBJ-1, after 1 day: inoculation of YZ-1-GFP1 microcapsules for 1 day followed by pathogenic bacteria XTBJ-1; SA+K-10: empty microcapsules, control: blank;
FIG. 17 shows the results of potting experiments for YZ-1 microcapsules. A is the disease condition of each treatment group after soil removal, wherein a-e is blank control, empty microcapsule treatment, YZ-1-GFP1 spore liquid treatment, YZ-1-GFP1 microcapsule treatment and positive control respectively, and B-G is each index measurement after the disease treatment, and the relative control effect, plant height, stem length, root length, fresh weight and dry weight are determined in sequence.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In the examples described below, the pathogenic fungi Botrytis cinerea (Botrytis cinerea), phytophthora capsici (Phytophthora capsica), phytophthora nicotianae (Rhizoctonia solani), rhizoctonia solani (Rhizoctonia solani), rhizoctonia cerealis (Colletotrichum scovillei) and Sclerotinia capsici (Sclerotium rolfsii) are all described in document （Qiu Z L , Liu S D , Li X G ,et al.Identification and mechanism characterization of Streptomyces griseoaurantiacus XQ-29 with biocontrol ability against pepper southern blight caused by Sclerotium rolfsii[J].Pesticide Biochemistry and Physiology, 2024, 202.DOI:10.1016/j.pestbp.2024.105956.）.
The culture media used in the following examples:
bengalia culture medium (RBA) comprises glucose 10 g, peptone 5g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5 g, agar 20g, bengalia 0.033 g, chloramphenicol 0.1g and distilled water 1L, pH 7.0-7.2;
Corn meal glucose medium (CMD) corn meal 40 g, agar 20g, glucose 20g and distilled water 1L;
The synthetic nutrient-free agar medium (SNA) comprises KH 2PO4 1 g、KNO3 1 g、MgSO4·7 H2 O0.5 g, KC 1.5 g, agar 20 g, glucose 0.2 g, sucrose 0.2 g and distilled water 1L.
In the examples below, all experimental data were analyzed by one-way variance analysis (one-way ANOVA analysis) using SPSS23.0 and multiple comparisons and significance analysis were performed by the base of the plot.
Example 1 identification of Trichoderma harzianum YZ-1
The invention obtains antagonistic fungus with inhibiting effect on capsicum southern blight germ (Sclerotium rolfsii) (XTBJ-1 strain for short) through screening, and the strain is identified as trichoderma harzianum (Trichoderma afroharzianum) and named YZ-1 through morphological observation and molecular biological identification.
1. Isolation and screening of Trichoderma harzianum
Trichoderma harzianum strain was isolated from capsicum rhizosphere soil by dilution plating method using Bengalia red medium and purified. The method comprises the steps of screening Trichoderma harzianum strains with antibacterial effect by counter culture with pepper southern blight XTBJ-1 as target pathogenic bacteria.
As shown in the figure 1, the invention screens a strain of Trichoderma harzianum with obvious inhibition effect on the growth of the hyphae of the pepper southern blight fungus, and the plate inhibition rate is 53.04 +/-0.08%, which is named YZ-1.
2. Morphological observation
YZ-1 was inoculated onto PDA, CMD and SNA medium. The strain growth rate and colony morphology were observed. Tender hyphae at the edge of YZ-1 plates cultured for 3 days were taken, and conidium peduncles were observed under an optical microscope.
As can be seen from FIG. 2, YZ-1 has rich hyphae, cotton-like shape, yellow-green to green color, no smell, and yellow pigment generation (a, b in FIG. 2) on PDA medium, has rich hyphae, cotton-like shape, and a large number of conidium clusters growing on top of hyphae, distributed in a dot shape, yellow to dark green on front, white to yellow on back (c, d in FIG. 2), and sparse hyphae, white to light green (e, f in FIG. 2) on SNA medium.
3. Molecular characterization
The genomic DNA of YZ-1 was extracted by CTAB method. ITS and tef-1 sequences of YZ-1 strain were amplified using the primers in Table 1. Each 50. Mu.L of the PCR reaction system contained 25. Mu. L FINETAQ PCR Super, 2. Mu.L forward primer (10. Mu.M), 2. Mu.L reverse primer (10. Mu.M), 4. Mu.L DNA template and 17. Mu. L H 2 O. The PCR reaction procedure was 95℃pre-denaturation for 3 min, 35 amplification cycles (95℃denaturation for 30 s,56℃annealing for 30 s,72℃extension for 1 min), 72℃extension for 5 min.
The amplified products were sequenced (qing biotechnology limited, beijing, china), the obtained sequences were aligned with NCBI database using BLASTN. Constructing a polygenic phylogenetic tree (figure 3) by adopting a maximum likelihood method in MEGA 7.0 software, and finally identifying YZ-1 as trichoderma harzianum (Trichoderma afroharzianum) by combining morphological characteristics of strains and molecular biological identification.
Primer sequences used in Table 1
The trichoderma harzianum YZ-1 has been preserved with the preservation number of CCTCC NO: M20241084, the classification of the trichoderma harzianum YZ-1 is Trichoderma afroharzianum, the preservation date of the trichoderma harzianum is 2024, 05 and 28 days, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation address of the trichoderma harzianum is China university.
Example 2 control effect of Trichoderma harzianum YZ-1
1. Antibacterial spectrum
The bacteriostasis spectrum of YZ-1 was measured by plate-counter culture. The method comprises the steps of respectively taking Botrytis cinerea (Botrytis cinerea), phytophthora capsici (Phytophthora capsica), tobacco target spot bacteria (Rhizoctonia solani), pepper rhizoctonia solani (Rhizoctonia solani) and pepper anthracnose bacteria (Colletotrichum scovillei) as target bacteria, inoculating a target bacteria cake (d=8 mm) to the left side of a PDA flat plate with the diameter of 7.5 cm, inoculating YZ-1 at a position 5 to cm away from the bacterial cake, taking a flat plate only receiving the target bacteria as a blank control, repeating each treatment for 3 times, placing the blank control in a 28 ℃ constant temperature incubator for culture, recording the colony diameters of the treatment group and the control group when the colony of the control group is about to grow up the flat plate, and determining the antibacterial rate of YZ-1 to the pathogenic fungi to be tested.
The antagonistic activity of YZ-1 against the above 5 test plant pathogenic fungi was examined by the plate-facing method (FIG. 4), and the inhibition rate of YZ-1 against these strains was found to be 51.12.+ -. 1.20% -81.09.+ -. 1.28% (Table 2). Wherein, the growth of the tobacco target spot bacteria and the botrytis cinerea bacteria is obviously inhibited.
TABLE 2 YZ-1 determination of the bacteriostasis spectra of 5 pathogenic bacteria
Note that the data in the tables are mean ± standard deviation, and that the different lower case letters after the same column of data indicate significant differences at P <0.05 levels by Tukey's HSD test.
2. In vivo control effect on pepper southern blight
Culturing YZ-1 bacterial cake in PD for 7 days, filtering with gauze to obtain mycelium, centrifuging the liquid obtained by filtering at 6000 rpm for 10 min, collecting supernatant to obtain YZ-1 fermentation filtrate, collecting precipitate, dissolving with sterile water, adding mycelium, mixing, and crushing to obtain YZ-1 bacterial suspension with concentration of 10 8 spores/mL.
The YZ-1 bacterial cake is placed in PD for 7 days, and the cultured bacterial liquid is crushed completely after 7 days, so that YZ-1 fermentation liquid with the concentration of 10 8 spores/mL is obtained.
And (3) taking YZ-1 fermentation filtrate, YZ-1 bacterial suspension and YZ-1 fermentation liquor, respectively carrying out root irrigation treatment on the pepper plants after 21 days of transplanting, 10 mL each plant, inoculating four fresh XTBJ-1 mycelium blocks (d=8 mm) on the base of the plant stems after 1 day, covering with moist soil, and carrying out moisture preservation culture at 28 ℃. 0.4% thifluzamide was used as positive control. After 5 days, observing the disease condition of each treatment, removing matrixes inoculated with the root of the capsicum for more intuitively observing the disease condition, washing redundant impurities with water, naturally airing, measuring the disease spot length of the root of the capsicum stem, and calculating the disease index.
The disease severity (disease grade) is defined according to the ratio of the length of the disease spots to the length of the pepper stems, and the standard is that grade 0 is X0, no symptoms exist, grade 1 is that the leaves are yellow, X is less than or equal to 1%, grade 3 is X is less than or equal to 10%, grade 5 is X is less than or equal to 20%, grade 7 is X is less than or equal to 30%, grade 9 is X is more than 30%, and the plants die. X (%) = [ (length of basal stem lesions)/(length of plant stems) ]x100.
The disease index is calculated by the formula of relative prevention effect (%) = [ (disease index of control group-disease index of control group)/disease index of control group ]. Times.100.
After the stem base of the control plant is inoculated with XTBJ-1 hypha blocks for 5 days, symptoms such as yellowing and wilting of leaves appear, and obvious brown lesions are visible on the stem after root soil is removed (A in figure 5). In contrast, plants treated with YZ-1 fermentation broths and bacterial suspensions did not show these symptoms. The disease index of YZ-1 fermentation broth and bacterial suspension treated plants was 1.23, the control plants were 50.62, and the relative control effect was 97.57% (B in FIG. 5). Notably, some diseases still exist in the plants treated by the YZ-1 fermentation filtrate, and the relative control efficiency is only 60.98%.
Taking YZ-1 fermentation filtrate, YZ-1 bacterial suspension and YZ-1 fermentation liquor, respectively carrying out root irrigation treatment on the pepper plants in a four-leaf and one-heart period, wherein each plant is subjected to root irrigation 10 mL, and the application is carried out once every 7: 7 d for three times, and the clear water treatment is used as a control. After 5 d, the growth was observed (fig. 6) and various indices of the pepper plants were measured for the different treatments (table 3). The results show that the YZ-1 fermentation liquor treated plants have no obvious difference in plant height and stem length, but the leaves are more enlarged, the roots are more dense, and the fermentation filtrate is more lean.
TABLE 3 measurement of various indices of Pepper plants treated differently
EXAMPLE 3 biocontrol mechanism study of Trichoderma harzianum YZ-1
1. Action of re-mailing hyphae of capsicum southern blight germ
After observing that the Trichoderma harzianum strain YZ-1 and XTBJ-1 are opposite, a clear overlapping area appears at the junction, and in order to further explore the plate bacteriostasis mechanism of Trichoderma, hypha at the junction is observed. YZ-1 and XTBJ-1 are respectively connected to two ends of the PDA flat plate, and a sterilized glass slide is placed in the middle of the PDA flat plate. When the mycelia contact with the glass slide, the glass slide is taken down, clean water is dripped on the surface, and the glass slide is covered and then observed under a microscope.
After YZ-1 and XTBJ-1 were co-cultured for 7 days, mycelium blocks at the junctions of the two were cut off by a blade, and the mycelium blocks were fixed in a 2.5% glutaraldehyde solution for SEM observation.
The overlapped hyphae of the strains YZ-1 and XTBJ-1 were observed by a microscope, and it was found that YZ-1 could be attached, entangled in the host hyphae, and the host hyphae were decomposed (A in FIG. 7). Scanning electron microscopy revealed that YZ-1 could be entangled and attached to the mycelia of XTBJ-1, growing in large amounts by depriving the host mycelia of nutrients, while causing the host mycelia to digest (B in FIG. 7). Indicating that YZ-1 can be used for re-mailing hyphae of capsicum southern blight germ.
2. Analysis of colonization in Capsici fructus plants
And (3) taking green fluorescent protein (green fluorescent protein, GFP) as a reporter gene, and observing the colonization condition of the strain YZ-1 on the root of the capsicum. A plasmid pCT74 carrying the hygromycin B phosphotransferase gene (hygr) was inserted into strain YZ-1 using the method described in Hua et al Oral vaccine against IPNV based on antibiotic-free resistance recombinant Lactobacillus casei expressing CK6-VP2 fusion protein (2021), and a YZ-1-GFP strain was constructed. Positive transformants were identified and selected on PDA plates containing 150. Mu.g/mL hygromycin B.
YZ-1-GFP strain was cultured on PDA plates for 7 days, scraped with a spreader, washed with sterile water, and filtered through gauze to give a conidium solution at a concentration of 1X 10 8 spores/mL. And irrigating roots of pepper plants by using the conidium solution in a four-leaf one-heart period, wherein each plant is 10mL. Pepper plants were sampled at various time points after root irrigation, carefully cleaned of soil, root rinsed with sterile water, and then sectioned with lancets to prepare slides. And observing each part of the pepper plant by adopting a fluorescence microscope.
Hygromycin was selected to obtain 33 transformants, 20 of which were used for observation of hyphal luminescence under a fluorescence microscope. Wherein, YZ-1-GFP-1, YZ-1-GFP-2 and YZ-1-GFP-3 are brightest and most uniform in fluorescence, and the colony morphology is not changed. YZ-1-GFP-1 was still significantly fluorescent after 10 passages (FIG. 8), so YZ-1-GFP-1 was selected for subsequent experiments. And collecting YZ-1-GFP-1 spores, and inoculating the spores to roots of pepper plants. Inoculated sterile water was also set as a control treatment group. YZ-1-GFP-1 spores adhered to the lateral root tip on day 1, aggregated in root epidermal cells on day 2, colonized in main roots on day 3, fluorescent light was seen in epidermal cells at the junction of the rhizomes on day 5, and mycelia had grown into stem epidermal cells on day 7 (FIG. 9). No fluorescence was detected at the roots of the pepper plants of the control treatment group.
Example 4 preparation of Trichoderma harzianum YZ-1 microcapsules and measurement of various indicators
1. Microcapsule preparation method
2.5 G of montmorillonite K-10 is dissolved in 300 mL of deionized water and stirred at room temperature. Adding 7.5g sodium alginate, stirring to dissolve completely, adding glycerol, and mixing to obtain montmorillonite-sodium alginate solution.
Taking YZ-1 spore liquid, centrifuging 10 min at 5000 rpm, and removing supernatant to obtain spores. Adding montmorillonite-sodium alginate solution to suspend spores, stirring thoroughly to make its concentration be 1×10 8 spores/mL, to obtain montmorillonite K-10-sodium alginate spore suspension. Adding 2% CaC1 2 solution into magnetic stirrer, and squeezing the montmorillonite K-10-sodium alginate spore suspension into the solution with a syringe to obtain calcium alginate-montmorillonite microcapsule. Filtering with gauze, collecting microcapsule, eluting with sterile water to obtain YZ-1 microcapsule, transferring into triangular flask, and preserving at 4deg.C.
2. Measurement of various indexes
2.1 Encapsulation efficiency
1ML of a 1X 10 8 spore/mL montmorillonite K-10-sodium alginate spore suspension was added dropwise to the sterilized 2% CaC1 2 solution. 1mL of the used CaC1 2 solution was pipetted to dilute the plate while the free spore concentration was calculated under a microscope. Encapsulation efficiency (%) = (N 0-Ne）/N0 x 100%, where N 0 and Ne represent the spore concentration used to make the microcapsule and the concentration of the free spores outside, respectively, the encapsulation efficiency was 99.98% as measured, and it can be seen that the preparation method provided by the invention can well encapsulate trichoderma harzianum in the microcapsule.
2.2 Sustained release rate
0.3G YZ-1 microcapsule was weighed, added to 7.5 mL of 0.9% physiological saline, and cultured at 27 ℃. 100 μl of the diluted plates were aspirated every 5 days and spore concentrations were calculated under the microscope for up to day 65.
The CaCl 2 solution for preparing YZ-1 microcapsule was sucked for plate coating, and the number of free colonies was calculated, and the slow release rate (EN%) was 99.98%. The release of YZ-1 microcapsules in physiological saline peaked at 25 days and then gradually decreased over time as shown in FIG. 10.
2.3 Particle size
The particle sizes of empty microcapsules (montmorillonite K-10-calcium alginate) and YZ-1 microcapsules (YZ-1 spores+montmorillonite K-10-calcium alginate) were calculated separately using a split microscope, 500 pieces each. The particle size of each of the two microcapsules was again calculated after drying 12 h at 27 ℃.
The wet empty microcapsules are spherical and white in color. The wet YZ-1 microcapsule is green and spherical. The dried empty microcapsules were yellowish, while the dried YZ-1 microcapsules were dark green. The diameter of the empty microcapsule and the African trichoderma harzianum YZ-1 microcapsule is mainly concentrated at 3200 mu m. The diameter of the dried microcapsules is concentrated to 1200-1400 μm due to water loss (fig. 11).
2.4 Microcapsule morphology
The YZ-1 microcapsules were dried with empty microcapsules at 27℃for 24 h. The external morphology of both microcapsules was observed using a Scanning Electron Microscope (SEM). The microcapsules were dehydrated at 50 ℃ for 24 hours before being coated with colloidal gold. Images were captured using SEM (hitachi SU 8010) run at 20 kV.
Scanning electron microscope shows that the empty microcapsule is approximately spherical and has smooth surface. In contrast, YZ-1 microcapsules exhibited slight protrusions and wrinkles, with the pore structure of the surface being more pronounced (fig. 12), which may promote water absorption and spore release after swelling.
2.5 Fourier transform infrared spectroscopy (FTIR)
Infrared spectra of microcapsules were recorded on FTIR (Thermo Nicolet IS, BRUKER TENSOR II) using KBr particle technology in the range 400-4000 cm -1. The method is divided into YZ-1 microcapsule treatment and empty microcapsule treatment.
The FTIR spectrum of the microcapsules is shown in fig. 13. The bands 3379.72, 2941.62, 1614.42 and 1036.46 cm -1 are O-H, C-H, O-C=O and C-O-C groups, respectively. There is a strong and broad absorption band at 3379.72 cm -1, belonging to O-H, which is related to the adsorbed water and the expansion ratio. A single peak at 2151.88cm -1 indicates that the microcapsules have an interaction with Trichoderma YZ-1.
2.6 Stability of
UV stability wet and dry YZ-1 microcapsules (1 g) were placed on a plate, respectively, with free YZ-1 spores as control. It was exposed to 253.7 wavelength ultraviolet light and emitted from 100 cm for UV treatment 0, 0.5, 1,2, 4, 9, 24, h. Each time 0.1 g was taken, suspended in 5 mL of 0.2. 0.2M NaHCO 3 and 0.06M sodium citrate solution and stirred for 15 hours to completely dissolve and dilute the plate. Survival is expressed as the percentage of viable conidia in the sample after uv irradiation relative to the conidia before irradiation. All treatments were repeated 3 times.
After 24 hours of UV irradiation, free YZ-1 spores were thoroughly killed, with mortality rates reaching 100%, whereas the dry and wet YZ-1 microcapsules were 87% and 91%, respectively (FIG. 14). The encapsulated YZ-1 is more resistant to ultraviolet light than the unencapsulated YZ-1. This result underscores the beneficial effect of the microcapsules in enhancing the uv resistance of biological control agents.
Temperature stability wet and dry YZ-1 microcapsules (1 g) were incubated at 7 ℃, 27 ℃ and 37 ℃ respectively, 0.1g per 15 d g was suspended in 5mL of 0.2M NaHCO 3 and 0.06M sodium citrate solution and stirred for 15 hours until complete dissolution and dilution plating was achieved until 60 days later. Viability is expressed as a percentage of the number of conidia in the samples at different time points relative to the number of conidia at day 0. All treatments were repeated 3 times.
The temperature stability results are shown in fig. 15, which shows that the stability at 7 ℃ is the highest for both dry and wet microcapsules and the stability at 37 ℃ is the worst. At the same time, the dried microcapsules have a higher storage stability.
2.7 Plate antibacterial effect
The bacteriostatic effect of YZ-1 microcapsules is observed by adopting opposite culture. The treatment was divided into four treatments, no-load microcapsules, YZ-1-GFP1 and blank. Wherein the treatment of YZ-1-GFP1 microcapsule is divided into two treatments of simultaneous inoculation with XTBJ-1 and inoculation of YZ-1-GFP1 microcapsule 1 day later and then inoculation of XTBJ-1.
When YZ-1-GFP1 microcapsules and XTBJ-1 were inoculated with PDA simultaneously, the inhibition was 37.24%. YZ-1-GFP1 microcapsules were inoculated 1 d followed by XTBJ-1 with an inhibition of 71.51%. The inhibition rate of YZ-1-GFP1 cake was 59.03% (FIG. 16).
2.8 Potted plant prevention effect
In order to determine the effect of YZ-1 microcapsules on controlling pepper southern blight, pepper seedlings were divided into 5 treatments, no-load microcapsules, YZ-1-GFP1, blank control and positive control. Empty microcapsules (3 g), YZ-1-GFP1 spore solution (10 mL), clear water (10 mL) and 0.4% thifluzamide (10 mL) were inoculated into the four leaves one heart of capsicum. All microcapsules are embedded in the root of the capsicum plant through soil, and all liquids are irrigated to the root. Each treatment was performed in 12 replicates. After 7 days, the treatment was repeated. After 14 days, four fresh XTBJ-1 mycelium pellets (d=8 mm) were inoculated at the base of the plant stem and covered with moist soil and cultivated with moisture at 28 ℃. After 5 days, the disease index is calculated, and indexes such as fresh weight, dry weight and the like are measured.
The prevention and treatment effect of YZ-1-GFP1 spore liquid is 42.19%. In contrast, the relative control efficiency of YZ-1-GFP1 microcapsules reached 74.13%. The pepper plants treated with the microcapsules showed better growth conditions (fig. 17).
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410973026.8A CN118667663B (en) | 2024-07-19 | 2024-07-19 | A strain of Trichoderma and its microcapsule preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410973026.8A CN118667663B (en) | 2024-07-19 | 2024-07-19 | A strain of Trichoderma and its microcapsule preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118667663A CN118667663A (en) | 2024-09-20 |
| CN118667663B true CN118667663B (en) | 2025-02-14 |
Family
ID=92721359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410973026.8A Active CN118667663B (en) | 2024-07-19 | 2024-07-19 | A strain of Trichoderma and its microcapsule preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118667663B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928673A (en) * | 2010-07-14 | 2010-12-29 | 洪亚辉 | Trichoderma harzianum |
| CN117327593A (en) * | 2023-11-23 | 2024-01-02 | 山东省花生研究所 | Trichoderma harzianum ZM1 and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117695253A (en) * | 2023-12-17 | 2024-03-15 | 仲景中医药产业研究院 | Preparation method and application of WGX-50/montmorillonite-sodium alginate microcapsule |
| CN117887589B (en) * | 2024-01-23 | 2024-09-20 | 海南大学 | Trichoderma FJ059 capable of parasitizing sclerotium of southern blight and application thereof |
-
2024
- 2024-07-19 CN CN202410973026.8A patent/CN118667663B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928673A (en) * | 2010-07-14 | 2010-12-29 | 洪亚辉 | Trichoderma harzianum |
| CN117327593A (en) * | 2023-11-23 | 2024-01-02 | 山东省花生研究所 | Trichoderma harzianum ZM1 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118667663A (en) | 2024-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104818216B (en) | One plant is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil | |
| CN102363750B (en) | Insecticidal fungus and application thereof | |
| PL175708B1 (en) | Application of streptomyces wyec 108 in fighting against plants pathogens | |
| CN102851219B (en) | Paecilomyces lilacinus and application thereof | |
| CN106701623B (en) | The atrophy bacillus of one plant of antagonism fructus lycii root rot and its application | |
| CN103243030B (en) | Lecanicilliumpsalliotae strain used for preventing and treating diaphorina citri | |
| CN117025466A (en) | Bacillus bailii and application thereof in controlling melon crop diseases | |
| CN106701624B (en) | The Te Jila bacillus of one plant of antagonism fructus lycii root rot and its application | |
| CN103333844B (en) | Strain of antagonistic poplar colletotrichumgloeosporioides and application thereof | |
| CN111286477A (en) | Pseudomonas syringae and application thereof in prevention and treatment of apple ring rot | |
| CN118667663B (en) | A strain of Trichoderma and its microcapsule preparation method and application | |
| CN111363695A (en) | A kind of apple tree rot disease biocontrol agent and preparation method and application thereof | |
| CN117701432A (en) | Acid-resistant bacillus pumilus and application thereof | |
| CN115851479B (en) | Bacteria with antagonism to botrytis cinerea and application thereof | |
| CN114196551B (en) | A kind of I. javanica strain for controlling citrus pests and its application | |
| CN113046249B (en) | Verticillium lecanii LL-01 and biocontrol application thereof | |
| CN112877220B (en) | Trichoderma harsii and application thereof | |
| CN100560716C (en) | Grass green streptomycete | |
| CN116463220A (en) | A Dark Septate DSE Fungus for Promoting Blueberry Growth and Its Application | |
| CN116410867A (en) | A kind of aspergillus flavus bacterial strain and application thereof | |
| CN116240126A (en) | Multifunctional bacillus belgium SB10 and application thereof | |
| CN108130293B (en) | Biocontrol bacteria, microbial inoculum and its application of one plant of antagonism Pestalotiopsis microspora bacterium | |
| CN111808785B (en) | Honghuadan endophytic Bacillus belgii and application thereof | |
| KR20080003084A (en) | Non-pathogenic strains Erwinia carobora 93 and culture method having cabbage control effect | |
| CN115029286B (en) | A kind of biocontrol bacteria compound bacteria agent and its application |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |