WO2022191689A1 - Endophytic bacterial strains, probiotic mixtures, formulation and method for stimulating plant seed germination - Google Patents
Endophytic bacterial strains, probiotic mixtures, formulation and method for stimulating plant seed germination Download PDFInfo
- Publication number
- WO2022191689A1 WO2022191689A1 PCT/MX2021/000010 MX2021000010W WO2022191689A1 WO 2022191689 A1 WO2022191689 A1 WO 2022191689A1 MX 2021000010 W MX2021000010 W MX 2021000010W WO 2022191689 A1 WO2022191689 A1 WO 2022191689A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- plants
- formulation
- cnrg
- bacterial strains
- plant seeds
- Prior art date
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 61
- 239000000203 mixture Substances 0.000 title claims abstract description 55
- 238000009472 formulation Methods 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 19
- 230000004936 stimulating effect Effects 0.000 title claims abstract description 11
- 239000006041 probiotic Substances 0.000 title claims abstract description 9
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 9
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 9
- 230000007226 seed germination Effects 0.000 title abstract description 5
- 241000196324 Embryophyta Species 0.000 claims abstract description 57
- 240000008042 Zea mays Species 0.000 claims abstract description 41
- 230000035784 germination Effects 0.000 claims abstract description 37
- 241000588696 Pantoea ananatis Species 0.000 claims abstract description 7
- 241000881810 Enterobacter asburiae Species 0.000 claims abstract description 6
- 241001245440 Enterobacter kobei Species 0.000 claims abstract description 6
- 235000013406 prebiotics Nutrition 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract description 5
- 235000013311 vegetables Nutrition 0.000 claims description 20
- 235000007244 Zea mays Nutrition 0.000 claims description 12
- 241000209149 Zea Species 0.000 claims description 9
- 241000209504 Poaceae Species 0.000 claims description 8
- 241000588914 Enterobacter Species 0.000 claims description 2
- 238000011282 treatment Methods 0.000 description 25
- 238000012360 testing method Methods 0.000 description 21
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 9
- 235000005822 corn Nutrition 0.000 description 9
- 230000009418 agronomic effect Effects 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 230000008635 plant growth Effects 0.000 description 8
- 229910019142 PO4 Inorganic materials 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 7
- 235000021317 phosphate Nutrition 0.000 description 7
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 6
- 235000009973 maize Nutrition 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000722363 Piper Species 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 108091000130 1-aminocyclopropane-1-carboxylate deaminase Proteins 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- 229930192334 Auxin Natural products 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 239000002363 auxin Substances 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 238000005063 solubilization Methods 0.000 description 4
- 230000007928 solubilization Effects 0.000 description 4
- 241001057636 Dracaena deremensis Species 0.000 description 3
- 241000520272 Pantoea Species 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 229930195732 phytohormone Natural products 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 244000291564 Allium cepa Species 0.000 description 2
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 235000002566 Capsicum Nutrition 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 241001393805 Pantoea ananatis B1-9 Species 0.000 description 2
- 239000006002 Pepper Substances 0.000 description 2
- 235000016761 Piper aduncum Nutrition 0.000 description 2
- 235000017804 Piper guineense Nutrition 0.000 description 2
- 235000008184 Piper nigrum Nutrition 0.000 description 2
- 239000003905 agrochemical Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 239000003617 indole-3-acetic acid Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 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
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 240000004160 Capsicum annuum Species 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
- 108010059892 Cellulase Proteins 0.000 description 1
- 241001214984 Crinum thaianum Species 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 241000147019 Enterobacter sp. Species 0.000 description 1
- 102000012330 Integrases Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 244000010815 Phlomis lychnitis Species 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 102000018819 Protein Translocation Systems Human genes 0.000 description 1
- 108010052646 Protein Translocation Systems Proteins 0.000 description 1
- 239000000589 Siderophore Substances 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 102000008579 Transposases Human genes 0.000 description 1
- 108010020764 Transposases Proteins 0.000 description 1
- 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 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000000003 effect on germination Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical class [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
- 235000021109 kimchi Nutrition 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000021749 root development Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 238000004162 soil erosion Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Definitions
- the present invention is related to the technical field of Biotechnology and Agriculture, because it provides endophytic bacterial strains from the corn plant (Zea mays L.); probiotic mixtures comprising endophytic strains; formulations and a method to stimulate the germination of vegetable seeds, through the use of said formulation, which may contain the bacterial strains.
- microorganisms as biofertilizers has emerged as an alternative, not only friendly to the environment, but also as an economic alternative to increase crop yield, as shown by the following scientific publications.
- the isolate grew well in the presence of 3% NaCl and 10 pg of streptomycin.
- the isolate promoted maize and rice seed germination as well as root growth, resulting in increased seedling weight over controls.
- Experiments to quantify the ability of the isolate to promote plant growth were performed using hydroponic solutions and, as appropriate, an inoculum of the isolate. Experiments in pots were also used. The results of these studies showed that the isolation significantly (P ⁇ 0.05) increased Nitrogen accumulation and improved seedling growth over controls.
- the isolate has potential for use as an inoculum for sustainable cereal production.
- NCOO1927-MR5 a strain of Enterobacter asburiae called NCOO1927-MR5
- NCOO1927-MR5 a strain of Enterobacter asburiae
- the tests to demonstrate its agronomic efficiency were carried out in the laboratory and in pots, that is, under controlled conditions; but it must be considered that it is the field conditions that have the real interaction of all the factors that a seed faces when it is put to germinate.
- the functional characterization study demonstrated the ability of B1-9 to function in phosphate solubilization, sulfur oxidation, nitrogen fixation, and indole-3-acetic acid production.
- B1-9 To track the colonization patterns of B1-9 in pepper plant tissues, they used the fluorescent dye DRAQ5TM, which stains the DNA of bacteria and plant cells. A large number of B1-9 bacteria were found on root and stem surfaces as well as in guard cells. In addition, several colonized B1-9 bacteria resided in inner cortical plant cells. Treatment of rhizosphere regions with strain B1-9 can result in efficient plant colonization and promote plant growth. from seedling to mature plant stage.
- the B1-9 strain can be successfully applied in pepper plantations due to its high colonization capacity in plant tissues, as well as its properties that promote efficient plant growth.
- this article describes the use of a bacterial strain of Pantoea annanatis isolated from the rhizosphere of onion plants to promote the growth of pepper plants.
- Figure 1 is a photograph illustrating a compatibility test between the three isolated and selected bacterial strains.
- Figure 2 is a graph illustrating the germination percentages obtained from the different bacterial treatments tested.
- Figure 3 is a graph of the average root length of the germinated seeds of the different bacterial treatments.
- Figure 4 is a graph showing the average seedling height in the different bacterial treatments.
- the object of the present invention is an isolated strain of Pantoea ananatis, which has the characteristics of the strain deposited under accession number CM-CNRG BT170.
- the subject of the invention is an isolated strain of Enterobacter kobei, which has the characteristics of the strain deposited with accession CM-CNRG BT172.
- This invention also has as its object an isolated strain of Enterobacter asbur ⁇ ae, whose characteristics are the same as the strain deposited with the accession number CM-CNRG BT173.
- CM-CNRG BT170, CM-CNRG BT172 and CM-CNRG BT173 are endophytes of Zea mays L.
- the plant seeds are from the Poaceae family; but in a more preferred embodiment when said plant seeds are of the Zea genus; although in a more preferred embodiment when such vegetable seeds are of the species Zea mays L.
- a further object of the present invention is a probiotic mixture of isolated bacteria, comprising at least two isolated bacterial strains, in accordance with the isolated bacterial strains described in the present invention.
- the bacterial strains exhibit stimulating activity on the germination of vegetable seeds; which are from the Poaceae family, or preferably from the Zea genus, or more preferably from the Zea mays L species, to name a few examples.
- One more embodiment of the probiotic mixture of the present invention is when the isolated bacterial strains are: the isolated strain of Enterobacter kobei CM-CNRG BT172, and the isolated strain of Enterobacter asburiae CM-CNRG BT173 in accordance with the present invention; and a preferred embodiment is when said strains are in a 2:1 ratio.
- the probiotic mixture of the present invention may have, is to prepare a formulation to stimulate the germination of vegetable seeds.
- one more object of the present invention is a formulation to stimulate the germination of vegetable seeds, which contains: i) at least one isolated bacterial strain, which is selected from the following group: CM-CNRG BT170, CM -CNRG BT172, CM-CNRG BT173, and their mixtures between them; ii) at least one prebiotic substance; and iii) at least one excipient.
- One embodiment of the formulation according to the present invention is when the isolated bacterial strain, either individually or mixed, is in an amount of 1%, the prebiotic substance in 1.5%, and the excipient in 97.5%, with respect to the total volume of the formulation.
- a preferred embodiment of the formulation of the present invention is when it comprises a mixture of the bacterial strains CM-CNRG BT172 and CM-CNRG BT173; and the most preferred embodiment is when said bacterial strains are in a ratio of 2:1.
- the plant seeds that can be stimulated to germinate with the formulation proposed by this invention can be seeds of plants of the Poaceae family; preferring seeds of the genus Zea; preferring even more seeds of the species Zea mays L.
- a method to stimulate the germination of vegetable seeds is also another object of the present invention, which comprises applying to vegetable seeds prior to their germination, a sufficient amount of a formulation to stimulate the germination of vegetable seeds, in accordance with the formulation described in this invention.
- a variant of the method to stimulate the germination of vegetable seeds, according to the present invention is when the sufficient quantity of the formulation is 1 L per 80,000 vegetable seeds.
- One more variant of the method in question is when the application of the formulation is at the moment of putting the vegetable seeds to germinate.
- said method is when the germination of seeds of plants of the Poaceae family is stimulated; being a preferred modality when the germination of seeds of plants of the genus Zea is stimulated; being a more preferred modality when the germination of seeds of plants of the species Zea mays L.
- Example 1 Isolation of endophytic bacterial strains from maize plants (Zea mays L.). Complete maize plants (Zea mays L.) with commercial maturity age from V4 to R6, were collected from 2 plots of maize cultivated conventionally with agrochemical products, located in the towns of Santa Maria and San Francisco de As ⁇ s, municipality of Atotonilco el Alto, Jalisco, Mexico; and from a plot of corn cultivated with organic products, located in the town of El Refugio, municipality of Tototlán, Jalisco, Mexico.
- a random sampling was carried out taking 10 complete maize plants, for each plot, of healthy appearance, trying to damage the root as little as possible. This collection was carried out at different strategic points of the plots, in order to cover the totality of its extensions in order to obtain representative samples.
- the plants were transported in a plastic bag to avoid possible damage to them. Once they arrived at the laboratory, they were rinsed with running water and soap to remove excess soil and allowed to drain until the water was eliminated. The leaves of the plants were sectioned into parts of 5 x 10 cm, and 100 g of seeds were taken from the cobs to subsequently follow the endophyte extraction protocol reported in the literature, which consisted of washing at 3% for 10 min.
- Example 2 Agronomic tests to which the endophytic bacterial strains of corn plants were subjected.
- the 42 bacterial strains isolated from corn in the previous example were subjected to the following agronomic tests: nitrogen fixation test (NFB), phosphate solubilization test (NBRIP Ca, NBRIP Al and NBRIP Fe), iron chelating test ( siderofbros), phytohormone (auxin) production test and enzyme acc deaminase (ACC) production test, as described by Dworkin (2006); Delvasto et al., (2006), Milagros et al. (1999); Gordon and Weber (1950); and Penrose and Glick (2003), respectively.
- NFB nitrogen fixation test
- NBRIP Ca phosphate solubilization test
- NBRIP Ca phosphate solubilization test
- siderofbros iron chelating test
- auxin phytohormone
- ACC enzyme acc deaminase
- phosphate solubilization For phosphate solubilization (NBRIP), the halo generated around the colony inoculated in the medium was measured, where bacterial strains M1 and M19 generated a 0.1 cm halo in the solubilization of Ca phosphates, while strain M20 managed to solubilize Ca phosphates but not enough to get an accurate measurement. With the aluminum and iron phosphates, none of the isolated strains managed to solubilize these phosphates. In the case of slderophores, strains M19 and M20 were clearly positive in the slderophore test, while M1 was only slightly positive.
- the strain that produced the greatest amount of this phytohormone was the M1 strain, obtaining 3 crosses out of 3, while the M19 and M20 strains obtained a production of 2 out of 3 crosses, that is, they produced it moderately.
- the production of the ACC deaminase enzyme as we observe in table 1, the M1 strain does not produced said enzyme by not being able to grow in the culture medium; while strains M19 and M20 produced it in low quantities (+).
- Example 3 Identification of the 3 endophytic bacterial strains of selected Zea mays L. plants.
- Bacterial identification was carried out using the MALDI-TOF technique, which consists of obtaining a protein profile of the microorganism to be analyzed and making a comparison with a database, resulting in a score, which corresponds to how successful the identification is. ID.
- the score ranges from 0 to 3, where a score of 0-1,599 indicates an unreliable Identity, a score of 1,600-1,999 indicates that there is a high probability that the identified gender is correct, a score of 2,000-2,299 is indicative that the identity of the genus is 100% certain, but the identity of the species has a certain probability that it is, and finally, a score of 2,300-3,000 is 100% certain that the identity of the genus and species do correspond.
- the identity results of the bacterial strains are shown in Table 2.
- Table 2 Identification by MALDI-TOF of the 3 endophytic bacterial strains of selected Zea mays L. plants.
- Example 4 Compatibility test between the endophytic bacterial strains of selected Zea may L. plants.
- the compatibility tests were carried out using the sandwich technique, where a bacterium is planted in the form of a lawn in a box of Casoy agar and a Casoy agar wafer is placed on top of the inoculated bacterium, where the rest of the bacteria are inoculated. try in the form of spots. If the bacteria on top of the agar wafer grow and grow, it is considered to be compatible with the bacteria on the bottom of the wafer. All this was done in triplicate.
- Example 5 Germination tests of Zea mays L. seeds treated with the 3 endophytic bacterial strains of selected Zea mays L. plants.
- corn seeds used were seeds that did not have any antifungal treatment, and care was taken that they were not broken or without the embryo, to ensure that the seeds were viable for germination.
- the seeds were submerged in falcon tubes containing a formulation to stimulate germination, which contains: the isolated bacterial strains, either individual and/or combined; and a prebiotic substance based on nitrogen and carbon sources.
- the seeds were submerged for 1 h in the formulation that stimulates germination; Later, with the help of tweezers, they were removed and placed in a substrate composed of soil, peatmoos and jal, previously prepared in a germination tray, and kept in the dark at room temperature, irrigated with 1mL of drinking water daily.
- the isolated bacterial strains M1, M19 and M20 have agronomic potential, they were deposited on October 19, 2020, in the Collection of Microorganisms of the National Center for Genetic Resources that belongs to the National Institute of Forestry, Agricultural and Agricultural Research. Livestock, with registration number before the World Federation of Culture Collection 1006 (CM-CNRG) and International Depositary Authority with notification 308 for patent procedure purposes in accordance with the Budapest Treaty; and who has his address at Boulevard de la Biodiversidad 400, CP 47600. Tepatitlán de Morelos, Jalisco, Mexico.
- the bacterial strain M1 Pantoea ananatis was assigned the deposit number CM-CNRG BT170
- the M19 Enterobacter kobei strain was assigned accession No. CM-CNRG BT172
- the M20 Enterobacter asburiae strain was assigned accession No. CM-CNRG BT173 .
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pretreatment Of Seeds And Plants (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Endophytic isolates from Zea mays L. plants: Pantoea ananatis, Enterobacter kobei, Enterobacter asburiae, which have the features of the strains deposited under accession numbers CM-CNRG BT170, CM-CNRG BT172, and CM-CNRG BT173, respectively. Probiotic mixtures, comprising at least two isolated bacterial strains, in accordance with the present invention. Formulation for stimulating plant seed germination, which contains: at least one isolated bacterial strain selected from tamong CM-CNRG BT170, CM-CNRG BT172, CM-CNRG BT173 and combinations thereof, at least one prebiotic substance; and at least one excipient. A method for stimulating plant seed germination, which comprises applying to plant seeds, prior to their germination, a sufficient quantity of a formulation for stimulating plant seed germination.
Description
CEPAS BACTERIANAS ENDÓFITAS, MEZCLAS PROBIÓTICAS, FORMULACIÓN Y MÉTODO, PARA ESTIMULAR GERMINACIÓN EN SEMILLAS VEGETALES ENDOPHYTIC BACTERIAL STRAINS, PROBIOTIC MIXTURES, FORMULATION AND METHOD, TO STIMULATE GERMINATION IN VEGETABLE SEEDS
CAMPO TÉCNICO DE LA INVENCIÓN TECHNICAL FIELD OF THE INVENTION
La presente invención se relaciona con el campo técnico de la Biotecnología y Agricultura, debido a que proporciona cepas bacterianas endófitas de planta de maíz (Zea mays L.); mezclas probióticas que comprenden a las cepas endófitas; formulaciones y un método para estimular la germinación de semillas vegetales, mediante el uso de dicha formulación, la cual puede contener a las cepas bacterianas. The present invention is related to the technical field of Biotechnology and Agriculture, because it provides endophytic bacterial strains from the corn plant (Zea mays L.); probiotic mixtures comprising endophytic strains; formulations and a method to stimulate the germination of vegetable seeds, through the use of said formulation, which may contain the bacterial strains.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El aumento en la demanda de los productos agroquímicos, principalmente en los productos para fertilización y su uso excesivo e indiscriminado han logrado no solo el aumento en el precio del producto, si no que tan bien provocan una erosión en los suelos, disminuyendo los nutrientes, la salud y fertilidad del suelo. El cultivo de maíz juega un papel importante a nivel mundial, donde México se encuentra entre los 5 primeros países con mayor producción. El crecimiento de dicho cultivo se ve limitado por la carencia de nutrientes en el suelo, la cual se refleja en el rendimiento, y en la susceptibilidad a patógenos, de dicho cultivo. The increase in the demand for agrochemical products, mainly in products for fertilization, and their excessive and indiscriminate use have achieved not only an increase in the price of the product, but also how well they cause soil erosion, reducing nutrients, soil health and fertility. Corn cultivation plays an important role worldwide, where Mexico is among the top 5 countries with the highest production. The growth of said crop is limited by the lack of nutrients in the soil, which is reflected in the yield, and in the susceptibility to pathogens, of said crop.
El uso de microorganismo como biofertilizantes ha surgido como una alternativa, no sólo amigable con el ambiente, si no también como una alternativa económica para incrementar el rendimiento del cultivo, tal como lo demuestran las siguientes publicaciones científicas. The use of microorganisms as biofertilizers has emerged as an alternative, not only friendly to the environment, but also as an economic alternative to increase crop yield, as shown by the following scientific publications.
Ogbo, F., y Okonkwo, J. (20-Ago-2012). Some Characteristics of a Plant Growth Promoting Enterobacter sp. Isolated from the Roots of Maize, emplearon una caracterización morfológica, bioquímica y filogenética utilizando la técnica de ADNr MicroSeq® 16S en la identificación de un aislado, que se reveló que era
muy cercano al 99.4% con Enterobacter asburiae. El aislado identificado como NCOO1927-MR5, posee propiedades de bacterias promotoras del crecimiento de las plantas. Así, produjo indol-3-acético, enzimas hidrolizantes de polímeros vegetales, pectinasa y celulasa, así como amoniaco in vitro. El aislado creció bien en presencia de NaCI al 3% y 10 pg de estreptomicina. En los bioensayos en placa, el aislamiento promovió la germinación de las semillas de maíz y arroz, así como el crecimiento de las raíces, lo que resultó en un aumento de peso de las plántulas sobre sus controles. Los experimentos para cuantificar la capacidad del aislado para promover el crecimiento de la planta se realizaron utilizando soluciones hidropónicas y, según corresponda, un inóculo del aislado. También se emplearon experimentos en macetas. Los resultados de estos estudios mostraron que el aislamiento aumentó significativamente (P<0.05) la acumulación de Nitrógeno y, mejoró el crecimiento de las plántulas sobre los controles. El aislado tiene potencial para su utilización como inóculo para la producción sostenible de cereales. Si bien es cierto que dichos autores también han aislado una cepa de Enterobacter asburiae denominada como NCOO1927- MR5, de Awka, Nigeria, para promover la germinación de semillas maíz y arroz, la cual aumentó el desarrollo radicular; pero eso no significa que se dicha cepa bacteriana tenga un buen desempeño en las regiones geográficas de nuestro país y similares; además, las pruebas para demostrar su eficiencia agronómica, fueron realizadas en laboratorio y en macetas, o sea en condiciones controladas; pero se debe considerar las que son las condiciones de campo las que tiene la real interacción de todos los factores a los que se enfrenta un semilla cuando es puesta a germinar. Ogbo, F., and Okonkwo, J. (20-Aug-2012). Some Characteristics of a Plant Growth Promoting Enterobacter sp. Isolated from the Roots of Maize employed morphological, biochemical, and phylogenetic characterization using the MicroSeq® 16S rDNA technique to identify one isolate, which was revealed to be very close to 99.4% with Enterobacter asburiae. The isolate identified as NCOO1927-MR5, has properties of plant growth promoting bacteria. Thus, it produced indole-3-acetic acid, plant polymer hydrolyzing enzymes, pectinase and cellulase, as well as ammonia in vitro. The isolate grew well in the presence of 3% NaCl and 10 pg of streptomycin. In plate bioassays, the isolate promoted maize and rice seed germination as well as root growth, resulting in increased seedling weight over controls. Experiments to quantify the ability of the isolate to promote plant growth were performed using hydroponic solutions and, as appropriate, an inoculum of the isolate. Experiments in pots were also used. The results of these studies showed that the isolation significantly (P<0.05) increased Nitrogen accumulation and improved seedling growth over controls. The isolate has potential for use as an inoculum for sustainable cereal production. Although it is true that these authors have also isolated a strain of Enterobacter asburiae called NCOO1927-MR5, from Awka, Nigeria, to promote the germination of maize and rice seeds, which increased root development; but that does not mean that said bacterial strain has a good performance in the geographical regions of our country and the like; In addition, the tests to demonstrate its agronomic efficiency were carried out in the laboratory and in pots, that is, under controlled conditions; but it must be considered that it is the field conditions that have the real interaction of all the factors that a seed faces when it is put to germinate.
Sheibani-Tezerji, Raheleh y col. (12-May-2015). The genomes of closely related Pantoea ananatis maize seed endophytes having different effects on the host plant differ in secretion system genes and mobile genetic elements, investigaron tres cepas de Pantoea ananatis estrechamente relacionadas (Hamadas S6, S7 y S8), que se aislaron de las semillas de maíz de plantas sanas. Los experimentos de inoculación de plantas revelaron que cada una de estas cepas exhibían un fenotipo diferente que variaba desde un patógeno débil (S7), comensal (S8), hasta un efecto beneficioso y promotor del crecimiento (S6) en el maíz.
Realizaron un análisis genómico comparativo para encontrar determinantes genéticos responsables de las diferencias observadas. Estudios recientes proporcionaron una visión emocionante de los impulsores genéticos de la adaptación del nicho y la diversificación funcional del género Pantoea. Sin embargo, informan aquí por primera vez sobre el análisis de cepas de P. ananatis que colonizan el mismo nicho ecológico pero que muestran distintas estrategias de interacción con la planta huésped. Dicho análisis comparativo reveló que los genomas de estas tres cepas son muy similares; pero se pudieron observar diferencias genómicas en genes que codifican sistemas de secreción de proteínas y podrían haber observado efectores putativos y genes relacionados con la transposasa/integrasas/fagos. Como se puede ver, en dicha publicación solamente se describe el análisis de tres cepas de Pantoea annanatis que, si bien son bacterias endófitas y fueron aisladas de semilla de maíz, pero no se divulga ni anticipa una aplicación o uso de dichas cepas. Sheibani-Tezerji, Raheleh et al. (12-May-2015). The genomes of closely related Pantoea ananatis maize seed endophytes having different effects on the host plant differ in secretion system genes and mobile genetic elements, investigated three closely related strains of Pantoea ananatis (Hamadas S6, S7 and S8), which were isolated from the seeds of corn from healthy plants. Plant inoculation experiments revealed that each of these strains exhibited a different phenotype ranging from a weak pathogen (S7), commensal (S8), to beneficial and growth promoting (S6) in maize. They performed a comparative genomic analysis to find genetic determinants responsible for the observed differences. Recent studies provided exciting insight into the genetic drivers of niche adaptation and functional diversification in the genus Pantoea. However, they report here for the first time on the analysis of P. ananatis strains that colonize the same ecological niche but display different interaction strategies with the host plant. Said comparative analysis revealed that the genomes of these three strains are very similar; but genomic differences could be observed in genes encoding protein secretion systems and could have observed putative effectors and transposase/integrases/phage-related genes. As can be seen, said publication only describes the analysis of three strains of Pantoea annanatis which, although they are endophytic bacteria and were isolated from corn seed, but does not disclose or anticipate an application or use of said strains.
Su-Mam K. y col. (septiembre 2012). Growth Promotion of Pepper Plants by Pantoea ananatis B1-9 and its Efficient Endophytic Colonization Capacity in Plant Tissues, describen una bacteria de Pantoea ananatis B1-9 que se aisló de la rizósfera de la cebolla verde, la cual podría promover el crecimiento de plantas de pimiento, pepino, tomate y melón. En particular, el rendimiento de pimiento después del tratamiento con B1-9 en las plántulas aumentó aproximadamente 3 veces más que el de las plantas de control en un experimento de campo. Las pruebas de patogenicidad mostraron que no son patógenos en el repollo kimchi, zanahoria y cebolla. El estudio de caracterización funcional demostró la capacidad de B1-9 de funcionar en la solubilización de fosfato, oxidación de azufre, fijación de nitrógeno y producción de ácido indol-3-acético. Para rastrear los patrones de colonización de B1-9 en tejidos de plantas de pimiento, utilizaron el colorante fluorescente DRAQ5™, que tiñe los ADN de bacterias y células vegetales. Se encontró una gran cantidad de bacterias B1-9 en las superficies de las raíces y tallos, así como en las células de guardia. Además, varias bacterias B1-9 colonizadas residían en células vegetales corticales internas. El tratamiento de las regiones de la rizósfera con la cepa B1-9 puede resultar en una colonización eficiente de las plantas y promover el crecimiento de las plantas
desde la plántula hasta la etapa de la planta madura. En resumen, la cepa B1-9 se puede aplicar con éxito en la plantación de pimiento debido a su alta capacidad de colonización en los tejidos vegetales, así como a las propiedades que promueven el crecimiento eficiente de la planta. Cómo se puede ver, este artículo describe el uso de una cepa bacteriana de Pantoea annanatis aislada de la rizósfera de plantas de cebolla para promover el crecimiento de plantas de pimiento. Su-Mam K. et al. (September 2012). Growth Promotion of Pepper Plants by Pantoea ananatis B1-9 and its Efficient Endophytic Colonization Capacity in Plant Tissues, describe a Pantoea ananatis B1-9 bacterium that was isolated from the rhizosphere of green onion, which could promote the growth of pepper plants. bell pepper, cucumber, tomato and melon. In particular, pepper yield after B1-9 treatment in seedlings increased about 3-fold higher than that of control plants in a field experiment. Pathogenicity tests showed that they are not pathogenic in kimchi cabbage, carrot and onion. The functional characterization study demonstrated the ability of B1-9 to function in phosphate solubilization, sulfur oxidation, nitrogen fixation, and indole-3-acetic acid production. To track the colonization patterns of B1-9 in pepper plant tissues, they used the fluorescent dye DRAQ5™, which stains the DNA of bacteria and plant cells. A large number of B1-9 bacteria were found on root and stem surfaces as well as in guard cells. In addition, several colonized B1-9 bacteria resided in inner cortical plant cells. Treatment of rhizosphere regions with strain B1-9 can result in efficient plant colonization and promote plant growth. from seedling to mature plant stage. In summary, the B1-9 strain can be successfully applied in pepper plantations due to its high colonization capacity in plant tissues, as well as its properties that promote efficient plant growth. As you can see, this article describes the use of a bacterial strain of Pantoea annanatis isolated from the rhizosphere of onion plants to promote the growth of pepper plants.
Por lo tanto, con el propósito de contribuir a la solución de los inconvenientes antes mencionados, se han asilados tres cepas bacterianas endófitas de plantas de maíz Zea mays L., para obtener mezclas probióticas que contienen a dichas cepas bacteriana, elaborar formulaciones que comprende a las cepas bacterianas, y un método para estimular la germinación de semillas vegetales. Therefore, with the purpose of contributing to the solution of the aforementioned drawbacks, three endophytic bacterial strains of Zea mays L. corn plants have been isolated, to obtain probiotic mixtures that contain said bacterial strains, to prepare formulations that comprise bacterial strains, and a method for stimulating the germination of vegetable seeds.
Los detalles característicos de la presente invención se muestran claramente en la siguiente descripción detallada, ejemplos y figuras, con la finalidad de ilustrar su concepción y algunas de sus realizaciones preferentes; por lo tanto, tal descripción detallada, ejemplos y figuras, no deben ser considerados como una limitante para los alcances de dicha invención. The characteristic details of the present invention are clearly shown in the following detailed description, examples and figures, with the purpose of illustrating its conception and some of its preferred embodiments; therefore, such detailed description, examples and figures should not be considered as limiting the scope of said invention.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
La figura 1 es una fotografía que ¡lustra una prueba de compatibilidad entre las tres cepas bacterianas aislada y seleccionada. Figure 1 is a photograph illustrating a compatibility test between the three isolated and selected bacterial strains.
La figura 2 es una gráfica que ilustra los porcentajes de germinación obtenidos de los diferentes tratamientos bacterianos probados. Figure 2 is a graph illustrating the germination percentages obtained from the different bacterial treatments tested.
La figura 3 es una gráfica de la longitud promedio de raíz de las semillas germinadas de los diferentes tratamientos bacterianos. Figure 3 is a graph of the average root length of the germinated seeds of the different bacterial treatments.
La figura 4 es un gráfico que muestra la altura promedio de la plántula en los diferentes tratamientos bacterianos.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN Figure 4 is a graph showing the average seedling height in the different bacterial treatments. DETAILED DESCRIPTION OF THE INVENTION
La presente Invención tiene como objeto una cepa aislada de Pantoea ananatis, la cual tiene las características de la cepa depositada con el número de acceso CM-CNRG BT170. The object of the present invention is an isolated strain of Pantoea ananatis, which has the characteristics of the strain deposited under accession number CM-CNRG BT170.
La invención en cuestión, tiene como objeto una cepa aislada de Enterobacter kobei, que tiene las características de la cepa depositada con el acceso CM- CNRG BT172. The subject of the invention is an isolated strain of Enterobacter kobei, which has the characteristics of the strain deposited with accession CM-CNRG BT172.
Esta invención también tiene como objeto una cepa aislada de Enterobacter asburíae, cuyas características son las mismas características de la cepa depositada con el número de acceso CM-CNRG BT173. This invention also has as its object an isolated strain of Enterobacter asburíae, whose characteristics are the same as the strain deposited with the accession number CM-CNRG BT173.
Las cepas bacterianas CM-CNRG BT170, CM-CNRG BT172 y CM-CNRG BT173, son endófitas de plantas de Zea mays L. The bacterial strains CM-CNRG BT170, CM-CNRG BT172 and CM-CNRG BT173 are endophytes of Zea mays L.
Dichas cepas CM-CNRG BT170, CM-CNRG BT172 y CM-CNRG BT173, exhiben actividad estimulante sobre la germinación de semillas vegetales, mejorando así dicha germinación. Said strains CM-CNRG BT170, CM-CNRG BT172 and CM-CNRG BT173, exhibit stimulating activity on the germination of vegetable seeds, thus improving said germination.
En una modalidad preferente de la presente invención es cuando las semillas vegetales son de la familia Poaceae; pero en una modalidad más preferente cuando dichas semillas vegetales son del género Zea; aunque en una modalidad más preferente cuando tales semillas vegetales son de la especie Zea mays L. In a preferred embodiment of the present invention, it is when the plant seeds are from the Poaceae family; but in a more preferred embodiment when said plant seeds are of the Zea genus; although in a more preferred embodiment when such vegetable seeds are of the species Zea mays L.
Un objeto más de la presente invención es una mezcla probiótica de bacterias aisladas, que comprende al menos dos cepas bacterianas aisladas, de conformidad con las cepas bacterianas aisladas y descritas en la presente invención. A further object of the present invention is a probiotic mixture of isolated bacteria, comprising at least two isolated bacterial strains, in accordance with the isolated bacterial strains described in the present invention.
En una realización de la mezcla de acuerdo con la presente invención, es que las cepas bacterianas exhiben actividad estimulante sobre la germinación de
semillas vegetales; las cuales son de la familia Poaceae, o preferentemente del género Zea, o más preferente de la especie Zea mays L, por citar algunos ejemplos. In one embodiment of the mixture according to the present invention, the bacterial strains exhibit stimulating activity on the germination of vegetable seeds; which are from the Poaceae family, or preferably from the Zea genus, or more preferably from the Zea mays L species, to name a few examples.
Una modalidad más de la mezcla probiótica de la presente invención, es cuando las cepas bacterianas aisladas son: la cepa aislada de Enterobacter kobei CM- CNRG BT172, y la cepa aislada de Enterobacter asburiae CM-CNRG BT173 de conformidad con la presente invención; y una modalidad preferente es cuando dichas cepas están en una proporción 2:1. One more embodiment of the probiotic mixture of the present invention is when the isolated bacterial strains are: the isolated strain of Enterobacter kobei CM-CNRG BT172, and the isolated strain of Enterobacter asburiae CM-CNRG BT173 in accordance with the present invention; and a preferred embodiment is when said strains are in a 2:1 ratio.
Entre los usos que puede tener la mezcla probiótica de la presente invención, es para preparar una formulación para estimular la germinación de semillas vegetales. Among the uses that the probiotic mixture of the present invention may have, is to prepare a formulation to stimulate the germination of vegetable seeds.
Por lo tanto, un objeto más de la presente invención es una formulación para estimular la germinación de semillas vegetales, la cual contiene: i) al menos, una cepa bacteriana aislada, la cual es seleccionada del siguiente grupo: CM-CNRG BT170, CM-CNRG BT172, CM-CNRG BT173, y sus mezclas entre ellas; ii) al menos, una sustancia prebiótica; y iii) al menos, un excipiente. Therefore, one more object of the present invention is a formulation to stimulate the germination of vegetable seeds, which contains: i) at least one isolated bacterial strain, which is selected from the following group: CM-CNRG BT170, CM -CNRG BT172, CM-CNRG BT173, and their mixtures between them; ii) at least one prebiotic substance; and iii) at least one excipient.
Una modalidad de la formulación según la presente invención es cuando la cepa bacteriana aislada, ya sea individual o mezclada, está en una cantidad de 1 %, la sustancia prebiótica en 1.5%, y el excipiente en 97.5%, con respecto al volumen total de la formulación. One embodiment of the formulation according to the present invention is when the isolated bacterial strain, either individually or mixed, is in an amount of 1%, the prebiotic substance in 1.5%, and the excipient in 97.5%, with respect to the total volume of the formulation.
Una realización preferida de la formulación de la presente invención, es cuando comprende una mezcla de las cepas bacterianas CM-CNRG BT172 y CM-CNRG BT173; y la realización más preferida es cuando dichas cepas bacterianas están en una proporción de 2:1.
Las semillas vegetales que pueden estimularse su germinación con la formulación que propone esta invención, a manera de ejemplo mencionamos que pueden ser semillas de plantas de la familia Poaceae; prefiriendo semillas del género Zea; prefiriendo aún más semillas de la especie Zea mays L. A preferred embodiment of the formulation of the present invention is when it comprises a mixture of the bacterial strains CM-CNRG BT172 and CM-CNRG BT173; and the most preferred embodiment is when said bacterial strains are in a ratio of 2:1. The plant seeds that can be stimulated to germinate with the formulation proposed by this invention, by way of example we mention that they can be seeds of plants of the Poaceae family; preferring seeds of the genus Zea; preferring even more seeds of the species Zea mays L.
Un método para estimular la germinación de semillas vegetales, también es un objeto más de la presente invención, el cual comprende, aplicar a semillas vegetales previas a su germinación, una cantidad suficiente de una formulación para estimular la germinación de semillas vegetales, de conformidad con la formulación descrita en esta invención. A method to stimulate the germination of vegetable seeds is also another object of the present invention, which comprises applying to vegetable seeds prior to their germination, a sufficient amount of a formulation to stimulate the germination of vegetable seeds, in accordance with the formulation described in this invention.
Una variante del método para estimular la germinación de semillas vegetales, de acuerdo con la presente invención, es cuando la cantidad suficiente de la formulación es de un 1 L por cada 80,000 de semillas vegetales. A variant of the method to stimulate the germination of vegetable seeds, according to the present invention, is when the sufficient quantity of the formulation is 1 L per 80,000 vegetable seeds.
Una variante más del método en cuestión, es cuando la aplicación de la formulación es al momento de poner a germinar las semillas vegetales. One more variant of the method in question is when the application of the formulation is at the moment of putting the vegetable seeds to germinate.
En una modalidad más de dicho método, es cuando se estimula la germinación de semillas de plantas de la familia Poaceae; siendo una modalidad preferida cuando se estimulan la germinación de semillas de plantas del género Zea; siendo una modalidad más preferida cuando se estimula la germinación de semillas de plantas de la especie Zea mays L. In one more modality of said method, it is when the germination of seeds of plants of the Poaceae family is stimulated; being a preferred modality when the germination of seeds of plants of the genus Zea is stimulated; being a more preferred modality when the germination of seeds of plants of the species Zea mays L.
EJEMPLOS EXAMPLES
Los siguientes ejemplos sólo tienen la finalidad de ilustrar la concepción de la presente Invención y algunas de sus realizaciones preferentes, los cuales no deben ser considerados como una limitante, para los alcances de la invención. The following examples are only intended to illustrate the conception of the present invention and some of its preferred embodiments, which should not be considered as limiting the scope of the invention.
Ejemplo 1. Aislamiento de cepas bacteriana endófitas de plantas de maíz (Zea mays L.).
Plantas completas de maíz (Zea mays L.) con edad de madurez comercial de V4 a R6, fueron colectadas de 2 parcelas de maíz cultivadas convencionalmente con productos agroquímico, ubicadas en las localidades de Santa Elena y San Francisco de Asís, municipio de Atotonilco el Alto, Jalisco, México; y de una parcela de maíz cultivada con productos orgánicos, ubicada en la localidad El Refugio, municipio de Tototlán, Jalisco, México. Example 1. Isolation of endophytic bacterial strains from maize plants (Zea mays L.). Complete maize plants (Zea mays L.) with commercial maturity age from V4 to R6, were collected from 2 plots of maize cultivated conventionally with agrochemical products, located in the towns of Santa Elena and San Francisco de Asís, municipality of Atotonilco el Alto, Jalisco, Mexico; and from a plot of corn cultivated with organic products, located in the town of El Refugio, municipality of Tototlán, Jalisco, Mexico.
Se realizó un muestreo al azar tomando 10 plantas completas de maíz, por cada parcela, de apariencia sana, intentando dañar lo menos posible la raíz. Esta colecta se llevó a cabo en diferentes puntos estratégicos de las parcelas, con el fin de cubrir el total de sus extensiones para de obtener muestras representativas. A random sampling was carried out taking 10 complete maize plants, for each plot, of healthy appearance, trying to damage the root as little as possible. This collection was carried out at different strategic points of the plots, in order to cover the totality of its extensions in order to obtain representative samples.
Las plantas se transportaron en una bolsa plástica para evitar posibles daños a las mismas. Una vez llegado al laboratorio se les dio un enjuague con agua corriente y jabón para quitar el exceso de tierra y se dejaron escurrir hasta que el agua se eliminó. Las hojas de las plantas se seccionaron en partes de 5 x 10 cm, y se tomaron 100 g de semillas de las mazorcas para posteriormente seguir el protocolo de extracción de endófitos reportado en literatura, que consistió en un lavado al 3% por 10 min de hipoclorito de sodios, seguido de 3 lavados con agua bidestilada estéril por 5 min cada uno, seguido de una molienda con mortero, pistilo y una solución estéril de cloruro de sodio 0.9% para formar una suspensión y que es diluida de manera serial, es decir se tomaron 100 pl de la suspensión y 900 pl de solución salina 0.9% estéril, se mezclaron y se tomaron 100 pl de esa mezcla y se pasan a otros 900 pl de solución salina 0.9% estéril, se mezclaron y se toman 100 pl de esta nueva mezcla y se pasan a otros 900 pl de solución salina 0.9% estéril; y así sucesivamente. Se realizaron 6 diluciones. Posteriormente de cada dilución se toman 100 pl y se esparcieron con ayuda de un asa en L en una caja Petri que contiene agar nutritivo. The plants were transported in a plastic bag to avoid possible damage to them. Once they arrived at the laboratory, they were rinsed with running water and soap to remove excess soil and allowed to drain until the water was eliminated. The leaves of the plants were sectioned into parts of 5 x 10 cm, and 100 g of seeds were taken from the cobs to subsequently follow the endophyte extraction protocol reported in the literature, which consisted of washing at 3% for 10 min. sodium hypochlorite, followed by 3 washes with sterile bidistilled water for 5 min each, followed by grinding with a mortar, pestle and a sterile 0.9% sodium chloride solution to form a suspension and which is serially diluted, that is, 100 μl of the suspension and 900 μl of sterile 0.9% saline solution were taken, they were mixed and 100 μl of that mixture were taken and passed to another 900 μl of 0.9% sterile saline solution, they were mixed and 100 μl of this were taken new mixture and another 900 pl of sterile 0.9% saline solution are transferred; and so on. 6 dilutions were made. After each dilution, 100 pl are taken and spread with the help of an L-shaped handle in a Petri dish containing nutrient agar.
La suspensión al ser diluida y plaqueada fue formando colonias cada vez menos concentradas. De dichas diluciones se seleccionaron las colonias que tuvieron
morfologías diferentes y fueron resembradas 2 a 3 veces en agar tripticaseína de soya hasta ser purificadas. When the suspension was diluted and plated, it formed increasingly less concentrated colonies. From these dilutions, the colonies that had different morphologies and were reseeded 2 to 3 times in soy trypticasein agar until purified.
Se lograron aislar un total de 42 colonias bacterianas con características y propiedades diferentes, las cuales se identificaron como se ilustra en el Cuadro 1 , y fueron sometidas a distintas pruebas agronómicas. A total of 42 bacterial colonies with different characteristics and properties were isolated, which were identified as illustrated in Table 1, and were subjected to different agronomic tests.
Ejemplo 2. Pruebas agronómicas a las que fueron sometidas las cepas bacterianas endófitas de plantas de maíz. Example 2. Agronomic tests to which the endophytic bacterial strains of corn plants were subjected.
Las 42 cepas bacterianas aisladas de maíz del ejemplo anterior, fueron sometidas a las siguientes pruebas agronómicas: prueba de fijación de nitrógeno (NFB), prueba de solubilización de fosfato (NBRIP Ca, NBRIP Al y NBRIP Fe), prueba de quelantes de hierro (siderófbros), prueba de producción de fitohormonas (auxinas) y prueba de producción de enzima acc desaminasa (ACC), como lo describe Dworkin (2006); Delvasto et al., (2006), Milagros et al. (1999); Gordon y Weber (1950); y Penrose y Glick (2003), respectivamente. En este caso, como nuestro principal objetivo era seleccionar microorganismos que indujeran la germinación de semillas vegetales, pusimos énfasis en aquellas cepas bacterianas que tuvieron producción de auxinas, ya que esta es una fitohormona relacionada con el crecimiento vegetal. Sin embargo, también consideramos si algunas de las demás pruebas agronómicas daban positivo o negativo. Los resultados de estas pruebas agronómicas se muestran en el Cuadro 1. The 42 bacterial strains isolated from corn in the previous example were subjected to the following agronomic tests: nitrogen fixation test (NFB), phosphate solubilization test (NBRIP Ca, NBRIP Al and NBRIP Fe), iron chelating test ( siderofbros), phytohormone (auxin) production test and enzyme acc deaminase (ACC) production test, as described by Dworkin (2006); Delvasto et al., (2006), Milagros et al. (1999); Gordon and Weber (1950); and Penrose and Glick (2003), respectively. In this case, as our main objective was to select microorganisms that induce the germination of plant seeds, we put emphasis on those bacterial strains that produced auxin, since this is a phytohormone related to plant growth. However, we also considered whether any of the other agronomic tests were positive or negative. The results of these agronomic tests are shown in Table 1.
Al analizar los resultados que arrojaron las cepas bacterianas aisladas (Cuadro 1 ), seleccionamos 3 cepas bacterianas, las cuales fueron M1 , M19 y M20, porque fueron positivas para la prueba de fijación de nitrógeno (NFB), debido al cambio de coloración de verde a azul que se presentó en el medio de cultivo donde fueron sembradas.
Cuadro 1. Resultados de las pruebas agronómicas de 42 cepas bacterianas endófitas aisladas de plantas de Zea mays L.
When analyzing the results of the isolated bacterial strains (Table 1), we selected 3 bacterial strains, which were M1, M19 and M20, because they were positive for the nitrogen fixation test (NFB), due to the color change from green to blue that was presented in the culture medium where they were sown. Table 1. Results of the agronomic tests of 42 endophytic bacterial strains isolated from Zea mays L.
Para la solubilización de fosfatos (NBRIP) se midió el halo generado alrededor de la colonia inoculada en el medio, donde las cepas bacterianas M1 y M19 generaron un halo de 0.1 cm en la solubilización de fosfatos de Ca, mientras que la cepa M20 logró solubilizar los fosfatos de Ca pero no lo suficiente como para lograr una medición exacta. Con los fosfatos de aluminio y hierro, ninguna de las cepas aisladas logró solubilizar dichos fosfatos. En el caso de slderóforos, las cepas M19 y M20 dieron claramente positivas a la prueba de slderóforos, mientras que M1 sólo fue ligeramente positiva. En la producción de auxinas, la cepa que produjo mayor cantidad de esta fitohormona fue la cepa M1 , al obtener 3 cruces de 3, mientras que las cepas M19 y M20, obtuvieron una producción de 2 cruces de 3, es decir, la produjeron moderadamente. Por último, la producción de la enzima ACC desaminasa, como observamos en el cuadro 1 , la cepa M1 no
produjo dicha enzima al no ser capaz de crecer en el medio de cultivo; mientras que las cepas M19 y M20 la produjeron en bajas cantidades (+). For phosphate solubilization (NBRIP), the halo generated around the colony inoculated in the medium was measured, where bacterial strains M1 and M19 generated a 0.1 cm halo in the solubilization of Ca phosphates, while strain M20 managed to solubilize Ca phosphates but not enough to get an accurate measurement. With the aluminum and iron phosphates, none of the isolated strains managed to solubilize these phosphates. In the case of slderophores, strains M19 and M20 were clearly positive in the slderophore test, while M1 was only slightly positive. In the production of auxins, the strain that produced the greatest amount of this phytohormone was the M1 strain, obtaining 3 crosses out of 3, while the M19 and M20 strains obtained a production of 2 out of 3 crosses, that is, they produced it moderately. . Finally, the production of the ACC deaminase enzyme, as we observe in table 1, the M1 strain does not produced said enzyme by not being able to grow in the culture medium; while strains M19 and M20 produced it in low quantities (+).
De acuerdo a los resultados arrojados de las cepas bacterianas (Cuadro 1) se seleccionaron solamente las cepas bacterianas M1, M19 y M20, por presentar producción de auxinas, resultaron positivas para la fijación de N, lograron sdubilizar fosfatos de Ca, fueron positivas a la producción de sideróforos y lograron producir la enzima ACC desaminasa, aunque en poca cantidad. Por lo que se procedió a la identificación de estas 3 cepas bacterianas seleccionadas. According to the results obtained from the bacterial strains (Table 1), only the bacterial strains M1, M19 and M20 were selected, due to their auxin production, they were positive for N fixation, they were able to weaken Ca phosphates, they were positive for production of siderophores and managed to produce the enzyme ACC deaminase, although in small quantities. Therefore, these 3 selected bacterial strains were identified.
Ejemplo 3. Identificación de las 3 cepas bacterianas endófitas de plantas de Zea mays L. seleccionadas. Example 3. Identification of the 3 endophytic bacterial strains of selected Zea mays L. plants.
Una vez seleccionadas las 3 cepas bacterianas del ejemplo anterior, se procedió a hacer la identificación de éstas. La identificación bacteriana se llevó a cabo mediante la técnica de MALDI-TOF, que consiste en obtener un perfil proteico del microorganismo a analizar y hacer una comparación con una base de datos, arrojando como resultado un score, que corresponde a que tan acertada es la identificación. El score va de 0 a 3, donde un score de 0-1.599 indica una Identidad no confiable, un score de 1.600-1.999 indica que hay mucha probabilidad que el género identificado sea el correcto, un score de 2.000-2.299 es indicativo de que la identidad del género es 100% seguro, pero la identidad de la especie tiene cierta probabilidad que sea, y por último, un score de 2.300- 3.000 es 100% seguro de que la identidad del género y especie, sí correspondan. Los resultados de identidad de las cepas bacterianas se muestran en el Cuadro 2. Once the 3 bacterial strains of the previous example had been selected, they were identified. Bacterial identification was carried out using the MALDI-TOF technique, which consists of obtaining a protein profile of the microorganism to be analyzed and making a comparison with a database, resulting in a score, which corresponds to how successful the identification is. ID. The score ranges from 0 to 3, where a score of 0-1,599 indicates an unreliable Identity, a score of 1,600-1,999 indicates that there is a high probability that the identified gender is correct, a score of 2,000-2,299 is indicative that the identity of the genus is 100% certain, but the identity of the species has a certain probability that it is, and finally, a score of 2,300-3,000 is 100% certain that the identity of the genus and species do correspond. The identity results of the bacterial strains are shown in Table 2.
Cuadro 2. Identificación por MALDI-TOF de las 3 cepas bacterias endófitas de plantas de Zea mays L. seleccionadas.
Ejemplo 4. Prueba de compatibilidad entre las cepas bacterianas endófitas de plantas de Zea may L. seleccionadas. Table 2. Identification by MALDI-TOF of the 3 endophytic bacterial strains of selected Zea mays L. plants. Example 4. Compatibility test between the endophytic bacterial strains of selected Zea may L. plants.
Una vez identificadas las 3 cepas bacterianas seleccionadas en el ejemplo anterior, se procedió a realizar la prueba de compatibilidad entre ellas, para ver la viabilidad de poder hacer mezclas y medir el efecto en la germinación. Once the 3 bacterial strains selected in the previous example had been identified, a compatibility test was carried out between them, to see the feasibility of being able to make mixtures and measure the effect on germination.
Las pruebas de compatibilidad se realizaron mediante la técnica de sándwich, donde una bacteria se siembra en forma de césped en una caja de agar casoy y encima de la bacteria inoculada se pone una oblea de agar casoy, donde son inoculadas el resto de las bacterias a probar en forma de manchas. SI la bacteria que queda encima de la oblea de agar casoy crece, se considera que es compatible con la bacteria que esta debajo de la oblea. Todo esto se realizó por triplicado. The compatibility tests were carried out using the sandwich technique, where a bacterium is planted in the form of a lawn in a box of Casoy agar and a Casoy agar wafer is placed on top of the inoculated bacterium, where the rest of the bacteria are inoculated. try in the form of spots. If the bacteria on top of the agar wafer grow and grow, it is considered to be compatible with the bacteria on the bottom of the wafer. All this was done in triplicate.
Los resultados arrojados por esta prueba de compatibilidad se ilustran en la figura 1, donde podemos observar que tanto M1 que es la bacteria que esta debajo, como M19 y M20 que están por encima en la oblea, presentaron buen crecimiento, indicando que las 3 cepas son compatibles entre sí y que pueden utilizarse en mezclas para lograr potencializar su efecto de estimular la germinación en semillas vegetales. The results of this compatibility test are illustrated in Figure 1, where we can see that both M1, which is the bacteria below, and M19 and M20, which are above the wafer, showed good growth, indicating that the 3 strains They are compatible with each other and can be used in mixtures to potentiate their effect of stimulating germination in vegetable seeds.
Ejemplo 5. Pruebas de germinación de semillas de Zea mays L. tratadas con las 3 cepas bacterianas endófitas de plantas de Zea mays L. seleccionadas. Example 5. Germination tests of Zea mays L. seeds treated with the 3 endophytic bacterial strains of selected Zea mays L. plants.
Con la finalidad de saber si realmente las cepas aisladas M1, M19 y M20, promueven la germinación en semillas vegetales, ya sea de manera conjunta o separadas, se realizó un experimento en Atotonilco el Alto, Jalisco, México, del 17 al 27 de enero de 2021, bajo condiciones de Invernadero (ex vitro). Los tratamientos que se evaluaron se muestran en el Cuadro 3, donde por cada tratamiento se utilizó una muestra de 10 semillas de maíz (Zea mays L.) de la variedad Brevant, que corresponde a maíz blanco.
Cuadro 3. Tratamientos a los que fueron sometidas semillas de maíz (Zea mays L), con las cepas bacterianas aisladas.
In order to know if the isolated strains M1, M19 and M20 really promote germination in vegetable seeds, either together or separately, an experiment was carried out in Atotonilco el Alto, Jalisco, Mexico, from January 17 to 27. 2021, under greenhouse conditions (ex vitro). The treatments that were evaluated are shown in Table 3, where for each treatment a sample of 10 corn seeds (Zea mays L.) of the Brevant variety, which corresponds to white corn, was used. Table 3. Treatments to which corn seeds (Zea mays L) were subjected, with the isolated bacterial strains.
Las semillas de maíz utilizadas fueron semillas que no tuvieron ningún tratamiento antifúngico, y se cuidó que no estuvieran rotas o sin el embrión, para asegurar que las semillas fueran viables para germinación. The corn seeds used were seeds that did not have any antifungal treatment, and care was taken that they were not broken or without the embryo, to ensure that the seeds were viable for germination.
Las semillas fueron sumergidas en tubos falcón que contenían una formulación para estimular la germinación, la cual contiene: las cepas bacterianas aisladas, ya sea individuales y/o combinadas; y una sustancia prebiótica a base fuentes de nitrógeno y carbono. Las semillas estuvieron sumergidas durante 1 h en la formulación que estimula la germinación; posteriormente con la ayuda de unas pinzas se sacaron y se colocaron en un sustrato compuesto por tierra, peatmoos y jal, preparado previamente en una charola de germinación, y se mantuvieron en oscuridad a temperatura ambiente, regándose con 1mL de agua potable diariamente. Después un periodo de una semana y media (cuando se empezaron a ver los brotes sobresalir del sustrato), las semillas se sacaron del sustrato, se enjuagaron con agua corriente y se midió la longitud de raíz y la longitud del cotiledón si la semilla germinó, cuyos resultados se muestran en las figuras 2, 3 y 4.
En la figura 2, observamos el porcentaje de germinación de los diferentes tratamientos probados, donde el tratamiento con mayor porcentaje de germinación es el tratamiento 1, teniendo un 90% de germinación, superando al control de agua que tienen un porcentaje de germinación del 70. Mientras que los tratamientos con menor porcentaje de germinación son los tratamientos 5 y 7, que sólo germinaron un 50%. The seeds were submerged in falcon tubes containing a formulation to stimulate germination, which contains: the isolated bacterial strains, either individual and/or combined; and a prebiotic substance based on nitrogen and carbon sources. The seeds were submerged for 1 h in the formulation that stimulates germination; Later, with the help of tweezers, they were removed and placed in a substrate composed of soil, peatmoos and jal, previously prepared in a germination tray, and kept in the dark at room temperature, irrigated with 1mL of drinking water daily. After a period of one and a half weeks (when the shoots began to protrude from the substrate), the seeds were removed from the substrate, rinsed with running water and the length of the root and the length of the cotyledon were measured if the seed germinated, whose results are shown in figures 2, 3 and 4. In figure 2, we observe the germination percentage of the different treatments tested, where the treatment with the highest germination percentage is treatment 1, with 90% germination, surpassing the water control, which has a germination percentage of 70. While the treatments with the lowest percentage of germination are treatments 5 and 7, which only germinated 50%.
Por otro lado, en la figura 3, observamos la longitud promedio de las raíces de las semillas que lograron germinar. El tratamiento que logró una mayor longitud de raíz fue el tratamiento 9, con un promedio de 4.68 cm de raíz, seguido de los tratamientos 2, 8 y 7 que lograron una longitud de raíz de 4.58, 4.3 y 4.2 cm respectivamente. Mientras que el tratamiento que menor longitud de raíz tuvo fue el tratamiento 3 con solo 2.91 cm de longitud de raíz. On the other hand, in figure 3, we observe the average length of the roots of the seeds that managed to germinate. The treatment that achieved a greater root length was treatment 9, with an average root length of 4.68 cm, followed by treatments 2, 8 and 7, which achieved a root length of 4.58, 4.3 and 4.2 cm, respectively. While the treatment with the shortest root length was treatment 3 with only 2.91 cm of root length.
Por último, en la figura 4, observamos la longitud promedio de la plántula (crecimiento de la planta) obtenido de las semillas que germinaron de los diferentes tratamientos. En esta figura 4 observamos que el tratamiento que induce un mayor crecimiento en la plántula fue el tratamiento 9, logrando una altura promedio de 3.8 cm, seguido de los tratamientos 4 y 7 con una longitud de 3.5 cm para ambos, siendo mayores que el control con agua, que solo logró incrementar la altura de la planta en 2.85 cm. El tratamiento con menor inducción de crecimiento de la planta fue el tratamiento 5 con una altura promedio de 2.48 cm. Finally, in figure 4, we observe the average length of the seedling (plant growth) obtained from the seeds that germinated from the different treatments. In this figure 4 we observe that the treatment that induces a greater growth in the seedling was treatment 9, achieving an average height of 3.8 cm, followed by treatments 4 and 7 with a length of 3.5 cm for both, being greater than the control. with water, which only managed to increase the height of the plant by 2.85 cm. The treatment with the lowest induction of plant growth was treatment 5 with an average height of 2.48 cm.
DEPÓSITO DE LAS CEPAS BACTERIANAS ASILADAS DEPOSIT OF ISOLATED BACTERIAL STRAINS
Al ver que las cepas bacterianas aisladas M1, M19 y M20, tienen potencial agronómico, se procedió a depositarlas el 19 de octubre de 2020, en la Colección de Microorganismos del Centro Nacional de recursos Genéticos que pertenece al Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, con número de registro ante la World Federation of Culture Collection 1006 (CM- CNRG) y Autoridad Depositaria Internacional con notificación 308 con fines de procedimiento de patente conforme al Tratado de Budapest; y que tiene su
domicilio en Boulevard de la Biodiversidad 400, C. P. 47600. Tepatitlán de Morelos, Jalisco, México. La cepa bacteriana M1 Pantoea ananatis le correspondió el número de depósito CM-CNRG BT170, la cepa M19 Enterobacter kobei se le asignó el acceso No. CM-CNRG BT172, y a la cepa M20 Enterobacter asburiae le correspondió el acceso No. CM-CNRG BT173. Seeing that the isolated bacterial strains M1, M19 and M20 have agronomic potential, they were deposited on October 19, 2020, in the Collection of Microorganisms of the National Center for Genetic Resources that belongs to the National Institute of Forestry, Agricultural and Agricultural Research. Livestock, with registration number before the World Federation of Culture Collection 1006 (CM-CNRG) and International Depositary Authority with notification 308 for patent procedure purposes in accordance with the Budapest Treaty; and who has his address at Boulevard de la Biodiversidad 400, CP 47600. Tepatitlán de Morelos, Jalisco, Mexico. The bacterial strain M1 Pantoea ananatis was assigned the deposit number CM-CNRG BT170, the M19 Enterobacter kobei strain was assigned accession No. CM-CNRG BT172, and the M20 Enterobacter asburiae strain was assigned accession No. CM-CNRG BT173 .
LITERATURA CITADA LITERATURE CITED
Dworkin, M.; Falkow, S.; Rosemberg, E.; Schleifer, K.H. y Stackebrandt, E. (2006). Non-Symbiotic nitrogen fixers. The prokaryotes Vol. 5: Proteobacteria: Alpha and Beta subclasses, 3a ed., 69-89. Dworkin, M.; Falkow, S.; Rosenberg, E.; Schleifer, K. H. and Stackebrandt, E. (2006). Non-Symbiotic nitrogen fixers. The prokaryotes Vol. 5: Proteobacteria: Alpha and Beta subclasses, 3rd ed., 69-89.
Delvasto, P., Valverde, A., Ballester, A., Igual, J. M., Muñoz, J. A., González, F., Blázquez, M.L. y García, C. (2006). Characterization of brushite as a recrystallization product formed during bacterial solubilization of hydroxyapatite in batch cultures. Soil Biology and Biochemistry, 38, 2645- 2654. doi: 10.1016/j.soilbio.2006.03.020. Delvasto, P., Valverde, A., Ballester, A., Igual, J. M., Muñoz, J. A., González, F., Blázquez, M.L. and Garcia, C. (2006). Characterization of brushite as a recrystallization product formed during bacterial solubilization of hydroxyapatite in batch cultures. Soil Biology and Biochemistry, 38, 2645-2654. doi: 10.1016/j.soilbio.2006.03.020.
Milagres, A. M. F., Machuca, A. y Napoleao, D. (1999). Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. Journal of Microbiological methods, 37(1), 1-6. Milagres, A. M. F., Machuca, A. and Napoleao, D. (1999). Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. Journal of Microbiological methods, 37(1), 1-6.
Gordon, S. A., y Weber, R. P. (1950). Colorimetric estimation of indoleacetic acid. Plant Physiology, 192-195. Gordon, S.A., and Weber, R.P. (1950). Colorimetric estimation of indoleacetic acid. Plant Physiology, 192-195.
Penrose, D. M., & Glick, B. R. (2003). Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant, 118(1), 10-15.
fe
Penrose, D.M., & Glick, B.R. (2003). Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. PhysiolPlant, 118(1), 10-15. faith
Claims
1. Una cepa aislada de Pantoea ananatis, caracterizada por, que tiene las características de la cepa depositada con el número de acceso CM-CNRG BT170. 1. An isolated strain of Pantoea ananatis, characterized by, having the characteristics of the strain deposited under accession number CM-CNRG BT170.
2. Una cepa aislada de Enterobacter kobei, caracterizada por, que tiene las características de la cepa depositada con el acceso CM-CNRG BT172. 2. An isolated strain of Enterobacter kobei, characterized by, having the characteristics of the strain deposited with the accession CM-CNRG BT172.
3. Una cepa aislada de Enterobacter asburíae, caracterizada por, que tiene las características de la cepa depositada con el número de acceso CM- CNRG BT173. 3. An isolated strain of Enterobacter asburíae, characterized by, having the characteristics of the strain deposited under accession number CM-CNRG BT173.
4. Las cepas de las reivindicaciones anteriores, donde dichas cepas bacterianas son endófitas de plantas de Zea mays L. 4. The strains of the preceding claims, wherein said bacterial strains are endophytic plants of Zea mays L.
5. Las cepas según las reivindicaciones anteriores, donde las cepas bacterianas exhiben actividad estimulante en la germinación de semillas vegetales. 5. The strains according to the preceding claims, wherein the bacterial strains exhibit stimulating activity in the germination of plant seeds.
6. Las cepas de la reivindicación precedente, donde las semillas vegetales son de plantas de la familia Poaceae. 6. The strains of the preceding claim, wherein the plant seeds are from plants of the Poaceae family.
7. Las cepas según la reivindicación anterior, donde las semillas vegetales son de plantas del género Zea. 7. The strains according to the preceding claim, wherein the plant seeds are from plants of the Zea genus.
8. Las cepas de la reivindicación anterior, donde las semillas vegetales son de plantas la especie Zea mays L. 8. The strains of the preceding claim, where the plant seeds are from plants of the species Zea mays L.
9. Una mezcla probiótica, caracterizada por, que comprende, al menos dos cepas bacterianas aisladas, de conformidad con cualquiera de las reivindicaciones anteriores.
9. A probiotic mixture, characterized by, comprising, at least two isolated bacterial strains, according to any of the preceding claims.
10. La mezcla de la reivindicación anterior, donde dichas cepas bacterianas son endófitas de plantas de Zea mays L. 10. The mixture of the preceding claim, wherein said bacterial strains are endophytic plants of Zea mays L.
11. La mezcla según la reivindicación 9, donde tales cepas bacterianas exhiben actividad estimulante sobre la germinación de semillas vegetales. 11. The mixture according to claim 9, wherein said bacterial strains exhibit stimulating activity on the germination of vegetable seeds.
12. La mezcla de la reivindicación precedente, donde las semillas vegetales son de plantas de la familia Poaceae. 12. The mixture of the preceding claim, wherein the plant seeds are from plants of the Poaceae family.
13. La mezcla según la reivindicación anterior, donde las semillas vegetales son de plantas del género Zea. 13. The mixture according to the preceding claim, wherein the plant seeds are from plants of the Zea genus.
14. La mezcla de la reivindicación anterior, donde las semillas vegetales son de plantas de la especie Zea mays L. 14. The mixture of the preceding claim, where the vegetable seeds are from plants of the species Zea mays L.
15. La mezcla de la reivindicación 9, donde las cepas bacterianas aisladas son: la cepa aislada de Enterobacter kobei, de conformidad con la reivindicación 2; y la cepa aislada de Enterobacter asburiae, de conformidad con la reivindicación 3. 15. The mixture of claim 9, wherein the isolated bacterial strains are: the isolated strain of Enterobacter kobei, according to claim 2; and the isolated strain of Enterobacter asburiae, according to claim 3.
16. La mezcla de la reivindicación precedente, donde las cepas bacterianas están en una proporción de 2:1. 16. The mixture of the preceding claim, wherein the bacterial strains are in a ratio of 2:1.
17. Una formulación para estimular la germinación de semillas vegetales, caracterizada por, que comprende: 17. A formulation to stimulate the germination of vegetable seeds, characterized by, comprising:
¡) al menos, una cepa bacteriana aislada, la cual es seleccionada del siguiente grupo: CM-CNRG BT170, CM-CNRG BT172, CM-CNRG BT173, y sus mezclas entre ellas; ii) al menos, una sustancia prebiótica; y iii) al menos, un excipiente.
¡) at least one isolated bacterial strain, which is selected from the following group: CM-CNRG BT170, CM-CNRG BT172, CM-CNRG BT173, and their mixtures between them; ii) at least one prebiotic substance; and iii) at least one excipient.
18. La formulación de la reivindicación anterior, donde la cepa bacteriana, ya sea individual o mezclada, está en una cantidad de 1%, la sustancia prebiótica en 1.5%, y el excipiente en 97.5%, con respecto al volumen total de la formulación. 18. The formulation of the preceding claim, where the bacterial strain, whether individual or mixed, is in an amount of 1%, the prebiotic substance in 1.5%, and the excipient in 97.5%, with respect to the total volume of the formulation .
19. La formulación de la reivindicación 17, donde las semillas vegetales son de plantas de la familia Poaceae. 19. The formulation of claim 17, wherein the plant seeds are from plants of the Poaceae family.
20. La formulación según a la reivindicación precedente, donde las semillas vegetales son de plantas del género Zea. 20. The formulation according to the preceding claim, wherein the plant seeds are from plants of the Zea genus.
21. La formulación de la reivindicación anterior, donde las semillas vegetales son de plantas de la especie Zea mays L. 21. The formulation of the preceding claim, where the plant seeds are from plants of the species Zea mays L.
22. La formulación de la reivindicación 17, donde una de las mezclas de las cepas bacterianas contiene las cepas bacterianas: CM-CNRG BT172 y CM- CNRG BT173. 22. The formulation of claim 17, wherein one of the mixtures of the bacterial strains contains the bacterial strains: CM-CNRG BT172 and CM-CNRG BT173.
23. La mezcla de la reivindicación precedente, donde las cepas bacterianas están en una proporción de 2:1. 23. The mixture of the preceding claim, wherein the bacterial strains are in a ratio of 2:1.
24. Un método para estimular la germinación de semillas vegetales, caracterizado por, que comprende, aplicar a semillas vegetales previas a su germinación, una cantidad suficiente de una formulación para estimular la germinación de semillas vegetales, de conformidad con las reivindicaciones 17 a la 23. 24. A method for stimulating the germination of plant seeds, characterized by, comprising, applying to plant seeds prior to germination, a sufficient amount of a formulation to stimulate the germination of plant seeds, in accordance with claims 17 to 23 .
25. El método de la reivindicación anterior, donde la cantidad suficiente de la formulación es de un 1 L por cada 80,000 de semillas vegetales. 25. The method of the preceding claim, wherein the sufficient amount of the formulation is 1 L per 80,000 plant seeds.
26. El método según la reivindicación 24, donde la aplicación de la formulación es al momento de poner a germinar las semillas vegetales.
26. The method according to claim 24, wherein the application of the formulation is at the moment of putting the vegetable seeds to germinate.
27. El método de la reivindicación 24, donde las semillas vegetales son de plantas de la familia Poaceae. 27. The method of claim 24, wherein the plant seeds are from plants of the Poaceae family.
28. El método según a la reivindicación precedente, donde las semillas vegetales son de plantas del género Zea. 28. The method according to the preceding claim, wherein the plant seeds are from plants of the Zea genus.
29. El método de la reivindicación anterior, donde las semillas vegetales son de plantas de la especie Zea mays L.
29. The method of the preceding claim, wherein the plant seeds are from plants of the species Zea mays L.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MX2021002954A MX2021002954A (en) | 2021-03-11 | 2021-03-11 | Endophyte bacterial strains, probiotic mixtures, formulation, and method to stimulate germination in vegetable seeds. |
| MXMX/A/2021/002954 | 2021-03-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022191689A1 true WO2022191689A1 (en) | 2022-09-15 |
Family
ID=83226963
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/MX2021/000010 WO2022191689A1 (en) | 2021-03-11 | 2021-03-12 | Endophytic bacterial strains, probiotic mixtures, formulation and method for stimulating plant seed germination |
Country Status (2)
| Country | Link |
|---|---|
| MX (1) | MX2021002954A (en) |
| WO (1) | WO2022191689A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200131096A1 (en) * | 2018-10-29 | 2020-04-30 | Sustainable Community Development, Llc | Biofertilizer Composition and Method of Manufacture |
-
2021
- 2021-03-11 MX MX2021002954A patent/MX2021002954A/en unknown
- 2021-03-12 WO PCT/MX2021/000010 patent/WO2022191689A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200131096A1 (en) * | 2018-10-29 | 2020-04-30 | Sustainable Community Development, Llc | Biofertilizer Composition and Method of Manufacture |
Non-Patent Citations (5)
| Title |
|---|
| DA SILVA J.F., BARBOSA R.R., DE SOUZA A.N., DA MOTTA O.V., TEIXEIRA G.N., CARVALHO V.S., DE SOUZA A.L.S.R, DE SOUZA FILHO G.A.: "Isolation of Pantoea ananatis from sugarcane and characterization of its potential for plant growth promotion", GENETICS AND MOLECULAR RESEARCH, vol. 14, no. 4, 1 January 2015 (2015-01-01), pages 15301 - 15311, XP055968715, DOI: 10.4238/2015.November.30.6 * |
| FRANK OGBO, JULIUS OKONKWO: "Some Characteristics of a Plant Growth Promoting <i>Enterobacter</i> sp. Isolated from the Roots of Maize", ADVANCES IN MICROBIOLOGY, SCIENTIFIC RESEARCH PUBLISHING, INC., US, vol. 02, no. 03, 1 January 2012 (2012-01-01), US , pages 368 - 374, XP055512891, ISSN: 2165-3402, DOI: 10.4236/aim.2012.23046 * |
| KIM SU-NAM, CHO WON KYONG, KIM WON-IL, JEE HYEONG JIN, PARK CHANG-SEUK: "Growth Promotion of Pepper Plants by Pantoea ananatis B1-9 and its Efficient Endophytic Colonization Capacity in Plant Tissues", PLANT PATHOLOGY JOURNAL, HAN-GUG SIGMUL BYEONGRI HAG-HOE, KR, vol. 28, no. 20, 1 January 2012 (2012-01-01), KR , pages 270 - 281, XP055968719, ISSN: 1598-2254, DOI: 10.5423/PPJ.OA.02.2012.0026 * |
| SINGH RAMESH K, SINGH MEENA VIJAY, KUMAR MEENA RAJESH: "Can we use Maize ( Zea mays ) Rhizobacteria as Plant Growth Promoter ? ", INTERNATIONAL JOURNAL OF PLANT RESEARCH, vol. 28, no. 1, pages 86 - 99, XP055968741, DOI: 10.5958/2229-4473.2015.00012.9 * |
| TIAGO JOÃO, OLIVEIRA CORREIA, SILVA GILKA TALITA, PATRÍCIA WILLIANE, DINIZ SILVA, FERNANDES FIGUEREDO EVERTHON, BATISTA ISANELI, S: "Diazotrophic bacteria isolated from Brachiaria spp.: genetic and physiological diversity", CIENCIA E INVESTIGACIÓN AGRARIA, vol. 45, no. 3, 1 January 2018 (2018-01-01), pages 277 - 289, XP055968724, DOI: 10.7764/rcia.v45i3.1949 * |
Also Published As
| Publication number | Publication date |
|---|---|
| MX2021002954A (en) | 2023-02-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2427595T3 (en) | Biofertilizer Formulation | |
| Balderas-Ruíz et al. | Bacillus velezensis 83 increases productivity and quality of tomato (Solanum lycopersicum L.): Pre and postharvest assessment | |
| WO2017089641A1 (en) | Plant-biostimulating compositions comprising microorganism strains | |
| JP4955431B2 (en) | Rhizobia | |
| CN108795797B (en) | Corn root system endophytic enterobacter cloacae and application thereof | |
| Kontopoulou et al. | Responses of hydroponically grown common bean fed with nitrogen-free nutrient solution to root inoculation with N2-fixing bacteria | |
| WO2018027198A1 (en) | Compositions and methods for enhancing plant growth | |
| WO2022191689A1 (en) | Endophytic bacterial strains, probiotic mixtures, formulation and method for stimulating plant seed germination | |
| JPWO2018105722A1 (en) | Method for selecting plant symbiotic microorganism group, and microorganism mixture | |
| ES2293085T3 (en) | BIOLOGICAL CONTROL AND FORMULATIONS AGENT. | |
| WO2022191690A1 (en) | Endophytic bacterial strains, probiotic mixtures, formulation and method, for stimulating plant growth | |
| JP2012135300A (en) | Microbial strain and cultivation method which show yield increase and inhibitory effect on late blight disease in solanaceous plant and which show protective effect on yield decrease due to continuous cropping in leguminous plant | |
| KR101892471B1 (en) | Natural fertilizer | |
| ES2891354T3 (en) | Method to improve plant development | |
| El-Shouny et al. | Endophytic colonization of tomato plants by Gluconacetobacter diazotrophicus and its effect on crops improvement and yield promotion | |
| Chang | The use of plant growth-promoting rhizobacteria (PGPR) and an arbuscular mycorrhizal fungus (AMF) to improve plant growth in saline soils for phytoremediation | |
| ES2269004B2 (en) | USE OF ARTHROBACTER OXIDANS BB1 AS A PROTECTOR AGAINST SALINE STRESS. | |
| ES2286917B1 (en) | PROCEDURE FOR OBTAINING LIQUID ORGANIC LIQUID RICH IN HUMIC SUBSTANCES FROM COMPOST OF VEGETABLE ORIGIN. | |
| Fatria et al. | Seedling growth analysis of papaya cultivated on several planting media enriched by plant growth promotor microbes. | |
| Meriem et al. | Effect of abiotic factors on the Sinozhizobium sp growth isolated from Medicago sativa cultivated on northeastern Algeria soils and the selection of the highly tolerant strains | |
| WO2022191691A1 (en) | Strain isolated from trichoderma harzianum, mixture and method, for promoting plant seed germination | |
| Shipoh | Isolation, identification and characterization of Cleome Gynandra L. bacterial seed endophytes from Northern Namibia | |
| WO2022025745A1 (en) | Biofertilisers and soil improvers based on sargassum spp. macroalgae enriched with plant-growth promoting bacteria | |
| Mishra et al. | Comparative study on plant growth promotion by endophytic Pseudomonas spp. and Bacillus spp. of Solanum lycopersicum | |
| Stojanović et al. | Quantitative traits of white cabbage cultivars in association with two biofertilisers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21930496 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21930496 Country of ref document: EP Kind code of ref document: A1 |