EP4188894A1 - Microbial strains bacillus pumilus, bacillus megaterium and uses thereof - Google Patents
Microbial strains bacillus pumilus, bacillus megaterium and uses thereofInfo
- Publication number
- EP4188894A1 EP4188894A1 EP21850718.4A EP21850718A EP4188894A1 EP 4188894 A1 EP4188894 A1 EP 4188894A1 EP 21850718 A EP21850718 A EP 21850718A EP 4188894 A1 EP4188894 A1 EP 4188894A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- bacteria
- extract
- spp
- vmc30
- anyone
- 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
- 230000000813 microbial effect Effects 0.000 title claims abstract description 28
- 241000194107 Bacillus megaterium Species 0.000 title claims abstract description 24
- 241000194103 Bacillus pumilus Species 0.000 title claims abstract description 23
- 230000001580 bacterial effect Effects 0.000 claims abstract description 61
- 239000000203 mixture Substances 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 26
- 230000008635 plant growth Effects 0.000 claims abstract description 18
- 238000011161 development Methods 0.000 claims abstract description 10
- 235000021231 nutrient uptake Nutrition 0.000 claims abstract description 8
- 230000008121 plant development Effects 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims description 106
- 239000000284 extract Substances 0.000 claims description 56
- 239000002609 medium Substances 0.000 claims description 45
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 43
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 40
- 239000011701 zinc Substances 0.000 claims description 39
- 241000894006 Bacteria Species 0.000 claims description 37
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 37
- 239000002689 soil Substances 0.000 claims description 37
- 229910052725 zinc Inorganic materials 0.000 claims description 37
- 239000002207 metabolite Substances 0.000 claims description 26
- 235000015097 nutrients Nutrition 0.000 claims description 26
- 239000006228 supernatant Substances 0.000 claims description 25
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 24
- 239000011591 potassium Substances 0.000 claims description 24
- 229910052700 potassium Inorganic materials 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 239000006166 lysate Substances 0.000 claims description 23
- 239000004615 ingredient Substances 0.000 claims description 22
- 229910052742 iron Inorganic materials 0.000 claims description 22
- 244000005700 microbiome Species 0.000 claims description 20
- 235000013369 micronutrients Nutrition 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 20
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 17
- 239000011785 micronutrient Substances 0.000 claims description 17
- 241000195493 Cryptophyta Species 0.000 claims description 16
- 239000000725 suspension Substances 0.000 claims description 16
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 14
- 239000011574 phosphorus Substances 0.000 claims description 14
- 229910052698 phosphorus Inorganic materials 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 11
- 230000001965 increasing effect Effects 0.000 claims description 11
- 239000011777 magnesium Substances 0.000 claims description 11
- 229910052749 magnesium Inorganic materials 0.000 claims description 10
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 9
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 9
- 239000011575 calcium Substances 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 7
- 229910052791 calcium Inorganic materials 0.000 claims description 7
- 239000002509 fulvic acid Substances 0.000 claims description 7
- 239000004021 humic acid Substances 0.000 claims description 7
- PUKLDDOGISCFCP-JSQCKWNTSA-N 21-Deoxycortisone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)C)(O)[C@@]1(C)CC2=O PUKLDDOGISCFCP-JSQCKWNTSA-N 0.000 claims description 6
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 6
- FCYKAQOGGFGCMD-UHFFFAOYSA-N Fulvic acid Natural products O1C2=CC(O)=C(O)C(C(O)=O)=C2C(=O)C2=C1CC(C)(O)OC2 FCYKAQOGGFGCMD-UHFFFAOYSA-N 0.000 claims description 6
- 241000235070 Saccharomyces Species 0.000 claims description 6
- 230000036579 abiotic stress Effects 0.000 claims description 6
- 230000018109 developmental process Effects 0.000 claims description 6
- 229940095100 fulvic acid Drugs 0.000 claims description 6
- 235000010958 polyglycerol polyricinoleate Nutrition 0.000 claims description 6
- 241000233866 Fungi Species 0.000 claims description 5
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 5
- 240000003768 Solanum lycopersicum Species 0.000 claims description 5
- 235000021307 Triticum Nutrition 0.000 claims description 5
- 244000098338 Triticum aestivum Species 0.000 claims description 5
- 239000010949 copper Substances 0.000 claims description 5
- 241001474374 Blennius Species 0.000 claims description 4
- 241000207199 Citrus Species 0.000 claims description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 4
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 claims description 4
- 235000010469 Glycine max Nutrition 0.000 claims description 4
- 244000068988 Glycine max Species 0.000 claims description 4
- 240000008415 Lactuca sativa Species 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 4
- 241000209504 Poaceae Species 0.000 claims description 4
- 240000000111 Saccharum officinarum Species 0.000 claims description 4
- 235000007201 Saccharum officinarum Nutrition 0.000 claims description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 claims description 4
- 235000020971 citrus fruits Nutrition 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 238000003973 irrigation Methods 0.000 claims description 4
- 230000002262 irrigation Effects 0.000 claims description 4
- 239000011593 sulfur Substances 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- 241000512259 Ascophyllum nodosum Species 0.000 claims description 3
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 claims description 3
- 235000011331 Brassica Nutrition 0.000 claims description 3
- 241000219198 Brassica Species 0.000 claims description 3
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 3
- 240000007817 Olea europaea Species 0.000 claims description 3
- 241000635201 Pumilus Species 0.000 claims description 3
- 241000219094 Vitaceae Species 0.000 claims description 3
- 230000008901 benefit Effects 0.000 claims description 3
- 229910052796 boron Inorganic materials 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 235000021374 legumes Nutrition 0.000 claims description 3
- 239000006194 liquid suspension Substances 0.000 claims description 3
- 229910052750 molybdenum Inorganic materials 0.000 claims description 3
- 239000011733 molybdenum Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 241001571014 Aeromonas rivuli Species 0.000 claims description 2
- 241000134715 Agromyces fucosus Species 0.000 claims description 2
- 244000144730 Amygdalus persica Species 0.000 claims description 2
- 235000016425 Arthrospira platensis Nutrition 0.000 claims description 2
- 240000002900 Arthrospira platensis Species 0.000 claims description 2
- 241000228212 Aspergillus Species 0.000 claims description 2
- 241000589151 Azotobacter Species 0.000 claims description 2
- 241000194108 Bacillus licheniformis Species 0.000 claims description 2
- 241000194106 Bacillus mycoides Species 0.000 claims description 2
- 241000835167 Bacillus safensis Species 0.000 claims description 2
- 244000063299 Bacillus subtilis Species 0.000 claims description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 241000751139 Beauveria bassiana Species 0.000 claims description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 claims description 2
- 241000335053 Beta vulgaris Species 0.000 claims description 2
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 claims description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 2
- 241000222178 Candida tropicalis Species 0.000 claims description 2
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 2
- 240000007154 Coffea arabica Species 0.000 claims description 2
- 241001266001 Cordyceps confragosa Species 0.000 claims description 2
- 229920000742 Cotton Polymers 0.000 claims description 2
- 241000305506 Desmodesmus Species 0.000 claims description 2
- 241000195634 Dunaliella Species 0.000 claims description 2
- 241001392689 Ecklonia maxima Species 0.000 claims description 2
- 241000588914 Enterobacter Species 0.000 claims description 2
- 239000001653 FEMA 3120 Substances 0.000 claims description 2
- 235000016623 Fragaria vesca Nutrition 0.000 claims description 2
- 240000009088 Fragaria x ananassa Species 0.000 claims description 2
- 235000011363 Fragaria x ananassa Nutrition 0.000 claims description 2
- 241000004454 Fucus serratus Species 0.000 claims description 2
- 241000004455 Fucus spiralis Species 0.000 claims description 2
- 241001674906 Funneliformis Species 0.000 claims description 2
- 241000235503 Glomus Species 0.000 claims description 2
- 241000168525 Haematococcus Species 0.000 claims description 2
- 241000605016 Herbaspirillum Species 0.000 claims description 2
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 241000218228 Humulus Species 0.000 claims description 2
- 241001501885 Isochrysis Species 0.000 claims description 2
- 240000001046 Lactobacillus acidophilus Species 0.000 claims description 2
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 2
- 241000186679 Lactobacillus buchneri Species 0.000 claims description 2
- 241000186673 Lactobacillus delbrueckii Species 0.000 claims description 2
- 241001468157 Lactobacillus johnsonii Species 0.000 claims description 2
- 241000186871 Lactobacillus murinus Species 0.000 claims description 2
- 241000866650 Lactobacillus paraplantarum Species 0.000 claims description 2
- 241000186684 Lactobacillus pentosus Species 0.000 claims description 2
- 240000006024 Lactobacillus plantarum Species 0.000 claims description 2
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims description 2
- 241000194036 Lactococcus Species 0.000 claims description 2
- 235000003228 Lactuca sativa Nutrition 0.000 claims description 2
- 241001598113 Laminaria digitata Species 0.000 claims description 2
- 241000234280 Liliaceae Species 0.000 claims description 2
- 241001491708 Macrocystis Species 0.000 claims description 2
- 240000004658 Medicago sativa Species 0.000 claims description 2
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 claims description 2
- 241000223201 Metarhizium Species 0.000 claims description 2
- 241000500375 Microbacterium sp. Species 0.000 claims description 2
- 241000224474 Nannochloropsis Species 0.000 claims description 2
- 241000502321 Navicula Species 0.000 claims description 2
- 241000195644 Neochloris Species 0.000 claims description 2
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 claims description 2
- 241000196159 Parietochloris Species 0.000 claims description 2
- 241000206766 Pavlova Species 0.000 claims description 2
- 241001262104 Pelvetia canaliculata Species 0.000 claims description 2
- 241000206731 Phaeodactylum Species 0.000 claims description 2
- 241000199919 Phaeophyceae Species 0.000 claims description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 2
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 2
- 235000010582 Pisum sativum Nutrition 0.000 claims description 2
- 240000004713 Pisum sativum Species 0.000 claims description 2
- 235000006040 Prunus persica var persica Nutrition 0.000 claims description 2
- 241000589516 Pseudomonas Species 0.000 claims description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 claims description 2
- 241000218899 Pseudomonas fulva Species 0.000 claims description 2
- 241001647875 Pseudoxanthomonas Species 0.000 claims description 2
- 241000233639 Pythium Species 0.000 claims description 2
- 241001092473 Quillaja Species 0.000 claims description 2
- 235000009001 Quillaja saponaria Nutrition 0.000 claims description 2
- 241000589157 Rhizobiales Species 0.000 claims description 2
- 241000589180 Rhizobium Species 0.000 claims description 2
- 241001175028 Rhizophagus <beetle> Species 0.000 claims description 2
- 241001518128 Rhodococcus coprophilus Species 0.000 claims description 2
- 241000983746 Saccharina latissima Species 0.000 claims description 2
- 241000235072 Saccharomyces bayanus Species 0.000 claims description 2
- 241001123227 Saccharomyces pastorianus Species 0.000 claims description 2
- 241000195474 Sargassum Species 0.000 claims description 2
- 241000195663 Scenedesmus Species 0.000 claims description 2
- 241001060494 Septoglomus Species 0.000 claims description 2
- 241000208292 Solanaceae Species 0.000 claims description 2
- 241001464945 Sphingopyxis macrogoltabida Species 0.000 claims description 2
- 241000122971 Stenotrophomonas Species 0.000 claims description 2
- 241000187747 Streptomyces Species 0.000 claims description 2
- 235000021536 Sugar beet Nutrition 0.000 claims description 2
- 241000192584 Synechocystis Species 0.000 claims description 2
- 241000196321 Tetraselmis Species 0.000 claims description 2
- 241001491691 Thalassiosira Species 0.000 claims description 2
- 241000223259 Trichoderma Species 0.000 claims description 2
- 241000675573 Trichoderma afroharzianum Species 0.000 claims description 2
- 241000894120 Trichoderma atroviride Species 0.000 claims description 2
- 241000223261 Trichoderma viride Species 0.000 claims description 2
- 241001261506 Undaria pinnatifida Species 0.000 claims description 2
- 235000004552 Yucca aloifolia Nutrition 0.000 claims description 2
- 235000012044 Yucca brevifolia Nutrition 0.000 claims description 2
- 235000017049 Yucca glauca Nutrition 0.000 claims description 2
- 240000005780 Yucca gloriosa Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 239000004492 dustable powder Substances 0.000 claims description 2
- 239000004495 emulsifiable concentrate Substances 0.000 claims description 2
- 239000000839 emulsion Substances 0.000 claims description 2
- 239000012520 frozen sample Substances 0.000 claims description 2
- 239000000499 gel Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 235000021021 grapes Nutrition 0.000 claims description 2
- 229940039695 lactobacillus acidophilus Drugs 0.000 claims description 2
- 229940072205 lactobacillus plantarum Drugs 0.000 claims description 2
- 235000009973 maize Nutrition 0.000 claims description 2
- 239000004530 micro-emulsion Substances 0.000 claims description 2
- 239000003094 microcapsule Substances 0.000 claims description 2
- 235000012162 pavlova Nutrition 0.000 claims description 2
- 229960002181 saccharomyces boulardii Drugs 0.000 claims description 2
- 239000004550 soluble concentrate Substances 0.000 claims description 2
- 229940082787 spirulina Drugs 0.000 claims description 2
- 239000004560 ultra-low volume (ULV) liquid Substances 0.000 claims description 2
- 238000009736 wetting Methods 0.000 claims description 2
- 241000208838 Asteraceae Species 0.000 claims 2
- 240000007124 Brassica oleracea Species 0.000 claims 2
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims 2
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims 2
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims 2
- 241000220485 Fabaceae Species 0.000 claims 2
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims 2
- 241000219066 Actinidiaceae Species 0.000 claims 1
- 244000291564 Allium cepa Species 0.000 claims 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 claims 1
- 244000144725 Amygdalus communis Species 0.000 claims 1
- 241000208223 Anacardiaceae Species 0.000 claims 1
- 241000233788 Arecaceae Species 0.000 claims 1
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 claims 1
- 235000006008 Brassica napus var napus Nutrition 0.000 claims 1
- 240000000385 Brassica napus var. napus Species 0.000 claims 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 claims 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 claims 1
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 claims 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 claims 1
- 241000234670 Bromeliaceae Species 0.000 claims 1
- 241000218235 Cannabaceae Species 0.000 claims 1
- 235000002566 Capsicum Nutrition 0.000 claims 1
- 241000219172 Caricaceae Species 0.000 claims 1
- 235000003255 Carthamus tinctorius Nutrition 0.000 claims 1
- 244000020518 Carthamus tinctorius Species 0.000 claims 1
- 241000871189 Chenopodiaceae Species 0.000 claims 1
- 241000219112 Cucumis Species 0.000 claims 1
- 240000008067 Cucumis sativus Species 0.000 claims 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims 1
- 241000219130 Cucurbita pepo subsp. pepo Species 0.000 claims 1
- 235000003954 Cucurbita pepo var melopepo Nutrition 0.000 claims 1
- 241000219104 Cucurbitaceae Species 0.000 claims 1
- 241000219428 Fagaceae Species 0.000 claims 1
- 235000009419 Fagopyrum esculentum Nutrition 0.000 claims 1
- 240000008620 Fagopyrum esculentum Species 0.000 claims 1
- 241000219146 Gossypium Species 0.000 claims 1
- 244000020551 Helianthus annuus Species 0.000 claims 1
- 235000003222 Helianthus annuus Nutrition 0.000 claims 1
- 241000758791 Juglandaceae Species 0.000 claims 1
- 241000218195 Lauraceae Species 0.000 claims 1
- 235000004431 Linum usitatissimum Nutrition 0.000 claims 1
- 240000006240 Linum usitatissimum Species 0.000 claims 1
- 241000219991 Lythraceae Species 0.000 claims 1
- 241000220225 Malus Species 0.000 claims 1
- 235000011430 Malus pumila Nutrition 0.000 claims 1
- 235000015103 Malus silvestris Nutrition 0.000 claims 1
- 241000219071 Malvaceae Species 0.000 claims 1
- 241000218231 Moraceae Species 0.000 claims 1
- 241000234615 Musaceae Species 0.000 claims 1
- 241000207834 Oleaceae Species 0.000 claims 1
- 241000218995 Passifloraceae Species 0.000 claims 1
- 239000006002 Pepper Substances 0.000 claims 1
- 235000016761 Piper aduncum Nutrition 0.000 claims 1
- 240000003889 Piper guineense Species 0.000 claims 1
- 235000017804 Piper guineense Nutrition 0.000 claims 1
- 235000008184 Piper nigrum Nutrition 0.000 claims 1
- 241000758706 Piperaceae Species 0.000 claims 1
- 235000009827 Prunus armeniaca Nutrition 0.000 claims 1
- 244000018633 Prunus armeniaca Species 0.000 claims 1
- 235000014443 Pyrus communis Nutrition 0.000 claims 1
- 240000001987 Pyrus communis Species 0.000 claims 1
- 235000004789 Rosa xanthina Nutrition 0.000 claims 1
- 241000220222 Rosaceae Species 0.000 claims 1
- 241001107098 Rubiaceae Species 0.000 claims 1
- 241001093501 Rutaceae Species 0.000 claims 1
- 235000002597 Solanum melongena Nutrition 0.000 claims 1
- 244000061458 Solanum melongena Species 0.000 claims 1
- 235000002595 Solanum tuberosum Nutrition 0.000 claims 1
- 244000061456 Solanum tuberosum Species 0.000 claims 1
- 235000009337 Spinacia oleracea Nutrition 0.000 claims 1
- 244000300264 Spinacia oleracea Species 0.000 claims 1
- 241001122767 Theaceae Species 0.000 claims 1
- 241000234299 Zingiberaceae Species 0.000 claims 1
- 235000020224 almond Nutrition 0.000 claims 1
- 239000008199 coating composition Substances 0.000 claims 1
- 230000002708 enhancing effect Effects 0.000 claims 1
- 210000003692 ilium Anatomy 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 16
- 230000000694 effects Effects 0.000 description 33
- 210000004027 cell Anatomy 0.000 description 30
- 238000005063 solubilization Methods 0.000 description 30
- 238000004519 manufacturing process Methods 0.000 description 27
- 230000007928 solubilization Effects 0.000 description 27
- 229960003975 potassium Drugs 0.000 description 22
- 239000000589 Siderophore Substances 0.000 description 20
- 235000021073 macronutrients Nutrition 0.000 description 20
- 235000013399 edible fruits Nutrition 0.000 description 18
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 15
- 229910019142 PO4 Inorganic materials 0.000 description 14
- 230000012010 growth Effects 0.000 description 14
- 239000010452 phosphate Substances 0.000 description 14
- 235000021317 phosphate Nutrition 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 239000006916 nutrient agar Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 238000000605 extraction Methods 0.000 description 10
- 238000011534 incubation Methods 0.000 description 10
- 239000002367 phosphate rock Substances 0.000 description 10
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 239000002028 Biomass Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 230000003381 solubilizing effect Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 235000013311 vegetables Nutrition 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000003337 fertilizer Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 235000001055 magnesium Nutrition 0.000 description 7
- 229940091250 magnesium supplement Drugs 0.000 description 7
- 241000894007 species Species 0.000 description 7
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 241000605014 Herbaspirillum seropedicae Species 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 229910052500 inorganic mineral Inorganic materials 0.000 description 6
- 235000010755 mineral Nutrition 0.000 description 6
- 239000011707 mineral Substances 0.000 description 6
- -1 roxitromycin Chemical compound 0.000 description 6
- 235000002639 sodium chloride Nutrition 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 235000011118 potassium hydroxide Nutrition 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 241000589938 Azospirillum brasilense Species 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 150000007513 acids Chemical class 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 229910001447 ferric ion Inorganic materials 0.000 description 4
- 125000001475 halogen functional group Chemical group 0.000 description 4
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 4
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 description 4
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 239000011787 zinc oxide Substances 0.000 description 4
- LMSDCGXQALIMLM-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;iron Chemical compound [Fe].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O LMSDCGXQALIMLM-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 240000004160 Capsicum annuum Species 0.000 description 3
- 235000010523 Cicer arietinum Nutrition 0.000 description 3
- 244000045195 Cicer arietinum Species 0.000 description 3
- 244000241257 Cucumis melo Species 0.000 description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000012208 gluconic acid Nutrition 0.000 description 3
- 229940093915 gynecological organic acid Drugs 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 3
- 230000003050 macronutrient Effects 0.000 description 3
- 239000011572 manganese Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000002420 orchard Substances 0.000 description 3
- 235000005985 organic acids Nutrition 0.000 description 3
- 230000029553 photosynthesis Effects 0.000 description 3
- 238000010672 photosynthesis Methods 0.000 description 3
- 230000000243 photosynthetic effect Effects 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000000375 suspending agent Substances 0.000 description 3
- 239000002562 thickening agent Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 239000000080 wetting agent Substances 0.000 description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 description 2
- 239000004254 Ammonium phosphate Substances 0.000 description 2
- 241000206761 Bacillariophyta Species 0.000 description 2
- 241000195940 Bryophyta Species 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 241000195628 Chlorophyta Species 0.000 description 2
- 241000192700 Cyanobacteria Species 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 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
- 239000005955 Ferric phosphate Substances 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 2
- 229920001732 Lignosulfonate Polymers 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241000985694 Polypodiopsida Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical class [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 239000005819 Potassium phosphonate Substances 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 241000206572 Rhodophyta Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 2
- 206010048259 Zinc deficiency Diseases 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000006578 abscission Effects 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- ZRIUUUJAJJNDSS-UHFFFAOYSA-N ammonium phosphates Chemical class [NH4+].[NH4+].[NH4+].[O-]P([O-])([O-])=O ZRIUUUJAJJNDSS-UHFFFAOYSA-N 0.000 description 2
- 235000019289 ammonium phosphates Nutrition 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 235000021028 berry Nutrition 0.000 description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000013522 chelant Substances 0.000 description 2
- 230000009920 chelation Effects 0.000 description 2
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 244000038559 crop plants Species 0.000 description 2
- 238000012272 crop production Methods 0.000 description 2
- 230000008260 defense mechanism Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- YXXXKCDYKKSZHL-UHFFFAOYSA-M dipotassium;dioxido(oxo)phosphanium Chemical compound [K+].[K+].[O-][P+]([O-])=O YXXXKCDYKKSZHL-UHFFFAOYSA-M 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000009313 farming Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000000174 gluconic acid Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000003077 lignite Substances 0.000 description 2
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 2
- 239000004137 magnesium phosphate Substances 0.000 description 2
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 2
- 229960002261 magnesium phosphate Drugs 0.000 description 2
- 235000010994 magnesium phosphates Nutrition 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 229910052748 manganese Inorganic materials 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000010445 mica Substances 0.000 description 2
- 229910052618 mica group Inorganic materials 0.000 description 2
- 235000020802 micronutrient deficiency Nutrition 0.000 description 2
- 230000001483 mobilizing effect Effects 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 239000004323 potassium nitrate Substances 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 2
- NYBZAGXTZXPYND-GBIKHYSHSA-N pyochelin I Chemical compound S1C[C@@H](C(O)=O)N(C)[C@H]1[C@@H]1N=C(C=2C(=CC=CC=2)O)SC1 NYBZAGXTZXPYND-GBIKHYSHSA-N 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 229910052604 silicate mineral Inorganic materials 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 235000019731 tricalcium phosphate Nutrition 0.000 description 2
- 229940078499 tricalcium phosphate Drugs 0.000 description 2
- VMFOHNMEJNFJAE-UHFFFAOYSA-N trimagnesium;diphosphite Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])[O-].[O-]P([O-])[O-] VMFOHNMEJNFJAE-UHFFFAOYSA-N 0.000 description 2
- FUIZKNBTOOKONL-DPSBJRLESA-K trisodium;5-[(e)-(3-carboxy-5-methyl-4-oxocyclohexa-2,5-dien-1-ylidene)-(2,6-dichloro-3-sulfonatophenyl)methyl]-3-methyl-2-oxidobenzoate Chemical compound [Na+].[Na+].[Na+].C1=C(C([O-])=O)C(=O)C(C)=C\C1=C(C=1C(=C(C=CC=1Cl)S([O-])(=O)=O)Cl)\C1=CC(C)=C(O)C(C([O-])=O)=C1 FUIZKNBTOOKONL-DPSBJRLESA-K 0.000 description 2
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 description 2
- 229910000165 zinc phosphate Inorganic materials 0.000 description 2
- 229960001763 zinc sulfate Drugs 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- VKZRWSNIWNFCIQ-WDSKDSINSA-N (2s)-2-[2-[[(1s)-1,2-dicarboxyethyl]amino]ethylamino]butanedioic acid Chemical compound OC(=O)C[C@@H](C(O)=O)NCCN[C@H](C(O)=O)CC(O)=O VKZRWSNIWNFCIQ-WDSKDSINSA-N 0.000 description 1
- QJVHTELASVOWBE-AGNWQMPPSA-N (2s,5r,6r)-6-[[(2r)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;(2r,3z,5r)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound OC(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21.C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 QJVHTELASVOWBE-AGNWQMPPSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- KBPZVLXARDTGGD-UHFFFAOYSA-N 2,3-dihydroxybutanedioic acid;iron Chemical compound [Fe].OC(=O)C(O)C(O)C(O)=O KBPZVLXARDTGGD-UHFFFAOYSA-N 0.000 description 1
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical class [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- SFJPGSCMZIUEDJ-UHFFFAOYSA-N 2-[2-[[carboxy-(2-hydroxy-4-methylphenyl)methyl]amino]ethylamino]-2-(2-hydroxy-4-methylphenyl)acetic acid Chemical compound OC1=CC(C)=CC=C1C(C(O)=O)NCCNC(C(O)=O)C1=CC=C(C)C=C1O SFJPGSCMZIUEDJ-UHFFFAOYSA-N 0.000 description 1
- GOPWHIPESJZSFI-UHFFFAOYSA-N 2-[2-[[carboxy-(2-hydroxy-5-sulfophenyl)methyl]amino]ethylamino]-2-(2-hydroxy-5-sulfophenyl)acetic acid Chemical compound C=1C(S(O)(=O)=O)=CC=C(O)C=1C(C(=O)O)NCCNC(C(O)=O)C1=CC(S(O)(=O)=O)=CC=C1O GOPWHIPESJZSFI-UHFFFAOYSA-N 0.000 description 1
- URDCARMUOSMFFI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetic acid Chemical compound OCCN(CC(O)=O)CCN(CC(O)=O)CC(O)=O URDCARMUOSMFFI-UHFFFAOYSA-N 0.000 description 1
- GRUVVLWKPGIYEG-UHFFFAOYSA-N 2-[2-[carboxymethyl-[(2-hydroxyphenyl)methyl]amino]ethyl-[(2-hydroxyphenyl)methyl]amino]acetic acid Chemical compound C=1C=CC=C(O)C=1CN(CC(=O)O)CCN(CC(O)=O)CC1=CC=CC=C1O GRUVVLWKPGIYEG-UHFFFAOYSA-N 0.000 description 1
- VBUYCZFBVCCYFD-JJYYJPOSSA-N 2-dehydro-D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)C(O)=O VBUYCZFBVCCYFD-JJYYJPOSSA-N 0.000 description 1
- JLLRXSCNTCSLFX-UHFFFAOYSA-H 2-hydroxybutanedioate;iron(3+) Chemical compound [Fe+3].[Fe+3].[O-]C(=O)C(O)CC([O-])=O.[O-]C(=O)C(O)CC([O-])=O.[O-]C(=O)C(O)CC([O-])=O JLLRXSCNTCSLFX-UHFFFAOYSA-H 0.000 description 1
- YNVZDODIHZTHOZ-UHFFFAOYSA-K 2-hydroxypropanoate;iron(3+) Chemical compound [Fe+3].CC(O)C([O-])=O.CC(O)C([O-])=O.CC(O)C([O-])=O YNVZDODIHZTHOZ-UHFFFAOYSA-K 0.000 description 1
- XMIIGOLPHOKFCH-UHFFFAOYSA-N 3-phenylpropionic acid Chemical compound OC(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- WZRJTRPJURQBRM-UHFFFAOYSA-N 4-amino-n-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide;5-[(3,4,5-trimethoxyphenyl)methyl]pyrimidine-2,4-diamine Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1.COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 WZRJTRPJURQBRM-UHFFFAOYSA-N 0.000 description 1
- IZSRJDGCGRAUAR-MROZADKFSA-N 5-dehydro-D-gluconic acid Chemical compound OCC(=O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O IZSRJDGCGRAUAR-MROZADKFSA-N 0.000 description 1
- 241001133760 Acoelorraphe Species 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 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
- 241000203069 Archaea Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 241000219193 Brassicaceae Species 0.000 description 1
- 108010004032 Bromelains Proteins 0.000 description 1
- 241000371430 Burkholderia cenocepacia Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- WNBCMONIPIJTSB-BGNCJLHMSA-N Cichoriin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1c(O)cc2c(OC(=O)C=C2)c1 WNBCMONIPIJTSB-BGNCJLHMSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 108010061075 Enterobactin Proteins 0.000 description 1
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920002444 Exopolysaccharide Polymers 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 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 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 208000019025 Hypokalemia Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical class NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 1
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 description 1
- 238000007476 Maximum Likelihood Methods 0.000 description 1
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 description 1
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 108010020943 Nitrogenase Proteins 0.000 description 1
- 241000187653 Nocardia globerula Species 0.000 description 1
- YJQPYGGHQPGBLI-UHFFFAOYSA-N Novobiocin Natural products O1C(C)(C)C(OC)C(OC(N)=O)C(O)C1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- AYRXSINWFIIFAE-UHFFFAOYSA-N O6-alpha-D-Galactopyranosyl-D-galactose Natural products OCC1OC(OCC(O)C(O)C(O)C(O)C=O)C(O)C(O)C1O AYRXSINWFIIFAE-UHFFFAOYSA-N 0.000 description 1
- 235000002725 Olea europaea Nutrition 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- MEVBFXZVEAUMTO-UHFFFAOYSA-L P(=O)([O-])([O-])O.[K+].S(=S)(=O)(O)O.[K+] Chemical class P(=O)([O-])([O-])O.[K+].S(=S)(=O)(O)O.[K+] MEVBFXZVEAUMTO-UHFFFAOYSA-L 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010093965 Polymyxin B Proteins 0.000 description 1
- 239000004260 Potassium ascorbate Chemical class 0.000 description 1
- 239000005700 Putrescine Substances 0.000 description 1
- NYBZAGXTZXPYND-UHFFFAOYSA-N Pyochelin I Natural products S1CC(C(O)=O)N(C)C1C1N=C(C=2C(=CC=CC=2)O)SC1 NYBZAGXTZXPYND-UHFFFAOYSA-N 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229910000831 Steel Inorganic materials 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
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 229920001807 Urea-formaldehyde Polymers 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- INAPMGSXUVUWAF-GCVPSNMTSA-N [(2r,3s,5r,6r)-2,3,4,5,6-pentahydroxycyclohexyl] dihydrogen phosphate Chemical compound OC1[C@H](O)[C@@H](O)C(OP(O)(O)=O)[C@H](O)[C@@H]1O INAPMGSXUVUWAF-GCVPSNMTSA-N 0.000 description 1
- MCDLETWIOVSGJT-UHFFFAOYSA-N acetic acid;iron Chemical compound [Fe].CC(O)=O.CC(O)=O MCDLETWIOVSGJT-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000008055 alkyl aryl sulfonates Chemical class 0.000 description 1
- 229940045714 alkyl sulfonate alkylating agent Drugs 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- SXQXMCWCWVCFPC-UHFFFAOYSA-N aluminum;potassium;dioxido(oxo)silane Chemical compound [Al+3].[K+].[O-][Si]([O-])=O.[O-][Si]([O-])=O SXQXMCWCWVCFPC-UHFFFAOYSA-N 0.000 description 1
- DLHONNLASJQAHX-UHFFFAOYSA-N aluminum;potassium;oxygen(2-);silicon(4+) Chemical compound [O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[Al+3].[Si+4].[Si+4].[Si+4].[K+] DLHONNLASJQAHX-UHFFFAOYSA-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
- XYXNTHIYBIDHGM-UHFFFAOYSA-N ammonium thiosulfate Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=S XYXNTHIYBIDHGM-UHFFFAOYSA-N 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 229940072107 ascorbate Drugs 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 229960004099 azithromycin Drugs 0.000 description 1
- MQTOSJVFKKJCRP-BICOPXKESA-N azithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)N(C)C[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 MQTOSJVFKKJCRP-BICOPXKESA-N 0.000 description 1
- 230000037358 bacterial metabolism Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 229910052626 biotite Inorganic materials 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 235000019835 bromelain Nutrition 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical class OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 229960002129 cefixime Drugs 0.000 description 1
- OKBVVJOGVLARMR-QSWIMTSFSA-N cefixime Chemical compound S1C(N)=NC(C(=N\OCC(O)=O)\C(=O)N[C@@H]2C(N3C(=C(C=C)CS[C@@H]32)C(O)=O)=O)=C1 OKBVVJOGVLARMR-QSWIMTSFSA-N 0.000 description 1
- 229960000484 ceftazidime Drugs 0.000 description 1
- NMVPEQXCMGEDNH-TZVUEUGBSA-N ceftazidime pentahydrate Chemical compound O.O.O.O.O.S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC(C)(C)C(O)=O)C=2N=C(N)SC=2)CC=1C[N+]1=CC=CC=C1 NMVPEQXCMGEDNH-TZVUEUGBSA-N 0.000 description 1
- 229960004755 ceftriaxone Drugs 0.000 description 1
- VAAUVRVFOQPIGI-SPQHTLEESA-N ceftriaxone Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C(=O)NN1C VAAUVRVFOQPIGI-SPQHTLEESA-N 0.000 description 1
- 229960001668 cefuroxime Drugs 0.000 description 1
- JFPVXVDWJQMJEE-IZRZKJBUSA-N cefuroxime Chemical compound N([C@@H]1C(N2C(=C(COC(N)=O)CS[C@@H]21)C(O)=O)=O)C(=O)\C(=N/OC)C1=CC=CO1 JFPVXVDWJQMJEE-IZRZKJBUSA-N 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000004464 cereal grain Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 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
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 229960002626 clarithromycin Drugs 0.000 description 1
- AGOYDEPGAOXOCK-KCBOHYOISA-N clarithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@](C)([C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)OC)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 AGOYDEPGAOXOCK-KCBOHYOISA-N 0.000 description 1
- 229940047766 co-trimoxazole Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- YGANSGVIUGARFR-UHFFFAOYSA-N dipotassium dioxosilane oxo(oxoalumanyloxy)alumane oxygen(2-) Chemical compound [O--].[K+].[K+].O=[Si]=O.O=[Al]O[Al]=O YGANSGVIUGARFR-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- FGRVOLIFQGXPCT-UHFFFAOYSA-L dipotassium;dioxido-oxo-sulfanylidene-$l^{6}-sulfane Chemical compound [K+].[K+].[O-]S([O-])(=O)=S FGRVOLIFQGXPCT-UHFFFAOYSA-L 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000010459 dolomite Substances 0.000 description 1
- 229910000514 dolomite Inorganic materials 0.000 description 1
- 230000003503 early effect Effects 0.000 description 1
- PZZHMLOHNYWKIK-UHFFFAOYSA-N eddha Chemical compound C=1C=CC=C(O)C=1C(C(=O)O)NCCNC(C(O)=O)C1=CC=CC=C1O PZZHMLOHNYWKIK-UHFFFAOYSA-N 0.000 description 1
- SERBHKJMVBATSJ-BZSNNMDCSA-N enterobactin Chemical compound OC1=CC=CC(C(=O)N[C@@H]2C(OC[C@@H](C(=O)OC[C@@H](C(=O)OC2)NC(=O)C=2C(=C(O)C=CC=2)O)NC(=O)C=2C(=C(O)C=CC=2)O)=O)=C1O SERBHKJMVBATSJ-BZSNNMDCSA-N 0.000 description 1
- 230000005183 environmental health Effects 0.000 description 1
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 description 1
- 229940093496 esculin Drugs 0.000 description 1
- AWRMZKLXZLNBBK-UHFFFAOYSA-N esculin Natural products OC1OC(COc2cc3C=CC(=O)Oc3cc2O)C(O)C(O)C1O AWRMZKLXZLNBBK-UHFFFAOYSA-N 0.000 description 1
- 235000021112 essential micronutrients Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000010433 feldspar Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229940032958 ferric phosphate Drugs 0.000 description 1
- RAQDACVRFCEPDA-UHFFFAOYSA-L ferrous carbonate Chemical compound [Fe+2].[O-]C([O-])=O RAQDACVRFCEPDA-UHFFFAOYSA-L 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 229910052587 fluorapatite Inorganic materials 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 229960000308 fosfomycin Drugs 0.000 description 1
- YMDXZJFXQJVXBF-STHAYSLISA-N fosfomycin Chemical compound C[C@@H]1O[C@@H]1P(O)(O)=O YMDXZJFXQJVXBF-STHAYSLISA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000005078 fruit development Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- DLRVVLDZNNYCBX-CQUJWQHSSA-N gentiobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-CQUJWQHSSA-N 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 235000021384 green leafy vegetables Nutrition 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 208000037824 growth disorder Diseases 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- GFAZHVHNLUBROE-UHFFFAOYSA-N hydroxymethyl propionaldehyde Natural products CCC(=O)CO GFAZHVHNLUBROE-UHFFFAOYSA-N 0.000 description 1
- 208000006278 hypochromic anemia Diseases 0.000 description 1
- 229910052900 illite Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 239000003617 indole-3-acetic acid Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000000797 iron chelating agent Substances 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 235000014413 iron hydroxide Nutrition 0.000 description 1
- 229910000398 iron phosphate Inorganic materials 0.000 description 1
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- MVFCKEFYUDZOCX-UHFFFAOYSA-N iron(2+);dinitrate Chemical compound [Fe+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O MVFCKEFYUDZOCX-UHFFFAOYSA-N 0.000 description 1
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 1
- NCNCGGDMXMBVIA-UHFFFAOYSA-L iron(ii) hydroxide Chemical compound [OH-].[OH-].[Fe+2] NCNCGGDMXMBVIA-UHFFFAOYSA-L 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 description 1
- 229940116298 l- malic acid Drugs 0.000 description 1
- 229960003376 levofloxacin Drugs 0.000 description 1
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 description 1
- 229960005287 lincomycin Drugs 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical class [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 239000011654 magnesium acetate Chemical class 0.000 description 1
- 235000011285 magnesium acetate Nutrition 0.000 description 1
- 229940069446 magnesium acetate Drugs 0.000 description 1
- 229940074358 magnesium ascorbate Drugs 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 229960001708 magnesium carbonate Drugs 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229960002337 magnesium chloride Drugs 0.000 description 1
- 235000011147 magnesium chloride Nutrition 0.000 description 1
- 239000004337 magnesium citrate Chemical class 0.000 description 1
- 229960005336 magnesium citrate Drugs 0.000 description 1
- 235000002538 magnesium citrate Nutrition 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- CPLXHLVBOLITMK-UHFFFAOYSA-N magnesium oxide Inorganic materials [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 1
- 229960003390 magnesium sulfate Drugs 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940062135 magnesium thiosulfate Drugs 0.000 description 1
- ZSMRTTMJRRECKC-RJNTXXOISA-L magnesium;(2e,4e)-hexa-2,4-dienoate Chemical class [Mg+2].C\C=C\C=C\C([O-])=O.C\C=C\C=C\C([O-])=O ZSMRTTMJRRECKC-RJNTXXOISA-L 0.000 description 1
- AIOKQVJVNPDJKA-ZZMNMWMASA-L magnesium;(2r)-2-[(1s)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2h-furan-3-olate Chemical compound [Mg+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] AIOKQVJVNPDJKA-ZZMNMWMASA-L 0.000 description 1
- GMDNUWQNDQDBNQ-UHFFFAOYSA-L magnesium;diformate Chemical compound [Mg+2].[O-]C=O.[O-]C=O GMDNUWQNDQDBNQ-UHFFFAOYSA-L 0.000 description 1
- TZKHCTCLSRVZEY-UHFFFAOYSA-L magnesium;dioxido-oxo-sulfanylidene-$l^{6}-sulfane Chemical compound [Mg+2].[O-]S([O-])(=O)=S TZKHCTCLSRVZEY-UHFFFAOYSA-L 0.000 description 1
- AXZKOIWUVFPNLO-UHFFFAOYSA-N magnesium;oxygen(2-) Chemical compound [O-2].[Mg+2] AXZKOIWUVFPNLO-UHFFFAOYSA-N 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 229940099690 malic acid Drugs 0.000 description 1
- MIVBAHRSNUNMPP-UHFFFAOYSA-N manganese(2+);dinitrate Chemical compound [Mn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O MIVBAHRSNUNMPP-UHFFFAOYSA-N 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 240000004308 marijuana Species 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229960003987 melatonin Drugs 0.000 description 1
- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 239000012569 microbial contaminant Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 229960000931 miocamycin Drugs 0.000 description 1
- GQNZGCARKRHPOH-RQIKCTSVSA-N miocamycin Chemical compound C1[C@](OC(C)=O)(C)[C@@H](OC(=O)CC)[C@H](C)O[C@H]1O[C@H]1[C@H](N(C)C)[C@@H](O)[C@H](O[C@@H]2[C@H]([C@H](OC(=O)CC)CC(=O)O[C@H](C)C/C=C/C=C/[C@H](OC(C)=O)[C@H](C)C[C@@H]2CC=O)OC)O[C@@H]1C GQNZGCARKRHPOH-RQIKCTSVSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229910052627 muscovite Inorganic materials 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 229960000808 netilmicin Drugs 0.000 description 1
- ZBGPYVZLYBDXKO-HILBYHGXSA-N netilmycin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@]([C@H](NC)[C@@H](O)CO1)(C)O)NCC)[C@H]1OC(CN)=CC[C@H]1N ZBGPYVZLYBDXKO-HILBYHGXSA-N 0.000 description 1
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 1
- VGIBGUSAECPPNB-UHFFFAOYSA-L nonaaluminum;magnesium;tripotassium;1,3-dioxido-2,4,5-trioxa-1,3-disilabicyclo[1.1.1]pentane;iron(2+);oxygen(2-);fluoride;hydroxide Chemical compound [OH-].[O-2].[O-2].[O-2].[O-2].[O-2].[F-].[Mg+2].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[K+].[K+].[K+].[Fe+2].O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2 VGIBGUSAECPPNB-UHFFFAOYSA-L 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 229960002950 novobiocin Drugs 0.000 description 1
- YJQPYGGHQPGBLI-KGSXXDOSSA-N novobiocin Chemical compound O1C(C)(C)[C@H](OC)[C@@H](OC(N)=O)[C@@H](O)[C@@H]1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-KGSXXDOSSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 150000002903 organophosphorus compounds Chemical class 0.000 description 1
- 229910052652 orthoclase Inorganic materials 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 150000002913 oxalic acids Chemical class 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 229960004236 pefloxacin Drugs 0.000 description 1
- FHFYDNQZQSQIAI-UHFFFAOYSA-N pefloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCN(C)CC1 FHFYDNQZQSQIAI-UHFFFAOYSA-N 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000002686 phosphate fertilizer Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 238000004175 phosphorus cycle Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229960002292 piperacillin Drugs 0.000 description 1
- WCMIIGXFCMNQDS-IDYPWDAWSA-M piperacillin sodium Chemical compound [Na+].O=C1C(=O)N(CC)CCN1C(=O)N[C@H](C=1C=CC=CC=1)C(=O)N[C@@H]1C(=O)N2[C@@H](C([O-])=O)C(C)(C)S[C@@H]21 WCMIIGXFCMNQDS-IDYPWDAWSA-M 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920000024 polymyxin B Polymers 0.000 description 1
- 229960005266 polymyxin b Drugs 0.000 description 1
- ODGAOXROABLFNM-UHFFFAOYSA-N polynoxylin Chemical compound O=C.NC(N)=O ODGAOXROABLFNM-UHFFFAOYSA-N 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 235000007686 potassium Nutrition 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 235000019275 potassium ascorbate Nutrition 0.000 description 1
- 229940017794 potassium ascorbate Drugs 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- 239000001103 potassium chloride Chemical class 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 208000007645 potassium deficiency Diseases 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000004302 potassium sorbate Chemical class 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- CONVKSGEGAVTMB-RXSVEWSESA-M potassium-L-ascorbate Chemical class [K+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] CONVKSGEGAVTMB-RXSVEWSESA-M 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 108010025281 pyoverdin Proteins 0.000 description 1
- 108700022487 rRNA Genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000026206 response to starvation Effects 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 1
- 229940120668 salicin Drugs 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 230000009291 secondary effect Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000008117 seed development Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229960002901 sodium glycerophosphate Drugs 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- REULQIKBNNDNDX-UHFFFAOYSA-M sodium;2,3-dihydroxypropyl hydrogen phosphate Chemical compound [Na+].OCC(O)COP(O)([O-])=O REULQIKBNNDNDX-UHFFFAOYSA-M 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000011044 succinic acid Nutrition 0.000 description 1
- 150000003444 succinic acids Chemical class 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- FKENQMMABCRJMK-RITPCOANSA-N sulbactam Chemical compound O=S1(=O)C(C)(C)[C@H](C(O)=O)N2C(=O)C[C@H]21 FKENQMMABCRJMK-RITPCOANSA-N 0.000 description 1
- 229960005256 sulbactam Drugs 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- JBQYATWDVHIOAR-UHFFFAOYSA-N tellanylidenegermanium Chemical compound [Te]=[Ge] JBQYATWDVHIOAR-UHFFFAOYSA-N 0.000 description 1
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical compound C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 1
- 229960000707 tobramycin Drugs 0.000 description 1
- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- XWKBMOUUGHARTI-UHFFFAOYSA-N tricalcium;diphosphite Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])[O-].[O-]P([O-])[O-] XWKBMOUUGHARTI-UHFFFAOYSA-N 0.000 description 1
- PLSARIKBYIPYPF-UHFFFAOYSA-H trimagnesium dicitrate Chemical class [Mg+2].[Mg+2].[Mg+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O PLSARIKBYIPYPF-UHFFFAOYSA-H 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical class [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- NCPXQVVMIXIKTN-UHFFFAOYSA-N trisodium;phosphite Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])[O-] NCPXQVVMIXIKTN-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000007492 two-way ANOVA Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000011667 zinc carbonate Substances 0.000 description 1
- 229910000010 zinc carbonate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- 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
- A01P21/00—Plant growth regulators
Definitions
- Microbial strains Bacillus pumilus, Bacillus megaterium and uses thereof Bacillus pumilus, Bacillus megaterium and uses thereof
- the present invention refers to a new bacterial strain, a microbial consortium comprising said strain and a composition comprising the bacterial strain or the consortium eventually in combination with a biostimulant for plants. Moreover, the present invention refers to the use of the bacterial strain, the microbial consortium or the composition in agriculture, preferably to improve plant processes, preferably nutrient up-take, and therefore to enhance plant growth and/or development.
- Background art In the past years, the heavy use of fertilizers, agro-chemicals and pesticides, which marked the First Green Revolution, strongly improved crop productivity, without taking in account adverse effects on environment and human health (Serpil, 2012), such as decreased soil fertility and increased susceptibility of plants to pests and disease (Jen-Hshuan Chen, 2006).
- Biofertilizers have been considered a good and eco-friendly solution for sustainable agriculture compared to chemical fertilizers and can significantly improve crop productivity, ameliorate nutrient up-take and make plants more tolerant to several biotic and abiotic stress in a sustainable manner (Deepak et al., 2014).
- the solution disclosed herein is based on a novel bacterial strain, a bacterial consortium or a composition thereof capable of increasing soil solubilization of main macronutrients and/or micronutrients, preferably phosphorus, zinc, iron, nitrogen, potassium and combinations thereof. Therefore, the strain, consortium or composition thereof is useful to improve micro and/or macronutrient uptake in plants and to ameliorate plant biology, in particular, the growth and/or the development of plants, nutrient use efficiency. Overall, the strain, consortium or composition disclosed herein is useful as biostimulant for plants, preferably to enhance in plants nutrient uptake, nutrient efficiency, tolerance to abiotic stress, crop quality and combination thereof.
- Figure 1 shows the phylogenetic tree obtained from the alignment of the full 16 rRNA gene sequences, with the “Maximum Likelihood” statistical method, “Tamura- Nei” substitution model and “Complete Deletion” mode (No. of bootstrap replications: 1000).
- strain Bacillus pumilus VMC30/195 is related to the species B. pumilus as it shares 97% of sequence similarity with the 16S rRNA sequence of Bacillus pumilus SH-B9.
- the strain Bacillus megaterium VMC30/195 is phylogenetically related to the species B. megaterium, as it shares 94,98% of sequence similarity with the 16S rRNA sequence of Bacillus megaterium CtST3.5.
- FIG. 2 shows protein mass spectra (in the range from 2 to about 12 KDa, peaks of a spectrum m/z values with a given intensity) of VMC30/195 (A) and VMC30/196 (B).
- Each peak in the graphs represents a different protein expressed by the microorganism and the intensity of the peaks represents the concentration of that proteins within the microbial cell. Since proteins are a direct expression of genome and genome of each strains is unique, thus the proteomic profile of each strain is unique and can be used as fingerprint differentiation and classification.
- FIG. 3 shows the P-solubilization index of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Herbaspitillum seropedicae ATCC 35893.
- FIG. 4 shows the Zinc Solubilization Efficiency (SE) of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Herbaspitillum seropedicae ATCC 35893.
- SE Zinc Solubilization Efficiency
- FIG. 5 shows siderophores production of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to
- FIG. 6 shows the potassium mobilization of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to an internal control.
- FIG. 7 shows IAA production of Bacillus pumilus VMC30/195 and Bacillus megaterium VMC30/196 compared to Azospirillum brasilense ATCC 29145.
- FIG. 8 shows the nitrogen fixation of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Azospirillum brasiliense ATCC 29145 and Herbaspirillum seropedicae ATCC 35893.
- the present invention refers to a novel strain of bacteria isolated and characterized for the ability to be used in agriculture, preferably for the purpose of solubilizing macro and/or micro-nutrients that are essential for plant biology, preferably for plant growth and development.
- strain disclosed herein is useful as biostimulant for plants and is preferably used to increase nutrient use efficiency in plants and/or resistance to abiotic stress of plants and/or plant growth and/or quality.
- strain refers to isolate or a group of isolates exhibiting phenotypic, physiologic, metabolic, and/or genotypic traits belonging to the same lineage and different/distinct from that of other strains belonging to the same species.
- isolated means a microorganism which has been removed from its original environment, including, but not limited to, soil, air, fresh water, sea water, algae, higher plants, seeds, roots, leaves, fruits, etc.
- the “isolate” has to be “pure”, meaning that it does not comprise other microbial contaminants in the isolate, according to the isolation method reported in the present invention.
- micronutrients or main nutrients refer - in general - to any nutrient that plays an essential role in any key physical, physiological and biochemical process of plants, providing a healthy and balanced growth and development during all lifecycle of plants.
- said nutrient is selected from: nitrogen, phosphorus, potassium (also known as NPK elements), calcium (Ca), magnesium (Mg), sulfur (S) and combinations thereof.
- Phosphorus (P) is one of the most indispensable macronutrients next to nitrogen for the growth and/or the development of plants.
- Deficiency or excess of the main nutrients have relevant consequences such as great imbalance of the crops, delay of the growth, leaves and/or flowers loss, reduced photosynthetic activity, with subsequent low fruit production and, overall, the crop productivity is heavily reduced.
- pathological consequences such as plant tissues and/or fruit necrosis, can also take place.
- plant biology refers to any physiological and/or pathological plant process, preferably selected from: growth and productivity of plants, comprising plant biomass (mainly N, but also S), plants metabolism and energy production, flowers, fruit and seeds development (mainly P, but also Ca and Mg), efficiency of the photosynthetic process, osmotic balance, and/or production and quality of the fruit (mainly K, but also Ca).
- micronutrients relate to the main nutrients absorbed in very small amounts grams per hectare (g/ha) compared with macronutrients (kg/ha).
- said micronutrient is selected from: boron (B), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), zinc (Zn) and combinations thereof.
- Zinc is an essential micronutrient, which means it is essential for plant growth and development but is required in very small quantities. It is crucial to plant development, as it plays a significant part in a wide range of processes, such as enzymes activation that are responsible for the synthesis of certain proteins.
- Zinc is essential in the growth hormone production and internode elongation. Zinc deficiency is probably the most common micronutrient deficiency in crops worldwide. Symptoms caused by zinc deficiency vary depending on the crop. Typically, they include one or some of the following: stunting-reduced height, interveinal chlorosis, brown spots on upper leaves and distorted leaves.
- the most common fertilizer sources of zinc are zinc chelates (contain approximately 14% zinc), zinc sulfate (25- 36% zinc) and zinc oxide (70-80% Zinc), where zinc sulfate is the most used source of zinc.
- Zinc solubilizing bacteria that are able to solubilize insoluble sources of zinc, preferably Zinc Oxide - ZnO, Zinc(ll) Carbonate -ZnCO 3 , or Zinc(ll) Phosphate - Zn 3 (PO 4 ) 2 ⁇
- Zinc solubilizer strains are rare to be isolated and consequently make available on the market for this activity. Therefore, the strain disclosed herein represents a valuable source to address Zinc solubilization need.
- Micronutrient deficiency affects crop yield and quality. While excess of some micronutrients, despite taking place in rare situation, can interfere with the uptake of other nutrients resulting in unbalanced plant development.
- “solubilization and/or mobilization of nutrients and/or micro - macronutrients” refers to the process allowing the conversion of the mineral forms of macro/micro-elements, which are present in the soil under unavailable forms for plants, into forms which can be dissolved in water and consequently taken up through their roots.
- the bacterial strains here disclosed can solubilize and mobilize, preferably in the soil, insoluble forms of macro/micro- nutrients, preferably starting from the inorganic forms of macro/micro-nutrients.
- the bacterial strains here disclosed are able to improve the solubilization and/or the dissolution and/or the mobilization of macro/micro-nutrients, preferably in the soil, and, consequently, they are able to improve the availability, preferably in the soil, of micro and/or macronutrients as defined above for plants.
- plant growth refers to the extension and/or expansion of plant tissues and/or organs, which bring to an increase of the vegetative biomass. An optimal plant growth results in balanced development of the whole plant and boosts processes of differentiation in flowers, fruits, and seeds.
- the isolated bacterial strain disclosed herein is a member of the genus Bacillus, species pumilus characterized by a DNA, more preferably the DNA of 16S rRNA gene coding comprising SEQ ID NO: 1 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 1.
- 16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea. The comparison of the 16S rRNA gene sequence of an isolate against sequences of type strains of all prokaryotic species provides an accurate and convenient way to routinely classify and identify prokaryotes.
- any pair of primers located in the DNA sequence of interest can be used.
- the pair of primers are the one disclosed in the examples and listed in Table I as SEQ ID NO: 3 and 4.
- the isolated bacterial strain disclosed herein has been deposited on February 21 st 2018 with the NCMR accession number MCC0530 and with IDA accession number MCC0195 (1 st July 2020) and the following name (denomination): Bacillus pumilus strain VMC30/195 (VMC30/195 from now on) at the International Depositary Authority: National Centre for Microbial Research, Pune, Maharashtra-411021 , India (hereinafter NCMR) according to the provisions of Budapest Treaty.
- VMC30/195 is gram positive and is preferably characterized by rod-shaped cells.
- the colony - when the bacteria are grown on nutrient agar - shows a yellowish color and is characterized by a size having an average diameter of 2-10 millimeters, preferably 3-5 millimeters.
- the colony’s shape of VMC30/195 is irregular.
- VMC30/195 can grow at a temperature up to 50C.
- VMC30/195 is able to use at least one of the carbon source selected from: glycerol, arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, methyl- ⁇ Dmannopyranoside, esculin, salicin, cellobiose, saccharose, trehalose, gentiobiose, tagatose, malic acid and any combination thereof.
- the carbon source selected from: glycerol, arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, methyl- ⁇ Dmannopyranoside, esculin, salicin, cellobiose, saccharose, trehalose, gentiobiose, tagatose, malic acid and any combination thereof.
- VMC30/195 is positive for: citrate utilization; and/or acetoin production and/or gelatinase.
- VMC30/195 is preferably resistant to at least one of the antibiotic agent, preferably present in a concentration ranging from 8-32 ⁇ g/ml and/or 15 ⁇ g/dish, selected from: ceftazidime, miokamycin, lincomycin and any combination therefore, and/or susceptible to at least one of the antibiotic agent, preferably present in a concentration ranging from 4 to 200 ⁇ g/ml and from 5 to 30 ⁇ g/disc and 300 U/disc, selected from: chloramphenicol, gentamycin, streptomycin, polymyxin B, netilmicin, tobramycin, amoxicillin+clavulanic acid, ampicillin, ampicillin+sulbactam, piperacillin, ceflacor, cefonidic, ceftriaxone, cefuroxime, ciprofloxacin, levofloxacin, pefloxacin, azithromycin, clarithromycin, erthromycin, rox
- VMC30/195 is characterized by a population doubling of 30-40 minutes.
- the invention refers also to a consortium comprising VMC30/195 and at least one further bacterial strain.
- said further bacterial strain is the isolated strain member of the genus Bacillus, species megaterium characterized by a DNA, more preferably the DNA of 16S rRNA gene coding, comprising SEQ ID NO: 2 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 2.
- said isolated strain member of the genus Bacillus, species megaterium has been deposited on February 21st 2018 at the NCMR with the accession number MCC0524 and with IDA accession number MCC0194 (1 st July 2020) and having the following name (denomination): Bacillus megaterium strain VMC30/196 (VMC 30/196 from now on).
- VMC30/196 is gram positive and is preferably characterized by rod-shaped cells.
- the colony - when the bacteria are grown on nutrient agar - shows a peach color and is characterized by a size having an average diameter of 2-10 millimeters, preferably 5-8 millimeters.
- the colony’s shape of VMC30/196 is circular.
- VMC30/196 can grow at a temperature up to 40°C, preferably up to 50°C.
- VMC30/196 is characterized by a population doubling of time 30-40 minutes.
- consortium means a group of different species and/or strains of microorganisms with different metabolic activities, preferably the consortium is defined “artificial” when it involves a multi-population system that can contain a diverse range of microbial species and is adjustable to serve a variety of industrial and ecological interests.
- consortium means a combination of VMC30/195 and at least one more bacterial strain, preferably a combination of VMC30/195 and VMC30/196, eventually comprising further bacterial species or strains.
- VMC30/195 and/or VMC30/196 and/or the consortium can be used as living microorganisms and/or dead cells and/or killed cells, preferably killed by heating, and/or as spores.
- VMC30/195 and/or VMC30/196 and/or the consortium may be used as a fresh or frozen sample.
- the strain(s) is(are) used dry, lyophilized or in liquid suspension, or encapsulated in the form of spores and/or living cells.
- VMC30/195 and/or VMC30/196 and/or the consortium may be cultivated continuously or discontinuously in any medium useful to grow bacteria, in liquid or solid form.
- Preferably VMC30/195 and/or VMC30/196 and/or the consortium is(are) cultivated on or in a medium(liquid/solid) that preferably comprises nutrient agar, meat extract, peptone, sodium chloride and yeast extract.
- a further aspect of the invention refers to a medium (broth) obtained/obtainable by culturing VMC30/195, eventually as consortium preferably with VMC30/196.
- the medium may contain the bacteria (the cells), that is the whole culture medium, or the medium may be a cell free medium (without the cells).
- the cell free medium may be obtained preferably by centrifuging the whole culture medium by using the common standard procedure useful for this purpose, to obtain a cell-free medium or supernatant.
- VMC30/195 eventually as consortium preferably with VMC30/196, may be used as culture medium (broth), preferably whole culture medium (comprising cells), or cell-free medium or supernatant (without cells).
- VMC30/195 eventually as consortium preferably with VMC30/196, is used as bacterial derivatives, preferably as lysate, as extract, preferably cell free extract, as fraction or as a metabolite(s) derived from said bacterium (a).
- culture medium means a solid, liquid or semi-solid nutritive matrix to support the growth of cells (prokaryotic or eukaryotic cells).
- culture medium preferably the following: proliferating medium, expansion medium, growth medium, nutrient medium.
- whole culture medium refers to a solution and/or a suspension containing bacterial cells, spores and derivatives thereof, such as nutrients, metabolites or cellular debris.
- supernatant refers to the liquid part of a culture broth free from the bacterial cells/spores and containing extracellular metabolites.
- lysate means the solution comprising all the intracellular and extracellular material released from the lysis of the bacterial cells.
- extract refers to a specific part of the culture broth, including or not living cells/spores.
- cell free extract means a solution containing all the microbial metabolites, without the presence of living cells/spores.
- fraction means a specific part of the whole culture medium, for example only the solution, only the cells or the like.
- metabolite means one or more intermediate(s) or final product(s) of the bacterial metabolism.
- a further aspect of the invention refers to mutants and/or edited strains derived/obtainable from VMC30/195.
- mutant means any microorganism obtained/obtainable by direct mutation, selection, or genetic recombination of VMC30/195. Mutant strains may be obtained by using any methods known in the art for this purpose, such as mutant selection, chemical mutagenesis, genetic manipulation physical mutagenesis (radiation) and biological mutagenesis.
- “edited” means preferably gene-edited strains wherein at least one gene of interest is edited/modified by using the common biological tools useful for this purpose, in particular biological tools based on the use of nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas).
- ZFNs zinc finger nucleases
- TALENs transcription activator-like effector nucleases
- CRISPR clustered regularly-interspaced short palindromic repeat
- Cas CRISPR-associated
- VVMC30/195 eventually as consortium preferably with VMC30/196, is(are) used in combination with further microorganisms, preferably selected from: bacteria, preferably PGPR (or rhizobacteria), yeasts, mycorrhizae, fungi, any derivatives as disclosed above and any combination thereof.
- bacteria preferably PGPR (or rhizobacteria)
- yeasts preferably yeasts, mycorrhizae, fungi, any derivatives as disclosed above and any combination thereof.
- the PGPR of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Aeromonas rivuli, Agromyces fucosus, Bacillus spp. Bacillus mycoides, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Bacillus safensis, Microbacterium sp., Nocardia globerula, Stenotrophomonas spp., Pseudomonas spp, Pseudomonas fluorescens, Pseudomonas fulva, Pseudoxanthomonas dajeonensis, Rhodococcus coprophilus, Sphingopyxis macrogoltabida, Streptomyces spp., Enterobacter spp., Azotobacter spp., Azospiriullum spp., Rhizobium spp., Herbas
- the yeasts of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Candida spp., Candida tropicalis, Saccharomyces spp., Saccharomyces bay anus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces exiguous, Saccharomyces pastorianus, Saccharomyces pombe, and combinations thereof.
- the mycorrhizae of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Glomus spp., Rhizophagus spp., Septoglomus spp., Funneliformis spp., and combinations thereof.
- the fungi of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Trichoderma spp., Trichoderma atroviride, Trichoderma viride, Trichoderma afroharzianum, Paecilomyces spp., Beauveria bassiana., Metarhizium spp., Lecanicillium lecanii, Penicillium spp., Aspergillus spp., Conythyrium minitans, Pythium spp, and combinations thereof.
- VMC30/195 eventually as consortium preferably with VMC30/196 can be used as plant biostimulant.
- VMC30/195 eventually as consortium preferably with VMC30/196 is(are) used in combination with an additional plant biostimulant (PB or PBS).
- PB or PBS additional plant biostimulant
- biostimulant refers to a substance(s) and/or micro-organisms or a composition comprising said substance(s) and/or micro-organisms whose function when applied to plants or to the rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality.
- said plant biostimulant comprises an extract of algae and/or an extract of microalgae and/or an extract of plant and/or a humic acid and/or a fulvic acid, and/or plant and/or animal byproducts.
- algae refer to a functional group of organisms that carry out oxygenic photosynthesis and are not embryophytes. They include both bacterial (cyanobacteria) and eukaryotic organism. The term encompasses organisms that are photoautotrophic, heterotrophic, or mixotrophic, and are typically found in freshwater and marine systems.
- the term algae include macroalgae (such as seaweed) and/or microalgae.
- said algae are brown algae, more preferably said algae are selected from: Ascophyllum nodosum, Ecklonia maxima, Laminaria saccharina, Laminaria digitata, Fucus spiralis, Fucus serratus, F.
- microalgae refer to any microscopic algae that are unicellular and simple multi-cellular microorganisms, including both prokaryotic microalgae, preferably, cyanobacteria (Chloroxybacteria), and eukaryotic microalgae, preferably green algae (Chlorophyta), red algae (Rhodophyta), or diatoms (Bacillariophyta).
- microalgae are selected from: Spirulina, Scenedesmus, Nannochloropsis, Haematococcus, Chlorella, Phaeodactylum, Arthrospyra, Tetraselmis, Isochrysis, Synechocystis, Clamydomonas, Parietochloris, Desmodesmus, Neochloris, Dunaliella, Thalassiosira, Pavlova, Navicula, Chaetocerous, and combinations thereof.
- plant means any one of the vast number of organisms within the biological kingdom Plantae.
- the term plant implies a taxon with characteristics of multicellularity, cell structure with walls containing cellulose, and organisms capable of photosynthesis.
- they include a host of familiar organisms including trees, forbs, shrubs, grasses, vines, ferns, mosses and crop plants as vegetables, orchards and row crops.
- the whole plant or part thereof may be used, preferably said part is selected from: leaves, roots, stems, fruits, flowers, seeds, seedlings, bark, berries, skins, and combinations thereof.
- the plant to be used as extract to add to the strains herewith disclosed is selected from: beet, sugar cane, alfalfa, maize, brassica, halophytes, soya, wheat, yucca, quillaja, hop, coffee, citrus, olive, and combinations thereof.
- said plants or algae or microalgae are extracted with common process, preferably said process comprising the following steps: (i) providing a sample of algae and/or a sample of microalgae and/or a sample of plants; and (ii) contacting said sample(s) with an aqueous solution comprising an extraction agent, in other words an extraction solution having an aqueous base.
- the extraction agent can be a base and/or an acid and/or an enzyme. These kind of extraction agents can be used in any combination or singly.
- the base is preferable an inorganic base, preferably selected from: NaOH, KOH, Na 2 CO 3 , K 2 CO 3 , NH 3 , salts thereof, and any combination thereof;
- the acid is preferably selected from: H 2 SO 4 , HNO 3 , HCI, H 3 PO 4 , and various acids of organic nature preferably selected from: acetic acid, citric acid, formic acid, butyric acid and ascorbic acid, gluconic acid, and any combination thereof;
- the enzyme is preferably selected from: papain, trypsin, amylase, pepsin, bromelain and specific enzymes that degrade organic polymers present in the algae, preferably alginases, and any combination thereof.
- the selection of the extracting agent to be used for the process depends upon the kind of algae/microalgae/plant to be extracted and/or the molecules/components to be extracted from them.
- the temperature of the extraction process ranges between -20 and 120°C, more preferably between 20 and 100°C.
- the extraction time ranges from a few minutes to several hours, more preferably between 30 minutes and 18 hours.
- the extraction process is realised at atmospheric pressure or at a pressure up to 10 Bar, more preferably at a pressure ranging from 1 to 8 Bar.
- the extraction process may be followed by a further step of separating/removing the non-solubilised and/or non-extracted component when it is desirable using only the extract in the formulation of the biostimulant.
- the removing/separating step is preferably performed by decantation, filtration or centrifugation. Alternatively, a suspension comprising both the extracted component and the non-extracted component can be used.
- the concentration of the extract from algae and/or microalgae in the biostimulant ranges from 1 to 60%, preferably it ranges from 5 to 50%, more preferably from 10 to 20%, still more preferably around 15%.
- the concentration of the plant extract in the biostimulant ranges from 1 to 60%, preferably it ranges from 5 to 50%, more preferably from 10 to 20%, still more preferably around 15%.
- the humic acid is extracted from leonardite, lignite, sub-products of the digestion of urban bio-waste and biochar. The extraction process is performed in water, in alkali, in acidic medium, or by pyrolysis.
- the concentration of the humic acid in the biostimulant ranges from 1 to 20%.
- the fulvic acid is extracted from peat, lignite, leonardite, digestion of urban bio-waste, biochar and vegetable materials. The extraction process is performed indifferently in water, in alkali, in acidic medium, and by pyrolysis.
- the concentration of the fulvic acid in the biostimulant ranges from 1 to 20%, preferably from 5 to 10%.
- VMC30/195 eventually as consortium preferably with VMC30/196
- said PBS is present in a concentration ranging from 5 to 50%, preferably from 10 to 40%, more preferably from 15 to 25 %, preferably around 20%.
- VMC30/195 eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 0.01 to 20%, preferably from 0.01 to 10%, more preferably from 0.05 to 5%, more preferably from 0.1 to 1%.
- VMC30/195 eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 10 to 20%.
- VMC30/195 eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 10M to 10 ⁇ 12 UFC/g each, preferably from 10 ⁇ 6 to 10 ⁇ 9 UFC/g, more preferably around 10 ⁇ 8 UFC/g.
- the PBS percentage refers to the sum of the percentage of each plant biostimulant component.
- the plant biostimulant component is preferably selected from: plant extracts, seaweed extracts, humic acid, fulvic acid, animal byproducts and combinations thereof.
- the percentage of the strain(s) refers to the sum of dried biomasses for each microorganism in 100 g of final composition.
- VMC30/195 eventually as consortium preferably with VMC30/196, or the medium or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite as disclosed above is/are mixed with the plant biostimulant as disclosed above and/or with a non- microbial component.
- Said non-microbial component comprises one or more components present in the mixture, preferably in the composition, with the exclusion of the microbial component, that is VMC30/195, eventually as consortium preferably with VMC30/196, or the medium or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite.
- the non-microbial component comprises preferably the PBS as disclosed above and/or at least one further ingredient, preferably a mineral component.
- Preferably said non-microbial component is present in a concentration up to 99,99%, 99,95 %, 99,90%, 99%, 95%, 90%, 85%, or 80% , and/or the bacteria are used in a concentration up to 20%, 15%, 10%, 5%, 1%, 0,1% 0.05% or 0.01 .
- said at least one further ingredient or mineral component is selected from:
- a nitrogen source preferably selected from: ammonium phosphates, ammonium nitrate, ammonium sulfate, ammonium thiosulfate, potassium thiosulfate, ammonia, urea, nitric acid, potassium nitrate, magnesium nitrate, calcium nitrate, sodium nitrate, protein hydrolisates of vegetal and animal origin, aminoacids, proteins, yeast lysate, manganese nitrate, zinc nitrate, slow release urea, preferably ureaformaldehyde, similar compounds and combinations thereof; and/or
- a phosphorus source preferably selected from: ammonium phosphates, potassium phosphates, phosphoric acid, sodium phosphates, calcium phosphate, magnesium phosphate, rock phosphate preferably hydroxyapatite and fluoroapatite, phosphorous acid, sodium phosphite, potassium phosphite, calcium phosphite, magnesium phosphite, organic phosphorus compounds, preferably inositol- phosphate, sodium glycerophosphate, ATP, similar compound and combinations thereof; and/or
- a potassium source preferably selected from: potassium acetate, potassium citrate, potassium sulfate preferably mixed salts of magnesium, potassium thiosulfate potassium phosphate, potassium phosphite, potassium carbonate, potassium chloride, potassium hydroxide, potassium nitrate, mixed salts of magnesium and potassium, potassium sorbate, potassium ascorbate, organic forms of potassium, and combinations thereof; and/or
- a magnesium and/or calcium source preferably selected from: magnesium nitrate, magnesium sulfate, magnesium chloride, magnesium phosphate, magnesium phosphite, magnesium thiosulfate, magnesium hydroxide, magnesium oxide, mixed salts of potassium and magnesium, mixed salts of magnesium and calcium (dolomite), magnesium acetate, magnesium citrate, magnesium sorbate, and organic forms of magnesium, magnesium carbonate, magnesium formate, magnesium ascorbate, and combinations thereof; and/or
- a sulfur source preferably selected from: sulfuric acid, sulfates, thiosulfate, sulfated aminoacids, and combinations thereof; and/or - Iron and/or manganese and/or zinc and/or copper source, preferably selected from: iron sulfate, iron oxide, iron hydroxide, iron chloride, iron carbonate, iron phosphate, iron nitrate, chelated iron with EDTA, DTPA, HEDTA, EDDHA, EDDHSA, EDDHCA, EDDHMA, HBED, EDDS; complexed iron with aminoacids, ligninsufonates, humic acids, fulvic acids, gluconic acid, heptagluconic acid, iron citrate, iron malate, iron tartrate, iron acetate, iron lactate, iron ascorbate, organic form of iron, and combinations thereof .
- VMC30/195 and/or VMC30/196 is(are) used in combination with further molecules, preferably proteins, protein hydrolisates, peptides, oligopeptides, peptidoglycans, low-molecular weight peptides, synthetic and natural occurring aminoacids; molasses, polysaccharides, lypopolysaccharides, monosaccharides, disaccharides, oligosaccharides, sulfated oligosaccharides, exopolysaccharides, chitosan.
- further molecules preferably proteins, protein hydrolisates, peptides, oligopeptides, peptidoglycans, low-molecular weight peptides, synthetic and natural occurring aminoacids; molasses, polysaccharides, lypopolysaccharides, monosaccharides, disaccharides, oligosaccharides, sulfated oligosaccharides, exopolys
- VMC30/195 and/or VMC30/196 are selected from: stress protecting molecules, such as betaines, mannitol, and other polyols with similar effects, and hormones and hormone-like compounds, such as melatonin, auxins, auxin-like compounds, cytokinins, cytokinin-like compounds, gibberellins, gibberellin-like compounds, jasmonates, hormones precursors like polyamines spermine, spermidine, putrescine, and metabolism stimulating substances like vitamins.
- stress protecting molecules such as betaines, mannitol, and other polyols with similar effects
- hormones and hormone-like compounds such as melatonin, auxins, auxin-like compounds, cytokinins, cytokinin-like compounds, gibberellins, gibberellin-like compounds, jasmonates, hormones precursors like polyamines spermine, spermidine, putrescine, and metabolism stimulating substances like vitamins.
- nucleic acids selected from: nucleic acids, uronic acids and polymers thereof, glucuronic acids and polymers thereof, small organic acids, such as oxalic and succinic acids.
- said small molecules may be a synthetic and/or naturally derived nucleic acid molecules containing multiple nucleotides, preferably being defined an oligonucleotide when the molecule is 18-25 nucleotides in length and polynucleotides when the molecule is 26 or more nucleotides.
- oligonucleotides or polynucleotides or a mixture of both include RNA or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof.
- a further aspect of the invention refers to a composition, preferably an agricultural composition, more preferably a plant biostimulant composition, comprising VMC30/195, eventually as consortium preferably with VMC30/196 as disclosed above, eventually in combination with the further components/ingredients disclosed above and a carrier, preferably an agricultural compatible carrier.
- said further components/ingredients is the PBS and/or the non-microbial component and/or the further ingredients or mineral component as previously disclosed.
- agricultural compatible carrier refers to any synthetic or natural, organic or inorganic, derived compound able to deliver the product in an active form in the site of action, preferably said carrier is selected from the categories of surfactants, thickeners, suspension agents, wetting agents, and combinations thereof.
- surfactant means any molecule able to modify the surface tension of the water and allowing the product to impact a wider area of the leave and/or root and/or fruit, or any other part of the plant.
- said surfactant is selected from: ionic, non-ionic, cationic surfactants, synthetic or naturally derived, preferably alkyl sulfonates, alkylarylsulfonates, ethoxylated alcohols, alkoxylated ethers, ethoxylated esters, alkylpolyglucosides, block copolymers, lignosulfonates, saponins, and the like.
- thickener means any molecule able to modify the rheology of any given composition in the sense of improving the viscosity and stabilize it.
- said thickener is selected from: natural and synthetic gums, lignosulfonates, molasses and the like.
- suspension agent means any molecule able to surround insoluble particles avoiding settlement and allowing the creation of a stable suspension of insoluble.
- said suspension agent is selected from: natural and synthetic colloids, clays, and their derivatives and the like.
- wetting agent means any molecule able to avoid fast water evaporation on a given surface and retain moisture for a long time.
- said wetting agent is selected from: glycols, glycerin and their derivatives and the like.
- VMC30/195 preferably as consortium, preferably with VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is/are formulated as: solution, suspension, water- soluble concentrates, dispersable concentrates, emulsifiable concentrates, emulsions, suspensions, microemulsion, gel, microcapsules, granules, ultralow volume liquid, wetting powder, dustable powder, or seed coating or treatment formulations.
- VMC30/195 and/or VMC30/196 is(are) able to solubilize macronutrients and/or micronutrients as defined above.
- the strain(s) is(are) able to solubilize macronutrients and/or micronutrients present in the soil and therefore the strain(s) allow(s) said solubilized macronutrients and/or micronutrients being more available for plants that consequently show an improved capability of micro and/or macronutrient’s uptake.
- plants improve the up-take capability of said solubilized macronutrients and/or micronutrients, preferably from the soil, and show a better growth.
- VMC30/195 is good performer in Phosporous solubilization, and when used with VMC30/196 the consortium works even better.
- VMC30/195 has shown great potential in Zinc solubilization more than the strain of reference known to have this activity.
- VMC30/195 represent an interesting emerging alternative of selected strain for this biological function in plants.
- VMC30/195 shows promising potential also in nitrogen fixation and in this regard, it works better compared to known nitrogen fixator strains.
- VMC30/195 when used in combination with VMC30/196, the consortium shows complete framework of micro-macronutrients solubilization activity because besides the above disclosed properties the consortium shows also potassium and iron solubilization/mobilization activity.
- VMC30/196 is a good producer of siderophores and so it helps and increases iron solubilization and consequently plant iron uptake.
- VMC30/196 is good potassium solubilized. Therefore, VMC30/196 can complement the panel of solubilizing/mobilizing activity toward micro-macronutrients of VMC30/195.
- VMC30/196 is also able to produce IAA.
- This molecule is known to help plant in the production of longer roots with an increased number of root hairs and root laterals which are involved in nutrient uptake (Datta and Basu, 2000).
- IAA promotes cell elongation, inhibit or delay abscission of leaves and induce flowering and fruiting.
- IAA producing bacteria may improve the fitness of the plant-bacterium interaction, colonize plant roots better than other bacteria by increased root system, weaken plant defense mechanisms making colonization easier, loosen plant cell walls and as a result promotes an increasing amount of root exudation that provides additional nutrients to support the growth of rhizosphere bacteria and provides more number of active sites and access to colonization for other PGPRs (Etesami et al., 2015).
- VMC30/196 preferably as consortium with VMC30/195, may be also used for increasing number of root hairs and/or root laterals; and/or promoting cell elongation, inhibiting or delaying abscission of leaves and inducing flowering and fruiting; and/or improving the fitness of the plant-bacterium interaction, colonizing plant roots by increasing the root system, and/or weakening plant defense mechanisms making colonization easier, and/or loosening plant cell walls, and/or promoting an increase in root exudation and so additional nutrients to support the growth of rhizosphere bacteria, and/or increasing active sites and accesses to colonization for other PGPRs.
- VMC30/195 and/or VMC30/196 eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is/are preferably used to solubilize (mobilize/dissolve) or to improve the dissolution, preferably in the soil, of phosphate, preferably any inorganic source of phosphate, more preferably selected from: phosphate tricalcium phosphate (Ca 3 (PO 4 ) 2 ), ferric phosphate (FePO 4 ), and aluminum phosphate (AIPO 4 ).
- phosphate preferably any inorganic source of phosphate, more preferably selected from: phosphate tricalcium phosphate (Ca 3 (PO 4 ) 2 ), ferric phosphate (FePO 4 ), and aluminum phosphate (AIPO 4 ).
- insoluble forms of phosphorus preferably contained into soils, are converted, through several mechanisms, preferably by organic acids production, chelation, ion-exchange reaction or polymeric substances formation, into soluble forms preferably H 2 PO 4 - and/or HPO 4 2- that are easier to be taken up plants.
- VMC30/195 and/or VMC 30/196 eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to solubilize (mobilize/dissolve) zinc, preferably any inorganic source of zinc, more preferably selected from: Zinc Oxide (ZnO), Zinc(ll) Carbonate (ZnCO 3 ), and Zinc(ll) Phosphate (Zn 3 (PO 4 ) 2 ).
- ZnO Zinc Oxide
- ZnCO 3 Zinc(ll) Carbonate
- Zn 3 (PO 4 ) 2 Zinc(ll) Phosphate
- VMC30/195 and/or VMC 30/196 eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to convert into soluble forms, existing as free Zn 2+ ions and Zn chelates, by the bacterial production of organic acids such as 5-ketogluconic acid and 2-ketogluconic acid.
- gluconic acids and ketogluconates are sugar acids having multiple conformations, which chelate the metal cations coming from solubilization process.
- VMC30/195 and/or VMC30/196 eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to produce siderophores.
- VMC30/195 and/or VMC30/196 eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to chelate iron ions (Fe 3+ ), making iron more available for plants.
- siderophores mean small, high-affinity iron-chelating compounds generally secreted by microorganisms such as bacteria, fungi and grasses. Siderophores are amongst the strongest soluble Fe3+ binding agents known.
- microbial siderophores belong to at least one of class of molecules preferably selected from: catecholates, hydroxamates, and a-carboxylates, depending on the chemical nature of their coordination sites with iron.
- the strain(s) of the invention is able to produce hydroxamates type siderophores. These siderophores form iron chelates by the binding site that is mounted on an L-ornithine derivative.
- iron ion Fe 3+
- Iron privation in plants causes as main effect the reduction in photosynthetic activity and as secondary effect the reduction in fruit quality in terms of color, size, sugar content, fruit hardness and taste.
- siderophores support plant growth also by inhibition of soil-borne plant pathogens. Indeed, the siderophores - produced in iron-limited conditions -sequester the less- available iron from the environment and inhibit pathogens by depriving iron.
- VMC30/195 and/or VMC30/196 eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above can be used to improve the capability of plants to uptake iron preferably from soil.
- VMC30/195 and/or VMC30/196 eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to mobilize K making it more available for plants.
- Potassium (K) is an essential macronutrient and plays an important role in the growth and metabolism of plants.
- concentrations of soluble potassium in the soil are usually very low and more than 90% of potassium in the soil exists in the form of insoluble rocks and silicate minerals (e.g., biotite, feldspar, illite, muscovite, orthoclase, and mica), not available for plant uptake.
- a deficiency of potassium causes a delay in the root and plant growth, a small seed production and a lower yield.
- potassium deficiency is becoming one of the major constraints in crop production. This aspect highlights the importance to maximize the bioavailability of such mineral in the soils for sustaining crop production.
- Soil microbes can play a central role in the natural potassium cycle and therefore, the potassium solubilizing microorganism present in the soil, can dissolve silicate minerals and release K through the production of organic and inorganic acids, acidolysis, polysaccharides, complexolysis, chelation, and exchange reactions.
- VMC30/195 and/or VMC30/196 eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above can dissolve potassium from insoluble K-bearing minerals and make it available to plant uptake.
- VMC30/195 and/or VMC30/196 eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to produce IAA.
- VMC30/195 and/or VMC30/196 eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to fix nitrogen.
- VMC30/195 and/or VMC30/196 eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to convert atmospheric N 2 into plant-utilizable forms by biological N 2 fixation which changes nitrogen to ammonia by nitrogen fixing microorganisms using a complex enzyme system known as nitrogenase.
- VMC30/195 when used as consortium with at least a further strain, preferably with VMC30/196, VMC30/195 is used in the same amount (1 :1) of the further strain.
- the range between the amount of VMC30/195 and the additional strain, preferably VMC30/196 varies between 1 :1 and 1 :100, preferably 1 :3, 1 :5 or 1 :10.
- the strain or the consortium or the strain derivatives or the composition here disclosed is preferably applied into the soil, with any useful means or way, preferably through fertigation or irrigation systems and is preferably diluted in water, more preferably through overhead applications with appropriate volumes of water to let the solution spreading through the soil.
- the application of the strain (VMC30/195) or the consortium of the strain with at least one additional bacterial strain, preferably VMC30/196, or the composition comprising the strain or the consortium is performed with at least one of the following practice: drip, overhead irrigation, seed treatment, soil application, foliar treatment, indoor farming, vertical farming and combination thereof.
- the application is performed all over the crop cycle, preferably during the early stages of crop development.
- the strain or the consortium or the strain derivatives or the composition here disclosed is preferably applied at a rate ranging between 100g-10.000 g/hectare, preferably between 2 and 10 kg/hectare preferably up to 5 times/crop cycle.
- the biological properties discussed above are particularly enhanced as well as the crop yield.
- the synergism between the strains in the presence of the PBS may be due to their ability to degrade the PB’s components and to generate metabolites able to support their growth/activities.
- VMC30/195 and/or VMC30/196 when used as consortium is/are able to emphasize, preferably to synergize, plants responses to any biostimulant substance.
- a significant yield improvement has been observed when VMC30/195 and/or VMC30/196 is(are) used with a biostimulant as defined above.
- VMC30/195 and/or VMC30/196 preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) particularly useful in agriculture, preferably to improve, preferably into soil, macronutrients and/or micronutrients solubilization (mobilization/dissolution/fixation), preferably phosphate and/or zinc and/or potassium solubilization/mobilization, and/or atmospheric nitrogen fixation and/or siderophores production, preferably iron availability and/or IAA production.
- solubilization mobility/dissolution/fixation
- phosphate and/or zinc and/or potassium solubilization/mobilization preferably phosphate and/or zinc and/or potassium solubilization/mobilization
- atmospheric nitrogen fixation and/or siderophores production preferably iron availability and/or IAA production.
- VMC30/195 and/or VMC 30/196 preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) particularly useful to improve macronutrients and/or micronutrients plant uptake, phosphate and/or zinc and/or iron and/or nitrogen uptake from plants.
- VMC30/195 and/or VMC30/196 preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) useful to improve plant growth.
- said further components/ingredients is the PBS as previously disclosed.
- plant means any one of the vast number of organisms within the biological kingdom Plantae.
- the term plant implies a taxon with characteristics of multicellularity, cell structure with walls containing cellulose, and organisms capable of photosynthesis.
- Modern classification schemes are driven by somewhat rigid categorizations inherent in DNA and common ancestry. In general, these species are considered of limited motility and generally manufacture their own food.
- plant includes a host of familiar organisms including trees, forbs, shrubs, grasses, vines, ferns, mosses and crop plants as vegetables, orchards and row crops.
- More preferably plant includes leaf vegetables, preferably selected from: Solanaceae, Brassicaceae, Liliaceae, and combinations thereof; and/or orchard plants, preferably selected from: fruit trees, grapes, olive trees, citrus trees, and combinations thereof; and/or row crops, preferably selected from: cereals, preferably corn, soybean, wheat, winter wheat, rice, barley, sugar cane, sugar beet, legumes preferably peas, beans, chickpeas but are not limited to: brassica, bulb vegetables, cereal grains, citrus, cotton, curcurbits, fruiting vegetables, leafy vegetables, legumes, oil seed crops, peanut, nut trees, cocoa tree, herbs, cannabis, pome fruit, root vegetables, tuber vegetables, corm vegetables, stone fruit, tobacco, palm trees, musae spp., strawberry and other berries, and various ornamentals.
- leaf vegetables preferably selected from: Solanaceae, Brassicaceae, Liliaceae, and combinations thereof
- orchard plants preferably selected from: fruit trees
- microbes have been isolated from rhizosphere soil samples coming from India.
- a soil sample (10 grams) has been taken and diluted into 90 ml of distilled water. After homogenization, serial dilution up to 10 -5 have been done. One ml of each serial dilution has been smeared on a nutritive agar plate and incubated for 24 h at 30 °C. After incubation period, the most relevant colonies found on the highest dilution ( 10 -5 ), have been picked and plated again on nutrient agar plates, incubated for 24 h at 30 °C.
- Bacillus pumilus was isolated from Chickpea (Cicer arietinum) collected from Ramanthapur village (Sanga Reddy), while Bacillus megaterium was isolated from Paddy rhizoshere soil collected from Indra Karan village (Sangareddy).
- the isolated strains have been deposited in National Centre for Microbial Research (NCMR, Pune, India) on 21 st February 2008, with the following name: Bacillus pumilus VMC30/195 (VMC30/195) having the deposit number MCC 0530, and Bacillus megaterium VMC30/196 (VMC30/196 from now on) having the deposit number MCC 0524.
- strains VMC30/195 and VMC30/196 were typed by direct sequencing of PCR- amplified 16S rDNA.
- the amplification protocol is the following.
- the Sanger sequencing was followed by alignment and phylogenetically analysis of the obtained data with the 16S rRNA sequences from the Type Strains.
- the strain Bacillus pumilus VMC 30/195 is related to the species B. pumilus as it shares 97% and 97,74% of sequence similarity with the 16S rRNA sequences of the corresponding Type Strains (Table II and Fig.1.A). Table II
- the strain Bacillus megaterium VMC 30/196 is related to the species B. megaterium as it shares 94,98% and 94,98% of sequence similarity with the 16S rRNA sequence of the corresponding strains (Table III and Fig. 1 B).
- This technique allows identification of microbes as intact cells or cell extracts.
- intact cells were tested by using a MicroflexTM MALDI-TOF (Matrix-assisted laser desorption ionization-time-of-flight) mass spectrometer (Bruker Daltonics, Leipzig, Germany).
- Initial manual/visual estimation of the mass spectra was performed using the FlexAnalysis 2.4 software (Bruker Daltonik GmbH, Germany).
- raw spectra were processed using the MALDI BioTyper 1.1 software (Bruker Daltonik GmbH, Germany) with default settings. The smoothing, normalization, baseline subtraction and peak picking was carried out by the software, thereby creating a list of the most significant peaks of a spectrum (m/z values with a given intensity). Samples were prepared according to manufacturers’ instructions.
- strains were tested in triplicate (analyses were performed on three different days and starting from different cultures). For each measurement, at least 300 individual spectra (30 laser shots at 10 different spot positions) were collected and averaged. External calibration was performed with the Bruker bacterial test standard (Bruker Daltonics, Bremen, Germany).
- FIG. 2 shows MALDI-TOF MS mass spectra of VMC30/195 (A) andVMC 10/196 (B), respectively.
- Each bar in the graphs represents a different protein expressed by the microbes and the intensity of the bars represents the concentration of the proteins within the microbial cell. Since proteins are a direct expression of genome and genome of each strains is unique, thus the proteomic profile of each strain is unique and can be used as fingerprint. The most and unique representative signal for each one of the strains are in the range from 2 to about 12 KDa using spectrum m/z 10 ⁇ 3.
- the color and the colony morphology of the strains have been characterized by direct observation of the microbial strains after growing on Nutrient Agar (NA) plates.
- NA Nutrient Agar
- the bacterial strains have been cultured on NA plates for 24 h at 30 °C. After incubation period, bacterial colonies appear on the plates and color and morphology can be observed.
- the motility and the colony size have been characterized by using optical microscope with 100X magnitude.
- the bacterial strains have been cultured on NA plates for 24 h at 30 °C. After incubation period, one bacterial colony has been picked and smeared into 1 ml water drop on a glass slide. The glass slide is then observed under optical microscope with 100x magnitude.
- Gram staining is determined by using the Gram staining kit.
- the growth of the bacterial strains at different temperatures has been evaluated by streaking the microorganisms on NA plates (in triplicates) and by incubating them at different temperatures (4, 40 and 50 °C). After 24 h of incubation, the appearance of colonies on the plates indicates the ability of the strain to grow at a specific temperature.
- the ability of the bacterial strains to grow at different pH values is determined by streaking the microorganisms on NA plates adjusted at different pH values (5.5, 6.5 and 9.5). Experiment was done in triplicate. After 24 h of incubation, the appearance of bacterial colonies on the plates indicates the ability to grow at a specific pH value.
- the salinity tolerance of the strains is determined by streaking the microorganisms on NA plates, modified by the addition of different amount of NaCI (8, 10, 12 and 14 %). The experiment is performed in triplicate. After 24 h of incubation, the appearance of colonies on the plates, indicates the ability of the strain to grow at a specific NaCI concentration, thus, to tolerate a specific salinity level. The results are given in Table IV.
- the biomass production process of the strains is divided into four steps that are: inoculum preparation, fermentation, biomass recovery and biomass drying.
- Recovery of microbial biomass can be done through several processes such as centrifugation, micro-filtration or ultra-filtration.
- Drying process is preferably made by freeze-drying.
- the phosphate solubilization activity of the VMC30/195 and VMC30/196 was shown by using an in-vitro plate assay according to Pikovskaya method (Yasmin and Bano, 2011). Tricalcium phosphate (Ca 3 (PO 4 ) 2 ) was used as inorganic source of phosphorus.
- the seeding was done superficially using an aliquot (10 ⁇ I) of the bacterial suspension (10 6 CFU/ml).
- Seeded samples were incubated at 30 °C for 7 days and colonies with a clear halo on plate were considered positive for phosphate solubilization.
- P-solubilization activity was measured as solubilization index (SI), according to Yasmin and Bano (2011) after 7th days and compared one to each other to individuate the best phosphorus solubilizing bacteria (PSB).
- SI solubilization index
- Solubilization index was determined as follows:
- Bacterial strains have been tested in mixed cultures, by spotting on plate an aliquot of 5 ⁇ I of each bacterial suspension and following the methodology described above. Moreover, the phosphate solubilizing activity of the microbes was evaluated in liquid medium in order to measure the exact quantity of phosphorus liberated from inorganic form (Ca 3 (PO 4 ) 2 ).
- the samples tested were the same as reported above.
- the bacteria (2ml - 10 6 CFU/ml) was inoculated in liquid Pikovskaya medium (pH 6.5) and incubated at 30°C for 5 days, shaking constantly at 120 rpm. Samples were then centrifugated at 10,000 rpm for 10 minutes. An aliquot of the supernatant was taken to measure the soluble phosphorus (P) and the final pH value of the medium. The amount of soluble phosphorus in the supernatant was measured by molybdenum-blue method using a spectrophotometer at a wavelength of 600 nm (King, 1932). The amount of soluble phosphorus was determined from the standard curve of KH 2 PO 4 . Herbaspirillum seropedicae ATCC 35893 have been used as specific controls.
- the strain VMC30/195 keeps the P-solubilization activity even when is co- cultured with VMC30/196.
- Zinc-solubilizing capacity of the bacterial strains were assessed by a plate assay method using a modified Pikovskaya method (Ghevariya and Desai, 2014and zinc oxide (ZnO) as inorganic source of zinc.
- the isolates were inoculated into agar medium containing 0.1% insoluble zinc (ZnO) and incubated at 30 °C for seven days.
- the seeding was done superficially using an aliquot (10 ⁇ I) of each bacterial suspension, with a concentration of 10 6 CFU/ml.
- VMC 30/196 does not showed the zinc solubilization activity in the tested conditions.
- Khanghahi et al., 2018 reported the effect of pH on Zn-solubilizing ability of selected bacteria.
- siderophores of the strain of the invention has been determined according to Schwyn and Neilands (1987). Briefly, 10 ⁇ I of each bacterial suspension with a concentration of 10 6 CFU/ml was spotted in triplicate on Nutrient broth (NB) and plated were incubated at 28°C for 72 h. Ten ml of chrome azurol S (CAS) agar medium were poured over the plates. After 24 h the formation of an orange halo was considered as indicator of siderophore production. The halo was measured according to Omidvari et al. (2010). Moreover, bacterial strains have been tested as mixed cultures, by spotting on plate an aliquot of 5 ⁇ I of each bacterial suspension.
- the potassium-solubilizing capacity of the two bacterial strains used in the present invention were screened by a plate assay method using Alexandrov’s agar medium according to the method described by Parmar and Sindhu (2013).
- the potassium solubilizing activity was evaluated using mica powder (potassium aluminum silicate) as insoluble form of potassium.
- the isolates were inoculated into agar medium containing 0.5% insoluble potassium.
- the seeding was done superficially using an aliquot (10 ⁇ I) of each bacterial suspension, with a concentration of 1 ,00E+06 CFU/ml.
- Bacterial strains have been tested as single cultures as well as mixed cultures, by spotting on plate an aliquot of 5 ⁇ I of each bacterial suspension.
- the test organisms were inoculated on these media and incubated at 28 °C for three days. Colonies with a clear halo on plate were considered positive for potassium (K) mobilization.
- K-mobilization activity was measured as mobilization index (Ml), according to Parmar and Sindhu (2013).
- Mobilization index was determined as follows:
- MI Diameter of z one of clearance f Diameter of growth.
- VMC 30/195 and VMC 30/196 were screened by a liquid assay method according to Sarker et al. (2013). 1 ,00E+06 CFU/ml or each strain was inoculated into nutrient broth containing 0,1% of tryptophan. Inoculated samples were incubated in shaking incubator at 28°C (100- 110 rpm) for 48 hours (for obtaining log phase). Uninoculated broth was used as negative control. After incubation, the IAA production was determined mixing 2 ml of each sample (previously spun at 13000 g x 10 minutes and filtered with 0,2 m filters) with 4 ml of 0 incubated in the dark at room temperature for 30 minutes.
- the nitrogen fixation capacity of the strains of the present invention have been assessed according to Okon et al. (1977) with minor changes.
- the bacteria were inoculated using an aliquot (10 ⁇ I) of each bacterial suspension, with a concentration of 10 6 CFU/ml in a medium made up of in 1 I: D-L malic acid 5 g, KOH 4 g, K 2 HPO 4 0.5 g, MgSO 4 0.2 g, NaCI 0.1 g, CaCl 2 0.02 g, bromotymol blu (BTB) (0.5% dissolved in 0.2 N KOH) 2 ml, trace element solution 1 ml (NaMo04 200 mg, MnSO 4 253 mg, H 3 Bo 3 280 mg, CuSO 4 8 mg, ZnSO 4 24 mg in a final volume of 200 ml of distilled water), Fe EDTA (EDTA 22.8 g/l, 250 ml of KOH 1 N, FeCI 3 10 g) 4 ml,
- the final pH was adjusted to 6.8.
- Fe EDTA solution was sterilized by filtration with 0.2 ⁇ m filters.
- Fe EDTA, BTB and the trace solution were added to the medium after cooling.
- the blue colored zone producing isolates were marked as nitrogen fixers in the solid culture conditions.
- the coloring zone was calculated by deducting the colony diameter from the coloring zone diameter, according to Gothwal et al. (2008). Bacterial strains have been tested as mixed cultures, by spotting on plate an aliquot of 5 ⁇ I of each bacterial suspension.
- Plant growth promotion activity on Lettuce plants The plant growth promotion activity of the strains was tested in combination with a plant biostimulant (PBs).
- PBs plant biostimulant
- in vivo bioassays have been done on Lactuca sativa cv. Canasta plants.
- Canasta plantlets were obtained from a local nursery. Two-week-old plantlets were transplanted in 17 cm diameter plastic pots, 2 Liters volume each. 4 replications of eight pots/treatment were prepared, on a sandy substrate, in a glasshouse.
- the environmental conditions during the experimental period were 22-33°C, with a relative humidity ranging from 70-80%, under natural light conditions.
- Nutrients were directly added to the substrate during the crop cycle, by providing 40 Kg/ha of water-soluble nutrient mix containing NPK (13-40-13), every 10 days. The density was 10 plants/m2.
- T reatments conditions were:
- VMC 30/195+VMC 30/196 two applications every 10 days at 5 Kg/ha rate
- the irrigation was carried out considering the substrate moisture content and the amount of water was determined to maintain the 80% of substrate water availability. Lettuce plants were harvested when the plants reached the commercial maturity stage, after 40 days of cultivation. Plant fresh weight (g) was determined at harvest. Data were subjected to two-way ANOVA and SNK comparison test was used for evaluating the differences among means at p ⁇ 0,05. Different letters indicate statistical differences for p ⁇ 0,05. Foliar Zinc, Nitrogen, Phosphorous and Iron were assessed, only for UTC and complete prototype (VMC30/195+VMC30/196+PBS), at the end of the cycle by leaf analysis performed in a dedicated local laboratory.
- VMC30/195+VMC30/196 promote plant growth and this activity is more enhanced in the presence of a plant biostimulant.
- Arabidopsis thaliana (Ecotype Col-0) was used in this experiment. The seeds were washed and rehydrated with water for 15 minutes before sowing, to facilitate the germination. After washing, single seed was taken with tweezers and sown on each single well (50/50 of earth and perlite) of the "Arasystem trays". After that, the trays were transferred into a growth chamber with controlled conditions to allow optimal plant growth.
- the microarray analysis (SureScan, Agilent system) has been performed after 24h (early effect) and 72h (mid effect) from the application on 15- days old A. thaliana seedlings.
- GO-Term enrichment was performed for interpreting sets of genes making use of the Gene Ontology system classification, in which genes are assigned to a set of predefined bins depending on their functional characteristics and cut-off selected (0,01).
- the GO-Groups correlated to the microbial component are the following: biosynthetic process, biotic response, hormone response and metabolic process.
- the plant growth promotion activity of the strains was tested in combination with PBS.
- greenhouse experiment has been done on sweet pepper plants using sub-optimal nutrition.
- Sweet pepper plantlets were transplanted in 17 cm diameter plastic pots, 2 Liters volume each. 4 replications of eight pots/treatment (7 treatments x 12 blocks) were prepared, on a sandy substrate, in a glasshouse. The environmental conditions during the experimental period were 22-33°C, with a relative humidity ranging from 70-80%, under natural light conditions. Nutrients were directly added to the substrate during the crop cycle.
- Prototype three applications every 7 days at 5 Kg/ha rate, first application after transplant at 14 days.
- PBS three applications every 7 days at 5 Kg/ha rate, first application after transplant at 14 days.
- Prototype three applications every 7 days at 10 Kg/ha rate, first application after transplant at 14 days
- PBS three applications every 7 days at 10 Kg/ha rate, first application after transplant at 14 days
- Plant Biomass showed statistical significance (SNK and Duncan’s test p ⁇ 0,05 and p ⁇ 0, 10) for Prototype (5-10 kg/ha) vs UTC;
- the prototype was assessed on Melon and Tomato crops under open field conditions in Rabi season at Maharashtra, India.
- untreated control UTC
- first dose of application was done after 5 days of transplantation of seedlings with second dose after 20 days of planting and 3 rd dose after 40 days of planting.
- Soil samples were collected from all the plots before and after application treatments i.e @ 0, 1 , 30 and 60 days after application to estimate the total viable count of microbial load.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Wood Science & Technology (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Dentistry (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
Abstract
The present invention refers to a new bacterial strain Bacillus pumilus VMC30/195 and Bacillus megaterium VMC30/195, a microbial consortium comprising said strain and a composition comprising the bacterial strain or the consortium eventually in combination with a biostimulant for plants. Moreover, the present invention refers to the use of the bacterial strain Bacillus pumilus VMC30/195 and Bacillus megaterium VMC30/195, the microbial consortium or the composition in agriculture, preferably to improve plant processes, preferably nutrient uptake, and therefore to enhance plant growth and/or development.
Description
Microbial strains Bacillus pumilus, Bacillus megaterium and uses thereof
DESCRIPTION
Technical field
The present invention refers to a new bacterial strain, a microbial consortium comprising said strain and a composition comprising the bacterial strain or the consortium eventually in combination with a biostimulant for plants. Moreover, the present invention refers to the use of the bacterial strain, the microbial consortium or the composition in agriculture, preferably to improve plant processes, preferably nutrient up-take, and therefore to enhance plant growth and/or development. Background art In the past years, the heavy use of fertilizers, agro-chemicals and pesticides, which marked the First Green Revolution, strongly improved crop productivity, without taking in account adverse effects on environment and human health (Serpil, 2012), such as decreased soil fertility and increased susceptibility of plants to pests and disease (Jen-Hshuan Chen, 2006). Nowadays, sustainable agriculture is considered as the new way to produce more food to meet the increasing demand for food of an ever-growing population, while counteracting the adverse effects of the climate change on crop productivity. Biofertilizers have been considered a good and eco-friendly solution for sustainable agriculture compared to chemical fertilizers and can significantly improve crop productivity, ameliorate nutrient up-take and make plants more tolerant to several biotic and abiotic stress in a sustainable manner (Deepak et al., 2014).
In this context, the solution disclosed herein is based on a novel bacterial strain, a bacterial consortium or a composition thereof capable of increasing soil solubilization of main macronutrients and/or micronutrients, preferably phosphorus, zinc, iron, nitrogen, potassium and combinations thereof. Therefore, the strain, consortium or composition thereof is useful to improve micro and/or macronutrient uptake in plants and to ameliorate plant biology, in particular, the growth and/or the development of plants, nutrient use efficiency. Overall, the strain, consortium or composition disclosed herein is useful as biostimulant for plants, preferably to enhance in plants nutrient uptake, nutrient efficiency, tolerance to abiotic stress, crop quality and combination thereof.
Brief description of drawings
- Figure 1 shows the phylogenetic tree obtained from the alignment of the full 16 rRNA gene sequences, with the “Maximum Likelihood” statistical method, “Tamura- Nei” substitution model and “Complete Deletion” mode (No. of bootstrap replications: 1000).
A) The strain Bacillus pumilus VMC30/195 is related to the species B. pumilus as it shares 97% of sequence similarity with the 16S rRNA sequence of Bacillus pumilus SH-B9.
B) The strain Bacillus megaterium VMC30/195 is phylogenetically related to the species B. megaterium, as it shares 94,98% of sequence similarity with the 16S rRNA sequence of Bacillus megaterium CtST3.5.
- Figure 2 shows protein mass spectra (in the range from 2 to about 12 KDa, peaks of a spectrum m/z values with a given intensity) of VMC30/195 (A) and VMC30/196 (B). Each peak in the graphs represents a different protein expressed by the microorganism and the intensity of the peaks represents the concentration of that proteins within the microbial cell. Since proteins are a direct expression of genome and genome of each strains is unique, thus the proteomic profile of each strain is unique and can be used as fingerprint differentiation and classification.
- Figure 3 shows the P-solubilization index of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Herbaspitillum seropedicae ATCC 35893.
- Figure 4 shows the Zinc Solubilization Efficiency (SE) of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Herbaspitillum seropedicae ATCC 35893.
- Figure 5 shows siderophores production of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to
Herbaspitillum seropedicae ATCC 35893.
- Figure 6 shows the potassium mobilization of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to an internal control.
- Figure 7 shows IAA production of Bacillus pumilus VMC30/195 and Bacillus megaterium VMC30/196 compared to Azospirillum brasilense ATCC 29145.
- Figure 8 shows the nitrogen fixation of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to
Azospirillum brasiliense ATCC 29145 and Herbaspirillum seropedicae ATCC 35893. Detailed description of preferred embodiments of the invention The present invention refers to a novel strain of bacteria isolated and characterized for the ability to be used in agriculture, preferably for the purpose of solubilizing macro and/or micro-nutrients that are essential for plant biology, preferably for plant growth and development. Moreover, the strain disclosed herein is useful as biostimulant for plants and is preferably used to increase nutrient use efficiency in plants and/or resistance to abiotic stress of plants and/or plant growth and/or quality. As used herein, “strain” refers to isolate or a group of isolates exhibiting phenotypic, physiologic, metabolic, and/or genotypic traits belonging to the same lineage and different/distinct from that of other strains belonging to the same species.
As used herein, “isolate” means a microorganism which has been removed from its original environment, including, but not limited to, soil, air, fresh water, sea water, algae, higher plants, seeds, roots, leaves, fruits, etc. Preferably, the “isolate” has to be “pure”, meaning that it does not comprise other microbial contaminants in the isolate, according to the isolation method reported in the present invention.
As used herein, “macronutrients or main nutrients” refer - in general - to any nutrient that plays an essential role in any key physical, physiological and biochemical process of plants, providing a healthy and balanced growth and development during all lifecycle of plants. Preferably, said nutrient is selected from: nitrogen, phosphorus, potassium (also known as NPK elements), calcium (Ca), magnesium (Mg), sulfur (S) and combinations thereof. Phosphorus (P) is one of the most indispensable macronutrients next to nitrogen for the growth and/or the development of plants. A greater part of soil phosphorus, around 95-99%, is present in insoluble form complexed with cations like iron, aluminum, and calcium, all of them being chemical forms of unavailable P that, therefore, cannot be utilized by the plants. The use of natural phosphate-bearing materials, such as rock phosphate (RP), as fertilizer for P- deficient soils has received due attention in recent years since substantial deposits of cheaper and low-grade RP are locally available in many countries of the world. However, the solubilization of natural phosphate-bearing materials, such as rock phosphate (RP), rarely occurs in nonacidic soils with a pH greater than 5.5 to 6.0. Conventionally, RP is chemically processed by reacting with sulfuric acid or phosphoric acid to produce partially acidulated RP. The process incurs high cost and
makes the environmental health worse. A much cheaper and convenient alternative is reclamation of exhausted soil through use of P-solubilizing microorganisms that have opened the possibility for solubilization of RP in soils. In this scenario, soil microorganisms play a critical role in natural phosphorus cycle and recently microbial-based approaches have been proposed to improve the agronomic value of RP. Therefore, microbial-based products represent cheaper approaches compared to the higher cost of manufacturing phosphate fertilizer in industry and, at the same time, they avoid the environment pollution posed by a traditional chemical process. Deficiency or excess of the main nutrients have relevant consequences such as great imbalance of the crops, delay of the growth, leaves and/or flowers loss, reduced photosynthetic activity, with subsequent low fruit production and, overall, the crop productivity is heavily reduced. In some cases, pathological consequences, such as plant tissues and/or fruit necrosis, can also take place.
As used herein, “plant biology” refers to any physiological and/or pathological plant process, preferably selected from: growth and productivity of plants, comprising plant biomass (mainly N, but also S), plants metabolism and energy production, flowers, fruit and seeds development (mainly P, but also Ca and Mg), efficiency of the photosynthetic process, osmotic balance, and/or production and quality of the fruit (mainly K, but also Ca).
As used herein, “micronutrients” relate to the main nutrients absorbed in very small amounts grams per hectare (g/ha) compared with macronutrients (kg/ha). Preferably, said micronutrient is selected from: boron (B), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), zinc (Zn) and combinations thereof. Zinc is an essential micronutrient, which means it is essential for plant growth and development but is required in very small quantities. It is crucial to plant development, as it plays a significant part in a wide range of processes, such as enzymes activation that are responsible for the synthesis of certain proteins. It is used in the formation of chlorophyll and some carbohydrates, conversion of starches to sugars and its presence in plant tissue helps the plant to withstand cold temperatures. Zinc is essential in the growth hormone production and internode elongation. Zinc deficiency is probably the most common micronutrient deficiency in crops worldwide. Symptoms caused by zinc deficiency vary depending on the crop. Typically, they include one or some of the following: stunting-reduced height, interveinal chlorosis,
brown spots on upper leaves and distorted leaves. The most common fertilizer sources of zinc are zinc chelates (contain approximately 14% zinc), zinc sulfate (25- 36% zinc) and zinc oxide (70-80% Zinc), where zinc sulfate is the most used source of zinc. The major problem of the application of these fertilizers is that in most of the cases only a small part of the total applied Zn is available to plants while remaining gets fixed into soil. Thus, an alternative solution is to use zinc solubilizing bacteria that are able to solubilize insoluble sources of zinc, preferably Zinc Oxide - ZnO, Zinc(ll) Carbonate -ZnCO3, or Zinc(ll) Phosphate - Zn3(PO4)2· With reference to Zinc, it must be pointed out that Zinc solubilizer strains are rare to be isolated and consequently make available on the market for this activity. Therefore, the strain disclosed herein represents a valuable source to address Zinc solubilization need. Micronutrient deficiency affects crop yield and quality. While excess of some micronutrients, despite taking place in rare situation, can interfere with the uptake of other nutrients resulting in unbalanced plant development.
As used herein, “solubilization and/or mobilization of nutrients and/or micro - macronutrients” refers to the process allowing the conversion of the mineral forms of macro/micro-elements, which are present in the soil under unavailable forms for plants, into forms which can be dissolved in water and consequently taken up through their roots. Advantageously, the bacterial strains here disclosed can solubilize and mobilize, preferably in the soil, insoluble forms of macro/micro- nutrients, preferably starting from the inorganic forms of macro/micro-nutrients. In other words, the bacterial strains here disclosed are able to improve the solubilization and/or the dissolution and/or the mobilization of macro/micro-nutrients, preferably in the soil, and, consequently, they are able to improve the availability, preferably in the soil, of micro and/or macronutrients as defined above for plants.
As used herein, “plant growth” refers to the extension and/or expansion of plant tissues and/or organs, which bring to an increase of the vegetative biomass. An optimal plant growth results in balanced development of the whole plant and boosts processes of differentiation in flowers, fruits, and seeds.
The isolated bacterial strain disclosed herein is a member of the genus Bacillus, species pumilus characterized by a DNA, more preferably the DNA of 16S rRNA gene coding comprising SEQ ID NO: 1 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 1.
16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea. The comparison of the 16S rRNA gene sequence of an isolate against sequences of type strains of all prokaryotic species provides an accurate and convenient way to routinely classify and identify prokaryotes.
To amplify the taxonomic-specific genes reported above any pair of primers located in the DNA sequence of interest can be used. Preferably, the pair of primers are the one disclosed in the examples and listed in Table I as SEQ ID NO: 3 and 4.
The isolated bacterial strain disclosed herein has been deposited on February 21st 2018 with the NCMR accession number MCC0530 and with IDA accession number MCC0195 (1st July 2020) and the following name (denomination): Bacillus pumilus strain VMC30/195 (VMC30/195 from now on) at the International Depositary Authority: National Centre for Microbial Research, Pune, Maharashtra-411021 , India (hereinafter NCMR) according to the provisions of Budapest Treaty.
VMC30/195 is gram positive and is preferably characterized by rod-shaped cells. The colony - when the bacteria are grown on nutrient agar - shows a yellowish color and is characterized by a size having an average diameter of 2-10 millimeters, preferably 3-5 millimeters. Preferably, the colony’s shape of VMC30/195 is irregular. According to a preferred embodiment of the invention, VMC30/195 can grow at a temperature up to 50C.
According to a further preferred embodiment of the invention, VMC30/195 is able to use at least one of the carbon source selected from: glycerol, arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, methyl-αDmannopyranoside, esculin, salicin, cellobiose, saccharose, trehalose, gentiobiose, tagatose, malic acid and any combination thereof.
According to a further preferred embodiment of the invention, VMC30/195 is positive for: citrate utilization; and/or acetoin production and/or gelatinase.
According to a further preferred embodiment of the invention, VMC30/195 is preferably resistant to at least one of the antibiotic agent, preferably present in a concentration ranging from 8-32 μg/ml and/or 15 μg/dish, selected from: ceftazidime, miokamycin, lincomycin and any combination therefore, and/or susceptible to at least one of the antibiotic agent, preferably present in a concentration ranging from 4 to 200 μg/ml and from 5 to 30 μg/disc and 300 U/disc, selected from: chloramphenicol,
gentamycin, streptomycin, polymyxin B, netilmicin, tobramycin, amoxicillin+clavulanic acid, ampicillin, ampicillin+sulbactam, piperacillin, ceflacor, cefonidic, ceftriaxone, cefuroxime, ciprofloxacin, levofloxacin, pefloxacin, azithromycin, clarithromycin, erthromycin, roxitromycin, fosfomycin, rifampicin, co-trimoxazole, novobiocin and any combination thereof, and/or intermediately susceptible to cefixime preferably present in a concentration of 32 μg/ml.
According to a further preferred embodiment of the invention, VMC30/195 is characterized by a population doubling of 30-40 minutes.
The invention refers also to a consortium comprising VMC30/195 and at least one further bacterial strain. Preferably said further bacterial strain is the isolated strain member of the genus Bacillus, species megaterium characterized by a DNA, more preferably the DNA of 16S rRNA gene coding, comprising SEQ ID NO: 2 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 2.
Preferably said isolated strain member of the genus Bacillus, species megaterium has been deposited on February 21st 2018 at the NCMR with the accession number MCC0524 and with IDA accession number MCC0194 (1st July 2020) and having the following name (denomination): Bacillus megaterium strain VMC30/196 (VMC 30/196 from now on).
VMC30/196 is gram positive and is preferably characterized by rod-shaped cells. The colony - when the bacteria are grown on nutrient agar - shows a peach color and is characterized by a size having an average diameter of 2-10 millimeters, preferably 5-8 millimeters. Preferably, the colony’s shape of VMC30/196 is circular. According to a preferred embodiment of the invention, VMC30/196 can grow at a temperature up to 40°C, preferably up to 50°C.
According to a further preferred embodiment of the invention, VMC30/196 is characterized by a population doubling of time 30-40 minutes.
In this context, “consortium” means a group of different species and/or strains of microorganisms with different metabolic activities, preferably the consortium is defined “artificial” when it involves a multi-population system that can contain a diverse range of microbial species and is adjustable to serve a variety of industrial and ecological interests. Preferably, in this context consortium means a combination of VMC30/195 and at least one more bacterial strain, preferably a combination of VMC30/195 and VMC30/196, eventually comprising further bacterial species or
strains.
According to a preferred embodiment of the invention, VMC30/195 and/or VMC30/196 and/or the consortium can be used as living microorganisms and/or dead cells and/or killed cells, preferably killed by heating, and/or as spores.
Moreover, VMC30/195 and/or VMC30/196 and/or the consortium may be used as a fresh or frozen sample. Alternatively, the strain(s) is(are) used dry, lyophilized or in liquid suspension, or encapsulated in the form of spores and/or living cells. VMC30/195 and/or VMC30/196 and/or the consortium may be cultivated continuously or discontinuously in any medium useful to grow bacteria, in liquid or solid form. Preferably VMC30/195 and/or VMC30/196 and/or the consortium is(are) cultivated on or in a medium(liquid/solid) that preferably comprises nutrient agar, meat extract, peptone, sodium chloride and yeast extract. The temperature of the culturing process ranges between 25°C and 30°C. Moreover, the pH value of the medium ranges preferably between 5 and 8, more preferably the pH is around 7. Therefore, a further aspect of the invention refers to a medium (broth) obtained/obtainable by culturing VMC30/195, eventually as consortium preferably with VMC30/196.The medium may contain the bacteria (the cells), that is the whole culture medium, or the medium may be a cell free medium (without the cells). The cell free medium may be obtained preferably by centrifuging the whole culture medium by using the common standard procedure useful for this purpose, to obtain a cell-free medium or supernatant. Therefore, VMC30/195, eventually as consortium preferably with VMC30/196, may be used as culture medium (broth), preferably whole culture medium (comprising cells), or cell-free medium or supernatant (without cells). Alternatively, VMC30/195, eventually as consortium preferably with VMC30/196, is used as bacterial derivatives, preferably as lysate, as extract, preferably cell free extract, as fraction or as a metabolite(s) derived from said bacterium (a).
As used herein, culture medium means a solid, liquid or semi-solid nutritive matrix to support the growth of cells (prokaryotic or eukaryotic cells).
In this context, alternative names for culture medium are preferably the following: proliferating medium, expansion medium, growth medium, nutrient medium.
As used herein, “whole culture medium” refers to a solution and/or a suspension containing bacterial cells, spores and derivatives thereof, such as nutrients,
metabolites or cellular debris. As used herein, “supernatant” refers to the liquid part of a culture broth free from the bacterial cells/spores and containing extracellular metabolites. As used herein, “lysate” means the solution comprising all the intracellular and extracellular material released from the lysis of the bacterial cells. As used herein, “extract” refers to a specific part of the culture broth, including or not living cells/spores. As used herein, “cell free extract” means a solution containing all the microbial metabolites, without the presence of living cells/spores. As used herein, “fraction” means a specific part of the whole culture medium, for example only the solution, only the cells or the like. As used herein, “metabolite” means one or more intermediate(s) or final product(s) of the bacterial metabolism.
A further aspect of the invention refers to mutants and/or edited strains derived/obtainable from VMC30/195. As used herein, “mutant” means any microorganism obtained/obtainable by direct mutation, selection, or genetic recombination of VMC30/195. Mutant strains may be obtained by using any methods known in the art for this purpose, such as mutant selection, chemical mutagenesis, genetic manipulation physical mutagenesis (radiation) and biological mutagenesis. As used herein, “edited” means preferably gene-edited strains wherein at least one gene of interest is edited/modified by using the common biological tools useful for this purpose, in particular biological tools based on the use of nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas).
According to a preferred embodiment of the invention, VVMC30/195, eventually as consortium preferably with VMC30/196, is(are) used in combination with further microorganisms, preferably selected from: bacteria, preferably PGPR (or rhizobacteria), yeasts, mycorrhizae, fungi, any derivatives as disclosed above and any combination thereof.
The PGPR of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Aeromonas rivuli, Agromyces fucosus, Bacillus spp. Bacillus mycoides, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Bacillus safensis, Microbacterium sp., Nocardia globerula, Stenotrophomonas spp., Pseudomonas spp, Pseudomonas fluorescens, Pseudomonas fulva, Pseudoxanthomonas dajeonensis, Rhodococcus coprophilus,
Sphingopyxis macrogoltabida, Streptomyces spp., Enterobacter spp., Azotobacter spp., Azospiriullum spp., Rhizobium spp., Herbaspirillum spp., Lactobaccillus spp., Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactococcus tactis, and combinations thereof.
The yeasts of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Candida spp., Candida tropicalis, Saccharomyces spp., Saccharomyces bay anus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces exiguous, Saccharomyces pastorianus, Saccharomyces pombe, and combinations thereof.
The mycorrhizae of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Glomus spp., Rhizophagus spp., Septoglomus spp., Funneliformis spp., and combinations thereof.
The fungi of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Trichoderma spp., Trichoderma atroviride, Trichoderma viride, Trichoderma afroharzianum, Paecilomyces spp., Beauveria bassiana., Metarhizium spp., Lecanicillium lecanii, Penicillium spp., Aspergillus spp., Conythyrium minitans, Pythium spp, and combinations thereof.
As said before VMC30/195 eventually as consortium preferably with VMC30/196 can be used as plant biostimulant. However, according to a preferred embodiment of the invention, VMC30/195 eventually as consortium preferably with VMC30/196 is(are) used in combination with an additional plant biostimulant (PB or PBS).
As used herein and according to the current definition of European Biostimulant Industry Council, “biostimulant” refers to a substance(s) and/or micro-organisms or a composition comprising said substance(s) and/or micro-organisms whose function when applied to plants or to the rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality. Preferably, said plant biostimulant comprises an extract of algae and/or an extract of microalgae and/or an extract of plant and/or a humic acid and/or a fulvic acid, and/or plant and/or animal byproducts.
As used herein, algae refer to a functional group of organisms that carry out oxygenic photosynthesis and are not embryophytes. They include both bacterial
(cyanobacteria) and eukaryotic organism. The term encompasses organisms that are photoautotrophic, heterotrophic, or mixotrophic, and are typically found in freshwater and marine systems. The term algae include macroalgae (such as seaweed) and/or microalgae. Preferably said algae are brown algae, more preferably said algae are selected from: Ascophyllum nodosum, Ecklonia maxima, Laminaria saccharina, Laminaria digitata, Fucus spiralis, Fucus serratus, F. vesicuiosus, Macrocystis spp., Pelvetia canaliculata, Himantalia elongata, Undaria pinnatifida, Sargassum spp, and combinations thereof. Ascophyllum nodosum is particularly preferred for the purposes of the present invention. As used herein, microalgae refer to any microscopic algae that are unicellular and simple multi-cellular microorganisms, including both prokaryotic microalgae, preferably, cyanobacteria (Chloroxybacteria), and eukaryotic microalgae, preferably green algae (Chlorophyta), red algae (Rhodophyta), or diatoms (Bacillariophyta). Preferably said microalgae are selected from: Spirulina, Scenedesmus, Nannochloropsis, Haematococcus, Chlorella, Phaeodactylum, Arthrospyra, Tetraselmis, Isochrysis, Synechocystis, Clamydomonas, Parietochloris, Desmodesmus, Neochloris, Dunaliella, Thalassiosira, Pavlova, Navicula, Chaetocerous, and combinations thereof.
As used herein, plant means any one of the vast number of organisms within the biological kingdom Plantae. Conventionally the term plant implies a taxon with characteristics of multicellularity, cell structure with walls containing cellulose, and organisms capable of photosynthesis. Preferably, they include a host of familiar organisms including trees, forbs, shrubs, grasses, vines, ferns, mosses and crop plants as vegetables, orchards and row crops. For the purpose of the present invention, the whole plant or part thereof may be used, preferably said part is selected from: leaves, roots, stems, fruits, flowers, seeds, seedlings, bark, berries, skins, and combinations thereof. Preferably, the plant to be used as extract to add to the strains herewith disclosed is selected from: beet, sugar cane, alfalfa, maize, brassica, halophytes, soya, wheat, yucca, quillaja, hop, coffee, citrus, olive, and combinations thereof.
Preferably said plants or algae or microalgae are extracted with common process, preferably said process comprising the following steps: (i) providing a sample of algae and/or a sample of microalgae and/or a sample of plants; and (ii) contacting
said sample(s) with an aqueous solution comprising an extraction agent, in other words an extraction solution having an aqueous base.
As used herein, the extraction agent can be a base and/or an acid and/or an enzyme. These kind of extraction agents can be used in any combination or singly. For the purpose of the present invention: the base is preferable an inorganic base, preferably selected from: NaOH, KOH, Na2CO3, K2CO3, NH3, salts thereof, and any combination thereof; the acid is preferably selected from: H2SO4, HNO3, HCI, H3PO4, and various acids of organic nature preferably selected from: acetic acid, citric acid, formic acid, butyric acid and ascorbic acid, gluconic acid, and any combination thereof; and the enzyme is preferably selected from: papain, trypsin, amylase, pepsin, bromelain and specific enzymes that degrade organic polymers present in the algae, preferably alginases, and any combination thereof. The selection of the extracting agent to be used for the process depends upon the kind of algae/microalgae/plant to be extracted and/or the molecules/components to be extracted from them. Preferably, the temperature of the extraction process ranges between -20 and 120°C, more preferably between 20 and 100°C. Preferably, the extraction time ranges from a few minutes to several hours, more preferably between 30 minutes and 18 hours. Preferably, the extraction process is realised at atmospheric pressure or at a pressure up to 10 Bar, more preferably at a pressure ranging from 1 to 8 Bar. The extraction process may be followed by a further step of separating/removing the non-solubilised and/or non-extracted component when it is desirable using only the extract in the formulation of the biostimulant. The removing/separating step is preferably performed by decantation, filtration or centrifugation. Alternatively, a suspension comprising both the extracted component and the non-extracted component can be used.
Preferably, the concentration of the extract from algae and/or microalgae in the biostimulant ranges from 1 to 60%, preferably it ranges from 5 to 50%, more preferably from 10 to 20%, still more preferably around 15%. Preferably, the concentration of the plant extract in the biostimulant ranges from 1 to 60%, preferably it ranges from 5 to 50%, more preferably from 10 to 20%, still more preferably around 15%. Preferably, the humic acid is extracted from leonardite, lignite, sub-products of the digestion of urban bio-waste and biochar. The extraction process is performed in water, in alkali, in acidic medium, or by pyrolysis. Preferably,
the concentration of the humic acid in the biostimulant ranges from 1 to 20%. Preferably, the fulvic acid is extracted from peat, lignite, leonardite, digestion of urban bio-waste, biochar and vegetable materials. The extraction process is performed indifferently in water, in alkali, in acidic medium, and by pyrolysis. Preferably, the concentration of the fulvic acid in the biostimulant ranges from 1 to 20%, preferably from 5 to 10%.
Preferably, when VMC30/195, eventually as consortium preferably with VMC30/196, is used in combination with the PBS as disclosed above, said PBS is present in a concentration ranging from 5 to 50%, preferably from 10 to 40%, more preferably from 15 to 25 %, preferably around 20%. VMC30/195, eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 0.01 to 20%, preferably from 0.01 to 10%, more preferably from 0.05 to 5%, more preferably from 0.1 to 1%. Alternatively, VMC30/195, eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 10 to 20%.
Alternatively, VMC30/195, eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 10M to 10Λ12 UFC/g each, preferably from 10Λ6 to 10Λ9 UFC/g, more preferably around 10Λ8 UFC/g.
The PBS percentage refers to the sum of the percentage of each plant biostimulant component. In this context, the plant biostimulant component is preferably selected from: plant extracts, seaweed extracts, humic acid, fulvic acid, animal byproducts and combinations thereof. The percentage of the strain(s) refers to the sum of dried biomasses for each microorganism in 100 g of final composition.
According to a further preferred embodiment, VMC30/195, eventually as consortium preferably with VMC30/196, or the medium or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite as disclosed above is/are mixed with the plant biostimulant as disclosed above and/or with a non- microbial component. Said non-microbial component comprises one or more components present in the mixture, preferably in the composition, with the exclusion of the microbial component, that is VMC30/195, eventually as consortium preferably with VMC30/196, or the medium or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite. The non-microbial component comprises preferably the PBS as disclosed above and/or at least one further ingredient, preferably a mineral component. Preferably said non-microbial
component is present in a concentration up to 99,99%, 99,95 %, 99,90%, 99%, 95%, 90%, 85%, or 80% , and/or the bacteria are used in a concentration up to 20%, 15%, 10%, 5%, 1%, 0,1% 0.05% or 0.01 .
According to a preferred embodiment of the invention, said at least one further ingredient or mineral component is selected from:
- A nitrogen source, preferably selected from: ammonium phosphates, ammonium nitrate, ammonium sulfate, ammonium thiosulfate, potassium thiosulfate, ammonia, urea, nitric acid, potassium nitrate, magnesium nitrate, calcium nitrate, sodium nitrate, protein hydrolisates of vegetal and animal origin, aminoacids, proteins, yeast lysate, manganese nitrate, zinc nitrate, slow release urea, preferably ureaformaldehyde, similar compounds and combinations thereof; and/or
- A phosphorus source, preferably selected from: ammonium phosphates, potassium phosphates, phosphoric acid, sodium phosphates, calcium phosphate, magnesium phosphate, rock phosphate preferably hydroxyapatite and fluoroapatite, phosphorous acid, sodium phosphite, potassium phosphite, calcium phosphite, magnesium phosphite, organic phosphorus compounds, preferably inositol- phosphate, sodium glycerophosphate, ATP, similar compound and combinations thereof; and/or
- A potassium source, preferably selected from: potassium acetate, potassium citrate, potassium sulfate preferably mixed salts of magnesium, potassium thiosulfate potassium phosphate, potassium phosphite, potassium carbonate, potassium chloride, potassium hydroxide, potassium nitrate, mixed salts of magnesium and potassium, potassium sorbate, potassium ascorbate, organic forms of potassium, and combinations thereof; and/or
- A magnesium and/or calcium source, preferably selected from: magnesium nitrate, magnesium sulfate, magnesium chloride, magnesium phosphate, magnesium phosphite, magnesium thiosulfate, magnesium hydroxide, magnesium oxide, mixed salts of potassium and magnesium, mixed salts of magnesium and calcium (dolomite), magnesium acetate, magnesium citrate, magnesium sorbate, and organic forms of magnesium, magnesium carbonate, magnesium formate, magnesium ascorbate, and combinations thereof; and/or
- A sulfur source, preferably selected from: sulfuric acid, sulfates, thiosulfate, sulfated aminoacids, and combinations thereof; and/or
- Iron and/or manganese and/or zinc and/or copper source, preferably selected from: iron sulfate, iron oxide, iron hydroxide, iron chloride, iron carbonate, iron phosphate, iron nitrate, chelated iron with EDTA, DTPA, HEDTA, EDDHA, EDDHSA, EDDHCA, EDDHMA, HBED, EDDS; complexed iron with aminoacids, ligninsufonates, humic acids, fulvic acids, gluconic acid, heptagluconic acid, iron citrate, iron malate, iron tartrate, iron acetate, iron lactate, iron ascorbate, organic form of iron, and combinations thereof .
According to a preferred embodiment of the invention, VMC30/195 and/or VMC30/196 is(are) used in combination with further molecules, preferably proteins, protein hydrolisates, peptides, oligopeptides, peptidoglycans, low-molecular weight peptides, synthetic and natural occurring aminoacids; molasses, polysaccharides, lypopolysaccharides, monosaccharides, disaccharides, oligosaccharides, sulfated oligosaccharides, exopolysaccharides, chitosan. Other molecules that can be advantageously used in combination with VMC30/195 and/or VMC30/196 are selected from: stress protecting molecules, such as betaines, mannitol, and other polyols with similar effects, and hormones and hormone-like compounds, such as melatonin, auxins, auxin-like compounds, cytokinins, cytokinin-like compounds, gibberellins, gibberellin-like compounds, jasmonates, hormones precursors like polyamines spermine, spermidine, putrescine, and metabolism stimulating substances like vitamins. Other molecules that can be advantageously used in combinations with VMC30/195 and/or VMC 30/196 are selected from: nucleic acids, uronic acids and polymers thereof, glucuronic acids and polymers thereof, small organic acids, such as oxalic and succinic acids. Preferably, said small molecules may be a synthetic and/or naturally derived nucleic acid molecules containing multiple nucleotides, preferably being defined an oligonucleotide when the molecule is 18-25 nucleotides in length and polynucleotides when the molecule is 26 or more nucleotides. Preferably said oligonucleotides or polynucleotides or a mixture of both, include RNA or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof.
A further aspect of the invention refers to a composition, preferably an agricultural composition, more preferably a plant biostimulant composition, comprising VMC30/195, eventually as consortium preferably with VMC30/196 as disclosed above, eventually in combination with the further components/ingredients disclosed
above and a carrier, preferably an agricultural compatible carrier. Preferably, said further components/ingredients is the PBS and/or the non-microbial component and/or the further ingredients or mineral component as previously disclosed.
As used herein “agricultural compatible carrier” refers to any synthetic or natural, organic or inorganic, derived compound able to deliver the product in an active form in the site of action, preferably said carrier is selected from the categories of surfactants, thickeners, suspension agents, wetting agents, and combinations thereof.
As used herein, surfactant means any molecule able to modify the surface tension of the water and allowing the product to impact a wider area of the leave and/or root and/or fruit, or any other part of the plant. Preferably said surfactant is selected from: ionic, non-ionic, cationic surfactants, synthetic or naturally derived, preferably alkyl sulfonates, alkylarylsulfonates, ethoxylated alcohols, alkoxylated ethers, ethoxylated esters, alkylpolyglucosides, block copolymers, lignosulfonates, saponins, and the like.
As used herein, thickener means any molecule able to modify the rheology of any given composition in the sense of improving the viscosity and stabilize it. Preferably, said thickener is selected from: natural and synthetic gums, lignosulfonates, molasses and the like.
As used herein, suspension agent means any molecule able to surround insoluble particles avoiding settlement and allowing the creation of a stable suspension of insoluble. Preferably, said suspension agent is selected from: natural and synthetic colloids, clays, and their derivatives and the like.
As used herein, wetting agent means any molecule able to avoid fast water evaporation on a given surface and retain moisture for a long time. Preferably, said wetting agent is selected from: glycols, glycerin and their derivatives and the like. VMC30/195, preferably as consortium, preferably with VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is/are formulated as: solution, suspension, water- soluble concentrates, dispersable concentrates, emulsifiable concentrates, emulsions, suspensions, microemulsion, gel, microcapsules, granules, ultralow volume liquid, wetting powder, dustable powder, or seed coating or treatment formulations.
As shown in the experimental part, VMC30/195 and/or VMC30/196 is(are) able to solubilize macronutrients and/or micronutrients as defined above. Preferably, the strain(s) is(are) able to solubilize macronutrients and/or micronutrients present in the soil and therefore the strain(s) allow(s) said solubilized macronutrients and/or micronutrients being more available for plants that consequently show an improved capability of micro and/or macronutrient’s uptake. In other words, thanks to the macronutrients and/or micronutrients solubilizing (mobilizing/dissolving) activity of the strain(s) of the invention, plants improve the up-take capability of said solubilized macronutrients and/or micronutrients, preferably from the soil, and show a better growth. In this regard, the experimental evidence has shown that VMC30/195 is good performer in Phosporous solubilization, and when used with VMC30/196 the consortium works even better. In addition, VMC30/195 has shown great potential in Zinc solubilization more than the strain of reference known to have this activity. As mentioned before Zinc solubilizers are very difficult to be isolated and therefore VMC30/195 represent an interesting emerging alternative of selected strain for this biological function in plants. In addition, VMC30/195 shows promising potential also in nitrogen fixation and in this regard, it works better compared to known nitrogen fixator strains.
Finally, when VMC30/195 is used in combination with VMC30/196, the consortium shows complete framework of micro-macronutrients solubilization activity because besides the above disclosed properties the consortium shows also potassium and iron solubilization/mobilization activity. Indeed, VMC30/196 is a good producer of siderophores and so it helps and increases iron solubilization and consequently plant iron uptake. Moreover, VMC30/196 is good potassium solubilized. Therefore, VMC30/196 can complement the panel of solubilizing/mobilizing activity toward micro-macronutrients of VMC30/195.
Moreover, VMC30/196 is also able to produce IAA. This molecule is known to help plant in the production of longer roots with an increased number of root hairs and root laterals which are involved in nutrient uptake (Datta and Basu, 2000). Moreover, IAA promotes cell elongation, inhibit or delay abscission of leaves and induce flowering and fruiting. Overall, IAA producing bacteria may improve the fitness of the plant-bacterium interaction, colonize plant roots better than other bacteria by increased root system, weaken plant defense mechanisms making colonization
easier, loosen plant cell walls and as a result promotes an increasing amount of root exudation that provides additional nutrients to support the growth of rhizosphere bacteria and provides more number of active sites and access to colonization for other PGPRs (Etesami et al., 2015). Therefore, VMC30/196, preferably as consortium with VMC30/195, may be also used for increasing number of root hairs and/or root laterals; and/or promoting cell elongation, inhibiting or delaying abscission of leaves and inducing flowering and fruiting; and/or improving the fitness of the plant-bacterium interaction, colonizing plant roots by increasing the root system, and/or weakening plant defense mechanisms making colonization easier, and/or loosening plant cell walls, and/or promoting an increase in root exudation and so additional nutrients to support the growth of rhizosphere bacteria, and/or increasing active sites and accesses to colonization for other PGPRs.
Therefore, VMC30/195 and/or VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is/are preferably used to solubilize (mobilize/dissolve) or to improve the dissolution, preferably in the soil, of phosphate, preferably any inorganic source of phosphate, more preferably selected from: phosphate tricalcium phosphate (Ca3(PO4)2), ferric phosphate (FePO4), and aluminum phosphate (AIPO4). In other words, insoluble forms of phosphorus, preferably contained into soils, are converted, through several mechanisms, preferably by organic acids production, chelation, ion-exchange reaction or polymeric substances formation, into soluble forms preferably H2PO4- and/or HPO4 2- that are easier to be taken up plants.
According to a further embodiment of the invention, VMC30/195 and/or VMC 30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to solubilize (mobilize/dissolve) zinc, preferably any inorganic source of zinc, more preferably selected from: Zinc Oxide (ZnO), Zinc(ll) Carbonate (ZnCO3), and Zinc(ll) Phosphate (Zn3(PO4)2). Preferably, VMC30/195 and/or VMC 30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to convert into soluble forms, existing as free Zn2+ ions and Zn chelates, by the bacterial production of organic acids such as 5-ketogluconic acid and 2-ketogluconic acid. Indeed, gluconic acids and ketogluconates are sugar acids having multiple conformations, which chelate the metal cations coming from
solubilization process.
According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to produce siderophores. Preferably, VMC30/195 and/or VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to chelate iron ions (Fe3+), making iron more available for plants. As used herein, “siderophores” mean small, high-affinity iron-chelating compounds generally secreted by microorganisms such as bacteria, fungi and grasses. Siderophores are amongst the strongest soluble Fe3+ binding agents known. These compounds are small proteic molecules generally <1000 Da, although some siderophores are bigger. They are rapidly assembled through short, well-defined metabolic pathways. These molecules comprise lateral chains and functional groups that confer a strong affinity (usually with Kd>1030 M-1) to coordinate with the ferric ion (Fe3+). Typically, microbial siderophores belong to at least one of class of molecules preferably selected from: catecholates, hydroxamates, and a-carboxylates, depending on the chemical nature of their coordination sites with iron. Preferably, the strain(s) of the invention is able to produce hydroxamates type siderophores. These siderophores form iron chelates by the binding site that is mounted on an L-ornithine derivative. Through this mechanism iron ion (Fe3+) is taken from insoluble forms and became available for plants. Iron privation in plants causes as main effect the reduction in photosynthetic activity and as secondary effect the reduction in fruit quality in terms of color, size, sugar content, fruit hardness and taste. Moreover, siderophores support plant growth also by inhibition of soil-borne plant pathogens. Indeed, the siderophores - produced in iron-limited conditions -sequester the less- available iron from the environment and inhibit pathogens by depriving iron. Therefore, according to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above can be used to improve the capability of plants to uptake iron preferably from soil.
According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to mobilize K making it more
available for plants. Potassium (K) is an essential macronutrient and plays an important role in the growth and metabolism of plants. The concentrations of soluble potassium in the soil are usually very low and more than 90% of potassium in the soil exists in the form of insoluble rocks and silicate minerals (e.g., biotite, feldspar, illite, muscovite, orthoclase, and mica), not available for plant uptake. A deficiency of potassium causes a delay in the root and plant growth, a small seed production and a lower yield. With the intensification of agriculture and the imbalanced fertilizer application, potassium deficiency is becoming one of the major constraints in crop production. This aspect highlights the importance to maximize the bioavailability of such mineral in the soils for sustaining crop production. Soil microbes can play a central role in the natural potassium cycle and therefore, the potassium solubilizing microorganism present in the soil, can dissolve silicate minerals and release K through the production of organic and inorganic acids, acidolysis, polysaccharides, complexolysis, chelation, and exchange reactions. Therefore, according to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above can dissolve potassium from insoluble K-bearing minerals and make it available to plant uptake.
According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to produce IAA.
According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to fix nitrogen. Preferably, VMC30/195 and/or VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to convert atmospheric N2 into plant-utilizable forms by biological N2 fixation which changes nitrogen to ammonia by nitrogen fixing microorganisms using a complex enzyme system known as nitrogenase.
As already disclosed above, when used as consortium with at least a further strain, preferably with VMC30/196, VMC30/195 is used in the same amount (1 :1) of the further strain. Alternatively, the range between the amount of VMC30/195 and the additional strain, preferably VMC30/196, varies between 1 :1 and 1 :100, preferably
1 :3, 1 :5 or 1 :10.
According to a preferred embodiment, the strain or the consortium or the strain derivatives or the composition here disclosed is preferably applied into the soil, with any useful means or way, preferably through fertigation or irrigation systems and is preferably diluted in water, more preferably through overhead applications with appropriate volumes of water to let the solution spreading through the soil. In general, the application of the strain (VMC30/195) or the consortium of the strain with at least one additional bacterial strain, preferably VMC30/196, or the composition comprising the strain or the consortium is performed with at least one of the following practice: drip, overhead irrigation, seed treatment, soil application, foliar treatment, indoor farming, vertical farming and combination thereof.
According to a preferred embodiment, the application is performed all over the crop cycle, preferably during the early stages of crop development.
According to a preferred embodiment, the strain or the consortium or the strain derivatives or the composition here disclosed is preferably applied at a rate ranging between 100g-10.000 g/hectare, preferably between 2 and 10 kg/hectare preferably up to 5 times/crop cycle.
Indeed, when tested together, especially in presence of a plant biostimulant, the biological properties discussed above are particularly enhanced as well as the crop yield. In this regard, the synergism between the strains in the presence of the PBS may be due to their ability to degrade the PB’s components and to generate metabolites able to support their growth/activities.
According to a preferred embodiment, VMC30/195 and/or VMC30/196 when used as consortium is/are able to emphasize, preferably to synergize, plants responses to any biostimulant substance. Preferably, a significant yield improvement has been observed when VMC30/195 and/or VMC30/196 is(are) used with a biostimulant as defined above.
Therefore, VMC30/195 and/or VMC30/196, preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) particularly useful in agriculture, preferably to improve, preferably into soil, macronutrients and/or micronutrients solubilization (mobilization/dissolution/fixation), preferably phosphate and/or zinc and/or potassium solubilization/mobilization, and/or atmospheric nitrogen fixation and/or siderophores
production, preferably iron availability and/or IAA production. Consequently, VMC30/195 and/or VMC 30/196, preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) particularly useful to improve macronutrients and/or micronutrients plant uptake, phosphate and/or zinc and/or iron and/or nitrogen uptake from plants. In view of these effects, VMC30/195 and/or VMC30/196, preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) useful to improve plant growth. Preferably, said further components/ingredients is the PBS as previously disclosed.
As used herein, plant means any one of the vast number of organisms within the biological kingdom Plantae. Conventionally the term plant implies a taxon with characteristics of multicellularity, cell structure with walls containing cellulose, and organisms capable of photosynthesis. Modern classification schemes are driven by somewhat rigid categorizations inherent in DNA and common ancestry. In general, these species are considered of limited motility and generally manufacture their own food. Preferably, plant includes a host of familiar organisms including trees, forbs, shrubs, grasses, vines, ferns, mosses and crop plants as vegetables, orchards and row crops. More preferably plant includes leaf vegetables, preferably selected from: Solanaceae, Brassicaceae, Liliaceae, and combinations thereof; and/or orchard plants, preferably selected from: fruit trees, grapes, olive trees, citrus trees, and combinations thereof; and/or row crops, preferably selected from: cereals, preferably corn, soybean, wheat, winter wheat, rice, barley, sugar cane, sugar beet, legumes preferably peas, beans, chickpeas but are not limited to: brassica, bulb vegetables, cereal grains, citrus, cotton, curcurbits, fruiting vegetables, leafy vegetables, legumes, oil seed crops, peanut, nut trees, cocoa tree, herbs, cannabis, pome fruit, root vegetables, tuber vegetables, corm vegetables, stone fruit, tobacco, palm trees, musae spp., strawberry and other berries, and various ornamentals.
All the sequences disclosed in the present invention are listed in the following Table and are further submitted as Sequence Listing. Any sequence having at least 80- 99% of identity with the sequences herewith should be considered part of the present disclosure.
Table
EXAMPLE
The present invention will be described in detail by means of the following examples. The following examples are for illustrative purposes and are not intended to limit the scope of the invention.
Isolation of Novel Bacterial Strains
The microbes have been isolated from rhizosphere soil samples coming from India.
A soil sample (10 grams) has been taken and diluted into 90 ml of distilled water. After homogenization, serial dilution up to 10-5 have been done. One ml of each serial dilution has been smeared on a nutritive agar plate and incubated for 24 h at 30 °C. After incubation period, the most relevant colonies found on the highest
dilution ( 10-5), have been picked and plated again on nutrient agar plates, incubated for 24 h at 30 °C.
This process allowed isolate a pure colony from each soil sample said colony being later identified as the Bacillus pumilus and the Bacillus megaterium of the invention. In particular, Bacillus pumilus was isolated from Chickpea (Cicer arietinum) collected from Ramanthapur village (Sanga Reddy), while Bacillus megaterium was isolated from Paddy rhizoshere soil collected from Indra Karan village (Sangareddy). The isolated strains have been deposited in National Centre for Microbial Research (NCMR, Pune, India) on 21st February 2008, with the following name: Bacillus pumilus VMC30/195 (VMC30/195) having the deposit number MCC 0530, and Bacillus megaterium VMC30/196 (VMC30/196 from now on) having the deposit number MCC 0524.
Identification and Characterization of Bacterial Strains
16S-rRNA Sequencing
The strains VMC30/195 and VMC30/196 were typed by direct sequencing of PCR- amplified 16S rDNA.
For the amplification the following primers were used:
The amplification protocol is the following.
The 16S rDNA sequences of the strains are set forth in the sequence Listing as indicated in Table I.
The Sanger sequencing was followed by alignment and phylogenetically analysis of the obtained data with the 16S rRNA sequences from the Type Strains.
According the analysis of the 16S rRNA sequence:
The strain Bacillus pumilus VMC 30/195 is related to the species B. pumilus as it shares 97% and 97,74% of sequence similarity with the 16S rRNA sequences of the corresponding Type Strains (Table II and Fig.1.A).
Table II
The strain Bacillus megaterium VMC 30/196 is related to the species B. megaterium as it shares 94,98% and 94,98% of sequence similarity with the 16S rRNA sequence of the corresponding strains (Table III and Fig. 1 B).
Table III
This technique allows identification of microbes as intact cells or cell extracts. In this case, intact cells were tested by using a MicroflexTM MALDI-TOF (Matrix-assisted laser desorption ionization-time-of-flight) mass spectrometer (Bruker Daltonics, Leipzig, Germany). Initial manual/visual estimation of the mass spectra was performed using the FlexAnalysis 2.4 software (Bruker Daltonik GmbH, Germany). For automated data analysis, raw spectra were processed using the MALDI BioTyper 1.1 software (Bruker Daltonik GmbH, Germany) with default settings. The smoothing, normalization, baseline subtraction and peak picking was carried out by the software, thereby creating a list of the most significant peaks of a spectrum (m/z values with a given intensity). Samples were prepared according to manufacturers’ instructions.
Briefly, after 24 hours of cultivation on Nutrient Agar (NA) at 30°C, a single colony
was transferred with a toothpick onto the MALDI steel target plates in triplicate. Spots were overlaid with 1 μI of a saturated solution of -cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in organic solution (50% acetonitrile, 2.5% trifluoroacetic acid), air-dried within minutes at room temperature and directly screened. Spectra were recorded by Flex Control software (Bruker Daltonics, Bremen, Germany) in a linear positive mode at a laser frequency of 200 Hz in the range from 2 to 20 kDa. In order to assess the reproducibility of MALDI-TOF-MS identification, strains were tested in triplicate (analyses were performed on three different days and starting from different cultures). For each measurement, at least 300 individual spectra (30 laser shots at 10 different spot positions) were collected and averaged. External calibration was performed with the Bruker bacterial test standard (Bruker Daltonics, Bremen, Germany).
Figure 2 shows MALDI-TOF MS mass spectra of VMC30/195 (A) andVMC 10/196 (B), respectively. Each bar in the graphs represents a different protein expressed by the microbes and the intensity of the bars represents the concentration of the proteins within the microbial cell. Since proteins are a direct expression of genome and genome of each strains is unique, thus the proteomic profile of each strain is unique and can be used as fingerprint. The most and unique representative signal for each one of the strains are in the range from 2 to about 12 KDa using spectrum m/z 10Λ3.
Morphological and Physiological Characterization
The color and the colony morphology of the strains have been characterized by direct observation of the microbial strains after growing on Nutrient Agar (NA) plates. In particular, the bacterial strains have been cultured on NA plates for 24 h at 30 °C. After incubation period, bacterial colonies appear on the plates and color and morphology can be observed.
The motility and the colony size have been characterized by using optical microscope with 100X magnitude. In particular, the bacterial strains have been cultured on NA plates for 24 h at 30 °C. After incubation period, one bacterial colony has been picked and smeared into 1 ml water drop on a glass slide. The glass slide is then observed under optical microscope with 100x magnitude.
Gram staining is determined by using the Gram staining kit.
The growth of the bacterial strains at different temperatures has been evaluated by
streaking the microorganisms on NA plates (in triplicates) and by incubating them at different temperatures (4, 40 and 50 °C). After 24 h of incubation, the appearance of colonies on the plates indicates the ability of the strain to grow at a specific temperature. The ability of the bacterial strains to grow at different pH values is determined by streaking the microorganisms on NA plates adjusted at different pH values (5.5, 6.5 and 9.5). Experiment was done in triplicate. After 24 h of incubation, the appearance of bacterial colonies on the plates indicates the ability to grow at a specific pH value. The salinity tolerance of the strains is determined by streaking the microorganisms on NA plates, modified by the addition of different amount of NaCI (8, 10, 12 and 14 %). The experiment is performed in triplicate. After 24 h of incubation, the appearance of colonies on the plates, indicates the ability of the strain to grow at a specific NaCI concentration, thus, to tolerate a specific salinity level. The results are given in Table IV.
Table IV
Growth (Fermentabilitv) of Strains for In-Vivo Tests
The biomass production process of the strains is divided into four steps that are: inoculum preparation, fermentation, biomass recovery and biomass drying. Recovery of microbial biomass can be done through several processes such as centrifugation, micro-filtration or ultra-filtration.
Drying process is preferably made by freeze-drying.
In vitro Assays
For the assays different strains were used as control. They were chosen according to the literature. Molina et al. (2018) reports Azospirillum brasilense ATCC 29145 as
IAA producer and we used it as control in the indole-3-acetic acid production assay. Herbaspirillum seropedicae ATCC 35893 was used as positive control for several biostimulant activities reported in literature, inter alia, inorganic tricalcium phosphate ((Ca3(P04)2) solubilization (Estrada et al. (2012), Rodriguez et al.,2004), siderophores production (Rosconi et al., 2012; Tortora et al., 2011) and zinc solubilization according Gontijo et al. 2017. Both Azospirillum brasilense ATCC 29145 and Herbaspirillum seropedicae ATCC 35893 were used as positive control for Nitrogen fixation (Rosconi et al., 2012 and Zeffa et al., 2019).
Phosphate solubilization activity
The phosphate solubilization activity of the VMC30/195 and VMC30/196 was shown by using an in-vitro plate assay according to Pikovskaya method (Yasmin and Bano, 2011). Tricalcium phosphate (Ca3(PO4)2) was used as inorganic source of phosphorus.
The seeding was done superficially using an aliquot (10 μI) of the bacterial suspension (106 CFU/ml).
Seeded samples were incubated at 30 °C for 7 days and colonies with a clear halo on plate were considered positive for phosphate solubilization.
P-solubilization activity was measured as solubilization index (SI), according to Yasmin and Bano (2011) after 7th days and compared one to each other to individuate the best phosphorus solubilizing bacteria (PSB).
Solubilization index was determined as follows:
Bacterial strains have been tested in mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension and following the methodology described above. Moreover, the phosphate solubilizing activity of the microbes was evaluated in liquid medium in order to measure the exact quantity of phosphorus liberated from inorganic form (Ca3(PO4)2).
The samples tested were the same as reported above. The bacteria (2ml - 106 CFU/ml) was inoculated in liquid Pikovskaya medium (pH 6.5) and incubated at 30°C for 5 days, shaking constantly at 120 rpm. Samples were then centrifugated at 10,000 rpm for 10 minutes. An aliquot of the supernatant was taken to measure the soluble phosphorus (P) and the final pH value of the medium. The amount of soluble
phosphorus in the supernatant was measured by molybdenum-blue method using a spectrophotometer at a wavelength of 600 nm (King, 1932). The amount of soluble phosphorus was determined from the standard curve of KH2PO4. Herbaspirillum seropedicae ATCC 35893 have been used as specific controls.
The results are showed in Fig. 3A and B.
VMC30/195 showed ability to solubilize phosphate on Picovskaya’s plates with SI =1 ,20 after seven days of incubation at 30°C. On the contrary Bacillus megaterium VMC30/196 did not express this activity under the tested conditions. Values of solubilized P concentrations were recorded among the bacterial strains in 5 days of incubation. The highest mobilized phosphate value (497,67 mg/L) was recorded from VMC30/195 followed by Herbaspirillum seropedicae ATCC 35893 with 454,50 mg/L of soluble-P. VMC30/196 did not show this activity. Results are summarized in Table V.
Table V
Interestingly, when bacterial strains were tested in mixed cultures, the consortia of VMC30/195 and VMC30/196 showed an increase of the SI value (SI = 1 ,85) compared to the strains used alone where SI is Sl=1 ,20 for VMC30/195 and no activity for VMC30/196. Results are shown in Fig. 3B.
Therefore, the strain VMC30/195 keeps the P-solubilization activity even when is co- cultured with VMC30/196.
Zinc solubilization activity
Zinc-solubilizing capacity of the bacterial strains were assessed by a plate assay method using a modified Pikovskaya method (Ghevariya and Desai, 2014and zinc oxide (ZnO) as inorganic source of zinc.
The isolates were inoculated into agar medium containing 0.1% insoluble zinc (ZnO) and incubated at 30 °C for seven days. The seeding was done superficially using an aliquot (10 μI) of each bacterial suspension, with a concentration of 106 CFU/ml. Zinc solubilization efficiency (SE) was calculated as described by Sharma et al., 2014. Bacterial strains have been tested as single cultures as well as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension. A highly significant (P= 0.05) variation of SE was recorded among the bacterial strains.
The results are summarized in Fig. 4A and 4B and showed that VMC30/195 is better zinc solubilizer (SE=266,67) compared to the control Herbaspirillum seropedicae ATCC 35893 (SE=110,32). VMC 30/196 does not showed the zinc solubilization activity in the tested conditions. Apparently, at least in the tested condition, we found out that for zinc solubilization the consortium works not as well as the single strain. This could be due to secondary metabolites production by both that can affect medium pH, leading to higher values. Such aspect can decrease the efficacy of the solubilization and final result under these experimental conditions. In this regard, indeed, Khanghahi et al., 2018 reported the effect of pH on Zn-solubilizing ability of selected bacteria.
Siderophore’s production
The production of siderophores of the strain of the invention has been determined according to Schwyn and Neilands (1987). Briefly, 10 μI of each bacterial suspension with a concentration of 106 CFU/ml was spotted in triplicate on Nutrient broth (NB) and plated were incubated at 28°C for 72 h. Ten ml of chrome azurol S (CAS) agar medium were poured over the plates. After 24 h the formation of an orange halo was considered as indicator of siderophore production. The halo was measured according to Omidvari et al. (2010). Moreover, bacterial strains have been tested as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension. The results are summarized in the Fig.5A and B and show that VMC30/196 works better than control (35 vs 19mm) while VMC30/195 did not show siderophore production activity in the tested conditions. Interestingly, the consortium of VMC 30/195 and VMC30/196 of works better that the strains alone. A possible explanation is because of the secreted molecules can be shared between cells; indeed, siderophore production is often considered a form of cooperation. Siderophores are public goods and their production typically represents a mutualistic form of cooperation, providing direct benefits to both the producers and the recipients. Evidence to support this concept, emerged from studies on siderophores, including pyoverdine and pyochelin from P. aeruginosa, ornibactin from B. cenocepacia and enterochelin from Escherichia coli.
Potassium mobilization
In order to determine the potassium-solubilizing capacity of the two bacterial strains used in the present invention, they were screened by a plate assay method using
Alexandrov’s agar medium according to the method described by Parmar and Sindhu (2013). The potassium solubilizing activity was evaluated using mica powder (potassium aluminum silicate) as insoluble form of potassium. The isolates were inoculated into agar medium containing 0.5% insoluble potassium. The seeding was done superficially using an aliquot (10 μI) of each bacterial suspension, with a concentration of 1 ,00E+06 CFU/ml. Bacterial strains have been tested as single cultures as well as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension. The test organisms were inoculated on these media and incubated at 28 °C for three days. Colonies with a clear halo on plate were considered positive for potassium (K) mobilization.
K-mobilization activity was measured as mobilization index (Ml), according to Parmar and Sindhu (2013).
Mobilization index was determined as follows:
MI = Diameter of z one of clearance f Diameter of growth.
The results are summarized in Fig. 6A and B. Only VMC 30/196 shows good mobilization activity (Ml=2,06) under the tested conditions.
IAA measurement
To determine the IAA-producing capacity VMC 30/195 and VMC 30/196 were screened by a liquid assay method according to Sarker et al. (2013). 1 ,00E+06 CFU/ml or each strain was inoculated into nutrient broth containing 0,1% of tryptophan. Inoculated samples were incubated in shaking incubator at 28°C (100- 110 rpm) for 48 hours (for obtaining log phase). Uninoculated broth was used as negative control. After incubation, the IAA production was determined mixing 2 ml of each sample (previously spun at 13000 g x 10 minutes and filtered with 0,2 m filters) with 4 ml of 0 incubated in the dark at room temperature for 30 minutes. Finally, each sample has been analyzed by a spectrophotometer reading at 530 nm and value compared with IAA standard curve to record ppm of lAA.The results are summarized Fig. 7 and show that VMC 30/196 works better that the control used (ppm=24,88 vs 19,04 ppm). Culture broth not inoculated as negative control.
Nitrogen fixation
The nitrogen fixation capacity of the strains of the present invention have been assessed according to Okon et al. (1977) with minor changes. In particular, the
bacteria were inoculated using an aliquot (10 μI) of each bacterial suspension, with a concentration of 106 CFU/ml in a medium made up of in 1 I: D-L malic acid 5 g, KOH 4 g, K2HPO4 0.5 g, MgSO40.2 g, NaCI 0.1 g, CaCl2 0.02 g, bromotymol blu (BTB) (0.5% dissolved in 0.2 N KOH) 2 ml, trace element solution 1 ml (NaMo04 200 mg, MnSO4 253 mg, H3Bo3 280 mg, CuSO4 8 mg, ZnSO4 24 mg in a final volume of 200 ml of distilled water), Fe EDTA (EDTA 22.8 g/l, 250 ml of KOH 1 N, FeCI3 10 g) 4 ml, agar 15 g. The final pH was adjusted to 6.8. Fe EDTA solution was sterilized by filtration with 0.2 μm filters. Fe EDTA, BTB and the trace solution were added to the medium after cooling. After 24 hours of incubation at 30°C, the blue colored zone producing isolates were marked as nitrogen fixers in the solid culture conditions. The coloring zone was calculated by deducting the colony diameter from the coloring zone diameter, according to Gothwal et al. (2008). Bacterial strains have been tested as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension.
Results are summarized in Fig 8A and B and show that both strains are able of nitrogen fixation. In particular, VMC30/195 is the best performer working even better than the two control strains (34 mm vs 21 and 20.33 mm). VMC30/196 works too but with lower performance (18 mm of diameter). The consortium shows good performance too.
Plant growth promotion activity on Lettuce plants The plant growth promotion activity of the strains was tested in combination with a plant biostimulant (PBs). In particular, in vivo bioassays have been done on Lactuca sativa cv. Canasta plants. Canasta plantlets were obtained from a local nursery. Two-week-old plantlets were transplanted in 17 cm diameter plastic pots, 2 Liters volume each. 4 replications of eight pots/treatment were prepared, on a sandy substrate, in a glasshouse. The environmental conditions during the experimental period were 22-33°C, with a relative humidity ranging from 70-80%, under natural light conditions. Nutrients were directly added to the substrate during the crop cycle, by providing 40 Kg/ha of water-soluble nutrient mix containing NPK (13-40-13), every 10 days. The density was 10 plants/m2. T reatments conditions were:
• Untreated Control (UTC)
• VMC 30/195+VMC 30/196: two applications every 10 days at 5 Kg/ha rate;
• PBS: two applications every 10 days at 5 Kg/ha rate;
• VMC 30/195+ VMC 30/196+ PBS: two applications every 10 days at 5 Kg/ha rate.
The irrigation was carried out considering the substrate moisture content and the amount of water was determined to maintain the 80% of substrate water availability. Lettuce plants were harvested when the plants reached the commercial maturity stage, after 40 days of cultivation. Plant fresh weight (g) was determined at harvest. Data were subjected to two-way ANOVA and SNK comparison test was used for evaluating the differences among means at p < 0,05. Different letters indicate statistical differences for p < 0,05. Foliar Zinc, Nitrogen, Phosphorous and Iron were assessed, only for UTC and complete prototype (VMC30/195+VMC30/196+PBS), at the end of the cycle by leaf analysis performed in a dedicated local laboratory.
The results show that VMC30/195+VMC30/196 promote plant growth and this activity is more enhanced in the presence of a plant biostimulant.
Mode of Action
To investigate the overall molecular effect of a composition comprising the 2 strains of the invention and PBS compared to plants treated only with PBS, a microarray- based transcriptomic analysis has been performed.
The samples tested are as follow:
Arabidopsis thaliana (Ecotype Col-0) was used in this experiment. The seeds were washed and rehydrated with water for 15 minutes before sowing, to facilitate the germination. After washing, single seed was taken with tweezers and sown on each single well (50/50 of earth and perlite) of the "Arasystem trays". After that, the trays were transferred into a growth chamber with controlled conditions to allow optimal
plant growth. The microarray analysis (SureScan, Agilent system) has been performed after 24h (early effect) and 72h (mid effect) from the application on 15- days old A. thaliana seedlings.
GO-Term enrichment was performed for interpreting sets of genes making use of the Gene Ontology system classification, in which genes are assigned to a set of predefined bins depending on their functional characteristics and cut-off selected (0,01).
The results show that the following GO-Groups are shared between PBS and the prototype: carbohydrates, transport, and response to starvation; these are most probably modulated by the PBS.
The GO-Groups correlated to the microbial component (the 2 strains of the invention) instead are the following: biosynthetic process, biotic response, hormone response and metabolic process.
Effect on Sweet Pepper
The plant growth promotion activity of the strains was tested in combination with PBS. In particular, greenhouse experiment has been done on sweet pepper plants using sub-optimal nutrition.
Sweet pepper plantlets were transplanted in 17 cm diameter plastic pots, 2 Liters volume each. 4 replications of eight pots/treatment (7 treatments x 12 blocks) were prepared, on a sandy substrate, in a glasshouse. The environmental conditions during the experimental period were 22-33°C, with a relative humidity ranging from 70-80%, under natural light conditions. Nutrients were directly added to the substrate during the crop cycle.
Treatments conditions were:
• Untreated Control (UTC)
• Prototype: three applications every 7 days at 5 Kg/ha rate, first application after transplant at 14 days.
• PBS: three applications every 7 days at 5 Kg/ha rate, first application after transplant at 14 days.
• Prototype: three applications every 7 days at 10 Kg/ha rate, first application after transplant at 14 days
• PBS: three applications every 7 days at 10 Kg/ha rate, first application after transplant at 14 days
• Internal Valagro control: three applications every 7 days at 10 Lt/ha rate, first application after transplant at 14 days
Sweet pepper plants were harvested when the plants reached the commercial maturity stage. Plant biomass (g) and fruits (total weight in grams) was determined at harvest. Data were subjected to Duncan’s test and SNK comparison test was used for evaluating the differences among means at p < 0,05 and p<0, 10. Different letters indicate statistical differences for p < 0,05 and p<0,10.
Plant Biomass showed statistical significance (SNK and Duncan’s test p<0,05 and p<0, 10) for Prototype (5-10 kg/ha) vs UTC;
For Fruits weight, plants treated with product Prototype (5-10 kg/ha) produced more fruits, with a higher weight (g) in comparison with the UTC. Statistical significance (SNK and Duncan’s test p<0,05 and p<0, 10).
Field Trials
The prototype was assessed on Melon and Tomato crops under open field conditions in Rabi season at Maharashtra, India. The experiment was carried out using Split plot design under two different approaches, a] Reduction of Phosphorous content i.e- 30% reduction b] Remobilization of Phosphorous i.e no available
Phosphorous. In both the crops and in both the approaches, untreated control (UTC) was used.
In both the crops, first dose of application was done after 5 days of transplantation of seedlings with second dose after 20 days of planting and 3rd dose after 40 days of planting. Soil samples were collected from all the plots before and after application treatments i.e @ 0, 1 , 30 and 60 days after application to estimate the total viable count of microbial load.
Statistical analysis: LSD (a 0.05)
Under the conditions of standard NPK fertilizer application, the effect of the prototype on yield (mt/ha) was increased significantly in case of Tomato crops in 2 different experiments, whereas, in Melon significant difference in yield was observed in treated plot compared to UTC in one experiment. Moreover, regarding 2 different strategies tested, yield increase was significant in case of Tomato in both the strategies, indicating that prototype is effective when P is reduced to 30% or reduced completely.
Effect of prototype was significantly high in both the crops considering fruit weight in Melon and number of fruits in Tomato.
Moreover, it is clear from the results below that the 2 strains of bacteria present in prototype survived and maintained stable viable cells in all the treated plots after the application of the prototype.
Claims
1. A consortium of bacteria comprising at least one bacterial strain belonging to the genus Bacillus species pumilus characterized by a DNA of the 16S rRNA gene coding comprising SEQ ID NO: 1 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 1 and at least one bacterial strain belonging to the genus Bacillus species megaterium characterized by a DNA of the 16S rRNA gene coding comprising SEQ ID NO: 2 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 2.
2. A medium and/or an extract, preferably bacteria-free extract, or a supernatant or a lysate or a fraction or a metabolite wherein said medium or extract or supernatant or lysate or fraction or metabolite is obtained/obtainable by culturing at least one bacterial strain wherein said bacterial strain belongs: to the genus Bacillus species pumilus and is characterized by a DNA of the 16S rRNA gene coding comprising SEQ ID NO: 1 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 1 and/or to the genus Bacillus species megaterium and is characterized by a DNA of the 16S rRNA gene coding comprising SEQ ID NO: 2 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 2.
3. The consortium according to claim 1 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to claim 2, wherein the bacterial strain Bacillus pumilus has been deposited at the NCMR with IDA accession number MCC0195 under denomination Bacillus pumilus strain VMC30/195 and wherein the bacterial strain Bacillus megaterium has been deposited at the NCMR with IDA accession number MCC0194 under the denomination Bacillus megaterium strain VMC30/196.
4. The consortium of claim 1 or 3 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to claim 2 or 3, wherein said bacterial strain is in a form selected from: fresh bacteria, frozen bacteria, dry bacteria, lyophilized bacteria, liquid suspension of bacteria, encapsulated bacteria in the form of spores, living bacteria, culture medium, preferably whole culture medium comprising the bacteria or bacteria free medium, extract of bacteria, preferably cell free extract, supernatant, lysate of bacteria, fraction of bacteria, metabolites derived from said bacteria and any
combination thereof.
5. The consortium according to anyone of claims 1 , 3-4 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-4, wherein said bacterial strain is mutated and/or edited.
6. The consortium according to anyone of claims 1 , 3-5 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-5 in combination with further microorganisms, preferably selected from: bacteria, preferably PGPR or rhizobacteria, yeasts, mycorrhizae, fungi, any derivatives as disclosed above and any combination thereof.
7. The consortium according to anyone of claims 1 , 3-6 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-6 wherein: the PGPR is selected from; Aeromonas rivuli, Agromyces fucosus, Bacillus spp. Bacillus mycoides, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Bacillus safensis, Microbacterium sp., Nocard i a globerula, Stenotrophomonas spp., Pseudomonas spp, Pseudomonas fluorescens, Pseudomonas fulva, Pseudoxanthomonas dajeonensis, Rhodococcus coprophilus, Sphingopyxis macrogoltabida, Streptomyces spp., Enterobacter spp., Azotobacter spp., Azospiriullum spp., Rhizobium spp., Herbaspirillum spp., Lactobaccillus spp., Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactococcus tactis, and combinations thereof; and/or the yeast is selected from: Candida spp., Candida tropicalis, Saccharomyces spp., Saccharomyces bay anus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces exiguous, Saccharomyces pastorianus, Saccharomyces pombe, and combinations thereof, and/or the mycorrhiza is selected from: Glomus spp., Rhizophagus spp., Septoglomus spp., Funneliformis spp., and combinations thereof.; and/or the fungus is selected from: Trichoderma spp., Trichoderma atroviride, Trichoderma viride, Trichoderma afroharzianum, Paecilomyces spp., Beauveria
bassiana., Metarhizium spp., Lecanicillium lecanii, Penici Ilium spp., Aspergillus spp., Conythyrium minitans, Pythium spp, and combinations thereof.
8. The consortium according to anyone of claims 1 , 3-7 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-7, wherein said strain or strains is/are living and/or dead and/or killed cells and/or as spores or as a fresh or frozen sample or dry, lyophilized or in liquid suspension, or encapsulated in the form of living and/or dead and/or killed cells and/or as spores.
9. The consortium according to anyone of claims 1 , 3-8 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-8 in combination with a plant biostimulant, preferably said plant biostimulant comprising at least one of the following ingredients: an extract of algae, and/or an extract of microalgae and/or an extract of plant and/or a humic acid and/or a fulvic acid and/or plant and/or animal byproducts.
10. The consortium or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to claim 9, wherein the concentration of the extract from algae and/or microalgae and/or plant in the biostimulant preferably ranges from 1 to 60%, more preferably it ranges from 5 to 50%, still more preferably from 10 to 20%, still more preferably around 15%; and/or the concentration of the humic acid in the biostimulant ranges from 1 to 20%; and/or the concentration of the fulvic acid in the biostimulant ranges from 1 to 20%, preferably from 5 to 10%.
11. The consortium or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to claim 9 or 10, wherein said algae are brown algae, preferably seaweeds, more preferably said algae are selected from: Ascophyllum nodosum, Ecklonia maxima, Laminaria saccharina, Laminaria digitata, Fucus spiralis, Fucus serratus, F. vesicuiosus, Macrocystis spp., Pelvetia canaliculata, Himantalia elongata, Undaria pinnatifida, Sargassum spp, and combinations thereof; and/or said microalgae are selected from: Spirulina, Scenedesmus, Nannochloropsis, Haematococcus, Chlorella, Phaeodactylum, Arthrospyra, Tetraselmis, Isochrysis, Synechocystis, Clamydomonas, Parietochloris, Desmodesmus, Neochloris, Dunaliella,
Thalassiosira, Pavlova, Navicula, Chaetocerous, and combinations thereof; and/or said plant is selected from: beet, sugar cane, alfalfa, maize, brassica, halophytes, soya, wheat, yucca, quillaja, hop, coffee, citrus, olive, and combinations thereof.
12. The consortium according to anyone of claims 1 , 3-11 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-11 in combination with a non-microbial component said non-microbial component preferably comprising a plant biostimulant component, preferably the plant biostimulant according to anyone of claim 9-11 , wherein said non microbial component is present in a concentration up to 99,99%, 99,95 %, 99,90%, 99%, 95%, 90%, 85%, or 80%, and/or the bacteria are used in a concentration up to 20%, 15%, 10%, 5%, 1%, 0,1% 0.05% or 0.01 and wherein said plant biostimulant represents 1-60%, preferably 5-50%, more preferably 10-20%, still more preferably around 15% of the non-microbial component percentage.
13. A composition, preferably an agricultural composition comprising the consortium according to anyone of claims 1 , 3-12 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-12 and a carrier, preferably an agricultural compatible carrier.
14. The consortium according to anyone of claims 1 , 3-12 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-12 or the composition according to claim 13 formulated as water-soluble concentrates, dispersable concentrates, emulsifiable concentrates, emulsions, suspensions, microemulsion, gel, microcapsules, granules, ultralow volume liquid, wetting powder, dustable powder, or seed coating formulations.
15. Use of the consortium according to anyone of claims 1 , 3-12 and 14 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-12 and 14 or the composition according to claim 13 or 14 in agriculture, preferably as biostimulant for plants, to enhance/benefit in plants nutrient uptake, preferably nutrient uptake from the soil, and/or nutrient efficiency, and/or tolerance to abiotic stress, and/or crop quality and/or plant growth and/or plant development.
16. Use according to claim 15, wherein said nutrients are macro- micronutrients, preferably selected from: nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, boron, copper, iron, manganese, molybdenum, zinc and combination thereof.
17. Use according to claim 15 and 16, wherein said plant is_selected from: Solanaceae, Cucurbitaceae, Graminaceae (Poaceae), Pomaceae, Chenopodiaceae, Brassicaeae, Compositae, Liliaceae, Leguminosae, Rosaceae, Vitaceae, Rutaceae, Oleaceae, Moraceae, Malvaceae, Musaceae, Lauraceae, Anacardiaceae, Juglandaceae, Zingiberaceae, Labiateae, Piperaceae, Cannabaceae, Arecaceae, Punicaceae, Bromeliaceae, Rubiaceae, Theaceae, Caricaceae, Passifloraceae, Asteraceae, Actinidiaceae, Fagaceae, Fabaceae, Ginkoaceae, Simondsiaceae and combinations thereof, preferably said plant is selected from: tomato, melon, eggplant, pepper, cucumber, zucchini, potato, cauliflower, onion, lettuce, spinach, cabbage, savaoy cabbage, peach, apricot, plum, apple, pear, strawberry, grapes, cotton, almonds, various ornamentals, sunflower, canola, bean, pea, flax, safflower, buckwheat, sugar beets, sugar cane, corn wheat, barley, soybean, and combinations thereof
18. Use of the consortium according to anyone of claims 1 , 3-12 and 14 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-12 and 14 or the composition according to claim 13 or 14 in agriculture as source of IAA.
19. Method for enhancing/benefiting in plants nutrient uptake, preferably nutrient uptake from the soil, and/or nutrient efficiency, and/or tolerance to abiotic stress, and/or crop quality and/or plant growth and/or plant development, and/or for increasing the availability, preferably in the soil, of nutrients and/or macro- micronutrients, and/or for improving plant growth and/or plant yield wherein preferably selected from: nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, boron, copper, iron, manganese, molybdenum, zinc and combination thereof, said method comprising at least one step of applying to plants in need thereof (target plants) the consortium according to anyone of claims 1 , 3-12 and 14 or the medium and/or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite according to anyone of claims 2 or 3-12 and 14 or the composition according to claim 13 or 14.
20. Method according to claim 19, wherein the strain or the consortium or the strain
derivatives or the composition is applied into the soil, preferably through fertigation or irrigation systems, at a rate ranging between 100g-10.000 g/hectare, preferably between 2 and 10kg/hectare and preferably up to 5 times/crop cycle, wherein the application is performed all over the crop cycle, preferably during the early stages of crop development.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN202041032728 | 2020-07-30 | ||
PCT/IB2021/056948 WO2022024050A1 (en) | 2020-07-30 | 2021-07-30 | Microbial strains bacillus pumilus, bacillus megaterium and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4188894A1 true EP4188894A1 (en) | 2023-06-07 |
Family
ID=80036175
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP21850718.4A Pending EP4188894A1 (en) | 2020-07-30 | 2021-07-30 | Microbial strains bacillus pumilus, bacillus megaterium and uses thereof |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP4188894A1 (en) |
WO (1) | WO2022024050A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110272848B (en) * | 2019-07-10 | 2023-01-03 | 西北农林科技大学 | Rhizobium J16 with potassium-dissolving effect and application thereof |
FR3132614A1 (en) * | 2022-02-15 | 2023-08-18 | Her Majesty Gyalum Kesang Choeden Wangchuck’s Trust Fund | BLEND OF BACTERIA LYSAT AND METABOLITES ISOLATED FROM CURCUMA LONGA FROM BHUTAN AND TO INCREASE YIELD AND ANTI-INFLAMMATORY PROPERTIES IN AGRICULTURAL PRODUCTION. |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110637082B (en) * | 2017-01-04 | 2024-04-05 | 诺维信生物农业公司 | Bacillus isolates and uses thereof |
MX2017014720A (en) * | 2017-11-16 | 2019-05-17 | Jose Alfredo Suarez Rivero | Microbial consortium for agricultural use and formulation containing same. |
-
2021
- 2021-07-30 WO PCT/IB2021/056948 patent/WO2022024050A1/en active Application Filing
- 2021-07-30 EP EP21850718.4A patent/EP4188894A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2022024050A1 (en) | 2022-02-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111954712B (en) | Novel Paenibacillus polymyxa and application thereof | |
Sivasakthi et al. | Biocontrol potentiality of plant growth promoting bacteria (PGPR)-Pseudomonas fluorescens and Bacillus subtilis: A review | |
EP3105198B1 (en) | Soil bacteria for inoculating stress soils | |
Arzanesh et al. | Wheat (Triticum aestivum L.) growth enhancement by Azospirillum sp. under drought stress | |
Turan et al. | Effects of Bacillus FS-3 on growth of tomato (Lycopersicon esculentum L.) plants and availability of phosphorus in soil | |
Nakayan et al. | Phosphate-solubilizing soil yeast Meyerozyma guilliermondii CC1 improves maize (Zea mays L.) productivity and minimizes requisite chemical fertilization | |
Islam et al. | Plant probiotics in phosphorus nutrition in crops, with special reference to rice | |
Mehta et al. | Tricalcium phosphate solubilisation by new endophyte Bacillus methylotrophicus CKAM isolated from apple root endosphere and its plant growth-promoting activities | |
WO2019155252A1 (en) | Novel bacillus sp. simplex and uses thereof | |
Jamal et al. | Effect of plant growth-promoting bacteria Bacillus amylliquefaciens Y1 on soil properties, pepper seedling growth, rhizosphere bacterial flora and soil enzymes | |
Hegazi et al. | Influence of different cyanobacterial application methods on growth and seed production of common bean under various levels of mineral nitrogen fertilization | |
MX2014012524A (en) | Use of synergistic microorganisms and nutrients to produce signals that facilitate the germination and plant root colonization of mycorrhizal fungi in phosphorus rich environments. | |
Kumawat et al. | Co-inoculation of indigenous Pseudomonas oryzihabitans and Bradyrhizobium sp. modulates the growth, symbiotic efficacy, nutrient acquisition, and grain yield of soybean | |
HU230555B1 (en) | Environment-friend micro-organism produce and producing thereof | |
Egamberdiyeva et al. | Improvement of wheat and cotton growth and nutrient uptake by phosphate solubilizing bacteria | |
US10000427B2 (en) | Phosphate solubilizing rhizobacteria bacillus firmus as biofertilizer to increase canola yield | |
EP4188894A1 (en) | Microbial strains bacillus pumilus, bacillus megaterium and uses thereof | |
Fürnkranz et al. | Characterization of plant growth promoting bacteria from crops in Bolivia/Charakterisierung pflanzenwachstumsfördernder Bakterien von Kulturpflanzen in Bolivien | |
Horácio et al. | Co-inoculation of rhizobia, azospirilla and cyanobacteria for increasing common bean production | |
Constantino et al. | Effect of inoculation with rhizobacteria and arbuscular mycorrhizal fungi on growth and yield of Capsicum chinense Jacquin | |
Batra et al. | Endophytes: An environmental friendly bacteria for plant growth promotion | |
Dhiman et al. | Isolation and characterization of Rhizobium associated with root nodules of Dalbergia sissoo | |
Mofokeng et al. | Integrating biostimulants in agrosystem to promote soil health and plant growth | |
El-Aal et al. | Impact of PGPR and inorganic fertilization on growth and productivity of sweet ananas melon. | |
Egamberdiyeva et al. | Promotion of plant growth of maize by plant growth promoting bacteria in different temperatures and soils |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20230228 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
RAP3 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: VALAGRO BIOSCIENCES PRIVATE LIMITED |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) |