MICROBIAL FORMULATION FOR THE PROTECTION OF PLANTS AND AGRICULTURAL CROPS AGAINST ENVIRONMENTAL CONDITIONS AND METHODS OF MANUFACTURE AND USE THEREOF

Abstract

An organic formulation based on a mixture of microorganisms and other derived organic components, obtained from plants that inhabit different extreme environments, including the Chilean Antarctic territory, for protecting plants and agricultural crops against environmental conditions, especially with respect to cold stress, water stress, and plant infection by pests and insects, is provided. Due to the activity of the consortia of microorganisms that compose it and its derived components, it favours plant growth and reduces the damage associated with cold and water stress in agricultural crops.

Claims

1. A formulation for the protection of agricultural plants and crops against unfavorable environmental conditions for their growth comprising: a. a microorganism without ice-nucleating activity (NINA) isolated from rhizosphere of plants inhabiting different extreme environments, including the Antarctic territory, capable of growing in the presence of carbohydrates and/or carbon-based substrate, and is resistant to environmental stress, including water stress, cold stress, wherein the microorganism: belongs to the genus Pseudomonas including the GPI-1 strain, lacks nucleating activity of ice and IBP genes, and where the microorganism also comprises a unique mechanism of induction, production and secretion of a biopolymer in response to stressful situations, including cold stress, and where in addition, the microorganism grows very well in economically and environmentally sustainable carbon sources, including: glucose, technical glycerol, crude glycerol, and any other carbon source derived from biodiesel industry waste; b. an organic component derived from the micro-organism, including at least: a biopolymer, a mixture of volatile components and any combination thereof; c. a means of solubilizing the micro-organism and the organic component derived from the micro-organism; and d. a stabilizer.

2. The formulation of claim 1, wherein the formulation contains at least one concentration of the biopolymer in the range of 1-100 mg/L of formulation, wherein in said range there is also a relationship between the microorganisms and the biopolymer of the invention, between 10.sup.5 to 10.sup.8 of CFU/ug of biopolymer.

3. The formulation of claim 1, wherein the microorganism corresponds to a new species of the genus Pseudomonas.

4. The formulation of claim 3, wherein the microorganism is the isolated and identified strain GPI-1 (SEQ ID No 1), and wherein said strain comprises a unique mechanism of induction, production and secretion for high concentrations of at least one biopolymer and at least one volatile substance, both protective products in response to environmental stress, including cold stress.

5. The formulation of claim 4, wherein the selected microorganism grows very well in economically and environmentally sustainable carbon sources, including: glucose, technical glycerol, crude glycerol, and any other carbon source derived from biodiesel industry waste.

6. The formulation of claim 5, wherein the microorganism presents differentiated growth using carbon sources, wherein said organism does not present growth when carbon sources of structure similar to maltodextrin are used, including but not limited to: lactulose, maltotriose, maltose, xylose; and where, if it presents growth when using carbon sources of the list that includes: arabinose, N-acetyl glucosamine, succinate, galactose, aspartic acid, proline, alanine, trehalose, mannose, glucose-6-phosphate, malate, ribose, rhamnose, fructose, acetate, glucose, glucose, thymidine, glutamate, sorbitol, fucose, gluconic acid.

7. The formulation of claim 6, wherein in addition, the microorganism grows correctly in the presence of molecules associated with Krebs cycle and amino acids.

8. The formulation of claim 7, wherein the microorganism secretes an exopolysaccharide (EPS) in the presence of glycerol and proportional to the amount of glycerol present in the medium.

9. The formulation of claim 1, wherein the organic component is a biopolymer produced and secreted from the microorganism of the formulation, wherein there is a relationship between the amount of microorganisms and biopolymers of at least between 10.sup.5 to 10.sup.8 CFU/ug of biopolymer, and where the concentration of the biopolymer in the formulation is in the range of 1 to 100 mg/L.

10. The formulation of claim 11, wherein said biopolymer corresponds to a sugar polymer of high molecular weight similar to maltodextrin, with cryoprotective capacity in microorganisms and multicellular organisms, including bacteria and plants.

11. The formulation of claim 1, wherein the organic component are nanoparticles made from the biopolymer secreted by the microorganism of the formulation, wherein the size of said nanoparticles is in the range of 50 to 500 nm.

12. The formulation of claim 1, wherein the organic component is a volatile organic substance produced or derived from at least one microorganism of the invention with protective capacity for plants, with respect to adverse environmental conditions, including: water stress, low temperatures, freezing and pests.

13. The formulation of claim 12, wherein the volatile organic substance can be chosen from the list comprising: Dimethyldisulfide, 1-Nonene, 1-Undecanol, 2-Undecanon, 2-Pentane, 3-Methyl-Butanal, Methyldiselenuro, 2-Heptane, 2-Nonanona, 1-Undecanol, 2-Undecanone, any other volatile substance produced by a microorganism of the formulation and combinations thereof.

14. The formulation of claim 1, wherein the formulation comprises at least two components: i) the microorganism, ii) the organic component; where the microorganism corresponds to GPI-1 which is a strain that is of the genus Pseudomonas that produces an EPS that corresponds to a sugar polymer of high molecular weight of more than 15 glucose units, similar to maltodextrin, and where the organic component corresponds to the EPS secreted by GPI-1, and where said formulation generates cryoprotection and biostimulation in plants.

15. The formulation of claim 1, wherein the formulation comprises at least two organic components, which may be selected from the list comprising: EPS derived from the microorganism, nanoparticles derived from EPS derived from the microorganism, volatile substances derived from the microorganism, and any combination thereof.

16. The formulation of claim 1, wherein the formulation protects the plant, crop, fruit, or vegetable where it is applied, for a sustained period, including maintaining its protective activity on the given target for up to 18 months.

17. A method for the protection of agricultural plants and crops against unfavorable environmental conditions for their growth using a product which includes the formulation of claim 1, the method comprising: a. Preparation of the product, b. Initial application of the product, c. Repeating the application of the product, with a frequency of between 10-14 days depending on the crop and the season of the year in which it is applied.

18. The method of claim 17, wherein the preparation of the product comprises the preparation of the formulation containing at least: (i) a micro-organism without ice-nucleating activity (NINA) identified from plants inhabiting different extreme environments, including the Antarctic territory, capable of growing in the presence of carbohydrates and/or carbon-based substrate, and is resistant to environmental stress, including water stress, cold stress; (ii) an organic component derived from the microorganism; (iii) a means of solubilizing the micro-organism and the organic component derived from the micro-organism; and (iv) a stabilizer.

19. The method of claim 18, wherein the initial application of the product comprises between 1 to 10 L/Ha depending on the agricultural crop to be protected.

20. The method of claim 19, wherein the application of the product in crops can be: a) by spraying on the leaf tissue, and b) by irrigation directly on the roots.

21. The formulation of claim 1, where the bacterium is resistant to environmental stress and in which the bacterium can produce a biopolymer and volatile substances

22. The formulation of claim 1, wherein the formulation has a mechanism of action including i) action by contact, causing asphyxiation by blocking the respiratory spiracles or stigmas of the pest, and ii) action by adhesion, which prevents the mite or insect from adhering to the surface of the plant, thus the formulation can act on a wide spectrum of insects.

23. A composition for the protection of agricultural plants and crops against unfavorable environmental conditions for their growth, wherein the composition comprises: a. a micro-organism without ice-nucleating activity (NINA) isolated and identified from plants inhabiting different extreme environments, including the Antarctic territory, capable of growing in the presence of carbohydrates and/or carbon-based substrate, and is resistant to environmental stress, including water stress, cold stress; wherein the microorganism can be selected from the list comprising: microorganisms of the rhizosphere resistant to extreme conditions without ice nucleating activity and without IBP genes, NINA bacteria, recombinant microorganisms without ice nucleating activity, isolated strains of NINA bacteria, strains of Pseudomonas NINA, including the GPI-1 strain (SEQ ID No 1) and any other microorganism without ice-nucleating activity isolated from a rhizosphere sample of plants of different species, including environment under extreme conditions, and where the selected microorganism can grow on economically and environmentally sustainable carbon sources, including: glucose, technical glycerol and crude glycerol. b. an organic component derived from the micro-organism; where said organic component includes at least one of the following: a biopolymer and a volatile substance, both protective to environmental stress, including cold stress; wherein the biopolymer produced and secreted in the presence of glycerol and proportional to the amount of glycerol present in the medium from the microorganism of the formulation, corresponds to a sugar polymer of high molecular weight similar to maltodextrin, with cryoprotective capacity in microorganisms and multicellular organisms, including bacteria and plants; and where the volatile substance has protective capacity for plants, with respect to adverse environmental conditions, including: water stress, low temperatures, freezing and pests, where said volatile substance is a stimulator of root growth and where in addition, said volatile substance corresponds to a mixture of volatile substances and where said mixture contains at least two of the elements of the list comprising: Dimethyldisulfide, 1-Nonene, 1-Undecanol, 2-Undecanon, 2-Pentane, 3-Methyl-Butanal, Methyldiselenide, 2-Heptane, 2-Nonanona, 1-Undecanol, 2-Undecanon, any other volatile substance produced by a microorganism of the composition and combinations thereof. c. a means of solubilizing the micro-organism and the organic component derived from the micro-organism; and d. a stabilizer.

24. The composition of claim 23, wherein the microorganism is GPI-1 and is found in the formulation in a concentration of between 10.sup.6 and 10.sup.7 CFU/mL.

25. The composition of claim 24, wherein the organic component corresponds to nanoparticles made from the biopolymer secreted by the microorganism of the formulation, wherein the size of said nanoparticles is in the range of 50-500 nm and wherein the concentration of said nanoparticles is in the between 50-100 mg/L.

26. The composition of claim 23, wherein the formulation protects the plant, crop, fruit, or vegetable where it is applied, for a sustained period of time, including maintaining its protective activity on the given target for at least 6 to 18 months.

27. The composition of claim 23, wherein the formulation protects plants from consecutive freezing and thawing events, for at least 7 consecutive days.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS(S)

[0015] FIG. 1. Effect of the invention on the protection of plants against cold stress. 1A shows the effect of using the invention and 1B the effect without the invention.

[0016] FIG. 2. Effect of the invention on the protection of plants against water stress. 1A shows the effect without the invention and 1B the effect with the invention.

[0017] FIG. 3. Effect of the invention on the protection of plants against Insect Infection, after different times of exposure of the invention.

[0018] FIG. 4. Root growth stimulation assay in the presence/absence of GPI-1, (A) applying substrate with GPI-1 and (B) volatile compounds derived from GPI-1.

[0019] FIG. 5. Production of EPS nanoparticles produced by Pewman PGI-1. Result of the dynamic light scattering (DLS) analysis, using as a substrate the purified EPS of the GPI-1 strain.

[0020] FIG. 6. Protective effect of the formulation of the invention, applied in the field.

[0021] FIG. 7. Effect of the protection of the formulation of the invention, before a shock of extreme cold in potato cultivation.

[0022] FIG. 8. Effect of the protection of the formulation of the invention, in field trials in cherry trees.

[0023] FIG. 9. Resistance of GPI-1 to freeze-thaw cycles in the presence or absence of the biopolymer.

[0024] FIG. 10. MALDI-TOF of the biopolymer secreted by GPI-1

[0025] FIG. 11. Protective effect in plants of the biopolymer secreted by separate GPI-1 (purified biopolymer) and in combination with the microorganism of the invention (CRIOPROTECT).

DETAILED DESCRIPTION

Characterization of Pewman GPI-1 Bacteria (Strain Isolated and Identified from Extreme Environment)

[0026] The sequencing of the GPI-1 genome demonstrates that the strain of the invention does not contain ice-nucleating proteins (INP) or ice binding proteins (PPIs). The absence of INP classifies Pewman GPI-1 as a NINA bacterium and allows for greater tolerance to cold stress while helping to lower the freezing temperature in plant roots and foliage. In this sense, incorporating GPI-1 to the plant also decreases the proportion of INP+ bacteria, and thus decreases one of the main cold damage in tissues associated with ice nucleation previously identified and known in the agricultural industry. (F.J. Bigras and S.J. Colombo (eds.), Conifer Cold Hardiness, (2001), 89-120; Lindow Plant Physiology (1982), 70, 1084-1089).

[0027] The Pewman GPI-1 strain grows in glucose, technical glycerol and in crude glycerol, where the generation of biomass is favored in crops using glycerol as the only carbon source, and where the ability of Pewman GPI-1 to grow using crude glycerol as a carbon source decreases the costs of production of biomass and bioproducts derived from this strain.

[0028] Additionally, the GPI-1 strain does not show growth with carbon sources of structure similar to Maltodextrin (lactulose, maltotriose, maltose, xylose, etc.). It grows correctly in the presence of molecules associated with the Krebs cycle and amino acids, and in carbon sources that can be selected from the list comprising, but not limited to: arabinose, N-acetyl glucosamine, succinate, galactose, aspartic acid, proline, alanine, trehalose, mannose, glucose-6-phosphate, malate, ribose, rhamnose, fructose, acetate, glucose, thymidine, glutamate, sorbitol, fucose, gluconic acid.

[0029] GPI-1, produces organic components of biopolymer type (exopolysaccharide or EPS) and volatile type, including: Dimethyldisulfide, 1-Nonene, 1-Undecanol, 2-Undecanon, 2-Pentane, 3-Methyl-Butanal, Methyldiselenure, 2-Heptane, 2-Nonanona, 1-Undecanol, 2-Undecanone, where both organic components mentioned above have protective capacity in plants and crops, against adverse effects of climate, including drought, freezing and pests.

[0030] The EPS produced and secreted by Pewman GPI-1 is a high molecular weight sugar polymer similar to maltodextrin (more than 15 glucose units), and where there would also be some other dextrins in the mixture.

[0031] TLC analysis suggests a polysaccharide of approx. 16-19 dextrose equivalents. There is an enrichment in polymers of longer chain (close to 19 equivalents of dextrose) at longer cultivation times.

[0032] On the other hand, in the formulation the EPS of GPI-1 is in the form of polymeric nanoparticles (NPs), where these formed NPs presented sizes less than 100 nm, ideal for nanotechnological applications and the decrease of heat transfer on surfaces (such as the surface of leaf tissue).

[0033] One application of the invention is its use directly as a plant growth biostimulant, which additionally decreases damage to crops due to water and cold stress. Due to its unique operating mechanism, the invention has a broad-spectrum application effect, being able to act efficiently on any crop, fruits, and vegetables, at different scales of agribusiness, including small, medium and large producers.

[0034] In another embodiment of the invention, it corresponds to a mixture of polysaccharides purified from cultures of microorganisms grown from the rhizosphere of plants of Chile, including GPI-1, which allows the control of mites, aphids and whitefly in crops, vegetables and fruits.

[0035] In another embodiment of the invention, it corresponds to a mixture of nanoparticles derived from polysaccharides purified from cultures of the selected microorganisms, including GPI-1, which protects different plant crops against cold, water stress and insect infections.

[0036] In another embodiment of the invention, the formulation may include a substrate, wherein the substrate may comprise at least one of the following components: microorganisms, metabolites derived from microorganisms, inorganic elements and biomolecules, and any combination thereof, capable of reducing the damage caused by the environment, including: drought and freezing, especially preventing ice nucleation; where this substrate remains for at least 18 months in the plant where it was applied, and induces some modulation in the plant microbiome without negatively affecting the plant microbiome in the long term (positive modulation).

[0037] In another embodiment of the invention, the formulation comprises at least one species of bacteria of the genus Pseudomonas capable of resisting environmental problems and conditions such as cold stress, including a bacterium capable of producing a specific compound, which includes a cryoprotective biopolymer, in which the biopolymer can be a carbohydrate and/or a biomolecule composed of carbon, induced in response to a specific environmental condition, including cold stress and any other equivalent environmental condition, that adversely affects the growth and development of plants, crops, fruits and vegetables.

[0038] In another application of the invention, it comprises the identification of the microorganism or microorganisms of the formulation, including identification at the genome level, where identification can be made by elements that can be selected from the list comprising: plasmids, specific genes, parts of specific sequences of a gene and any other appropriate section of the genome.

[0039] In another application of the invention, the formulation containing bacteria may be effective against pests and/or different insect infections, wherein the formulation containing bacteria, the mechanism of action includes: i) action by contact, causing suffocation by blocking the respiratory spiracles or stigmas of the pest, or ii) action by adhesion, which prevents the mite or insect from adhering to the surface of the plant, The invention can act on a wide spectrum of insects.

[0040] Additionally or alternatively, bacteria capable of producing a cryoprotectant biopolymer, which may be part of the formulation of the invention, include (at least one of) the following characteristics: [0041] 1. Where the absence of ice nucleating proteins (INP) in the genome of the bacterium increases stress tolerance in the roots and foliage of the plant by reducing the starting point of the freezing temperature. In addition, by adding the bacteria and/or the formulation containing bacteria to the plant, the proportion of bacteria of Ice Nucleating Activity (INA +) is reduced, reducing and/or decreasing the effects of one of the main damages in the tissues due to the action of cold, associated with the nucleation of ice produced during frost; [0042] 2. In which the bacterium comprises a unique mechanism of induction, production and secretion for high concentrations of a biopolymer in response to cold stress; [0043] 3. Where the bacterium is able to survive cold stress, including the freeze-thaw cycles characteristic of frost, which favor its protective effect during and after frost. [0044] 4. Where, due to the characteristics mentioned in 1 to 3, the bacterium is able to prevail or endure in the plant and resist adverse conditions, generating sustained protection over time. [0045] 5. Where in addition, the ability of bacteria to produce volatile compounds favors plant growth (mainly the establishment of roots) [0046] 6. Where the growth of the bacterium and the production of high concentrations of the polymer can occur using a different set of carbon sources, including glycerol, glucose, any other suitable carbon source and any combination of them, and where one of the main characteristics of the bacterium is that it is able to grow and produce the polymer using crude glycerol (biodiesel residue) as the only carbon source with yields equal to of pure glycerol and superior to those of glucose.

[0047] The application of the product is suggested between 1-10 L/Ha depending on the agricultural crop to be protected; It is incorporated into crops by spraying on the leaf tissue and by irrigation on the roots, and with a frequency of between 10-14 days depending on the crop and the season of the year in which it is applied.

[0048] The concentration of the biopolymer in the formulation is in the range of 1-100 mg/L; wherein, the present formulation comprises at least one ratio of UFCs of microorganism/ug of biopolymer, unique characteristic of the present invention, and wherein the range of values of the concentration of microorganism comprises 10exp5-10exp8 CFU/ug of biopolymer.

[0049] The present invention may be developed or made as a formulation and/or a composition as long as it contains the essential constituent elements described above for the protection of plants with respect to adverse environmental conditions affecting the growth, development and production of crops and plants.

Cold Resistance: Cold-Shock Proteins (Csp) Family of Transcriptional Regulators.

[0050] As part of the characterization of the GPI-1 strain, in particular, it presents 2 copies of the cspA gene, the main transcriptional regulator of DNA binding of cold response, which has been described as initiating a signaling cascade of transcription of different topoisomerases. Experimental evidence in E. coli indicates that cspA transcription is not temperature dependent, however, its mRNA degrades rapidly above 37 C., so its action is cold-dependent. In addition, it has been experimentally demonstrated that the CspA protein acts as RNA chaperone at low temperatures, preventing the formation of secondary RNA structures. A copy of the cspD gene was also found, whose product has no function described beyond a helicase homologous to CspA.

TABLE-US-00001 >198P_05228MajorcoldshockproteinCspA MATRETGNVKWFNDAKGYGFIQREDGKDVFVHYRAIRGDGHRSLSEGQQ VEYAVVTGEKGLQAEDVVGL >198P_05353MajorcoldshockproteinCspA MAERQSGTVKWFNDEKGFGFITPESGPDLFVHFRAIQGNGFKSLKEGQK VTFIAVQGQKGMQADEVQAEG >198P_04298Coldshock-likeproteinCspD MASGKVKWFNNAKGYGFINEDGKEDDLFAHYSAIQMDGYKTLKAGQPVS FEIIQGPKGLHAVNIGAPVSLGTAKEDVAQKSEKQSA

Water Stress: RpoS and DskA (DksA)

[0051] In general, drought response pathways are not specifically studied, since all factors identified to date as drought tolerance in bacteria correspond to tolerance factors to general abiotic stress. However, a 2018 study determined 2 factors as critical for specific tolerance to water stress: Dsk y RpoS. (A network of regulators promotes dehydration tolerance in Escherichia coliPubMedhttps://pubmed.ncbi.nlm.nih.gov/29457688)

[0052] RpoS is a general stressor and is present with 1 copy in the genome of our bacterium and with 4 transcriptional regulators. On the other hand, DksA is an RNA-polymerase binding protein, and our bacteria have 3 copies of that gene. Additionally, the bacterium has a copy of DksD, a protein described as homologous to DksA.

TABLE-US-00002 >198P_05240RNApolymerasesigmafactorRpoS MALSKEAPEFDIDDEVLLMETGIATESMSNEGPAVPSVRTKSKNSTALKQHKYIDYTRAL DATQLYLNEIGFSPLLTPEEEVFFARLSQKGDPAGRKRMIESNLRLVVKIARRYVNRGLS LLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHVVKEL NVYLRAARELTQKLDHEPSPEEIANLLEKPVGEVKRMLGLNERVSSVDVSLGPDSDKTLL DTLTDDRPTDPCELLQDDDLSQSIDQWLSELTDKQREVVIRRFGLRGHESSTLEDVGLEI GLTRERVRQIQVEGLKRLREILEKNGLSSESLFQ >198P_00382RegulatorofRpoS MTAVDLPAVPRVLIAEADPWSRDLLKQVLLNVRCDARLDVCADGQQAAELLRDKPYDLII ADWELPGVDGLSLLHSVRQQRRSPLLPFILLGTRNDSASVHEVLPLAPTAYLTKPLNMES LTQRLQDLLLNEGETVYCEVPALAPGMTLPVFLERRREASDGAPLRVDVQAAVQYSLEPE GLDLKRLEEQVRMDPQITAVLIAAANSAGHHGSPVQTLAMALHKLAAGQSMNLILGLALK HNVVLSDPSLKDYAERYWQLSQRTADYARSLARMLDLDHERCYSAGILHRLGDLALLRCL QDWLQGGGELDDEAIGESLYTFGAAYGSALRTRWRLPLELRQLIAAIYSLEGGVYSREAL VVNLAAQLARLTEHEGVEALAKSKTARLLKVGLPELARMRKV >198P_00843RegulatorofRpoS MNKLTSEVKVLVVDDQPLIVEELCEFLESNGYRCVPCNSSQQAIERFRDDTEIGLVLCDL HMPEMDGIELVQALQRLAGKQRVFEAIMLTGRADKQDVIKALRAGIADYYQKPINLGELL EGLQRQVVALQDRQKNLDLGHLNQKLQFLSASIDDLYHDLDKVRSSPQTVQSNEADGEVS DTDRVEIPAIFNQLSPRQLDVARLVGKGQTNYQIACELGITENTVKLYVSQVLRLTHMHN RTQLALALSPNNSPARQRVTAH >198P_01240RegulatorofRpoS MAQPSILVLEDDEIIRSLMVDVLEDFGAVVTSFPSADEGMIFLERTSDPVDLIVSDIQMP GLLNGYDLSKVVAHRWPSLPVLLTSGNTAMASQLGSTVRFLPKPWSAERLLDCVQSALLK GPPLH >198P_02056RegulatorofRpoS MQKTSATLLIIDDDEVVRASLAAYLEDSGFSVLQASNGLQGLQVFERDKPDLVICDLRMP QVGGLELIRQVTDLSPQTPVIVVSGAGVMNDAVEALRLGAADYLIKPLEDLAVLEHSVRR ALDRARLLLENQRYREKLETANRELEASLNLLQEDQNAGRQVQMNMLPVSPWSIDEFKFA HQIIPSLYLSGDFVDYFRVDERRVAFYLADVSGHGASSAFVTVLLKFMTTRLLFESKRNG TLPEFTPSQVLGHINRGLISCKLGKHVTMVGGVIDEETGLLTYSIGGHLPLPVLYTPDSV RYLEGRGLPVGLFNEATYEDHILELPPTFSLTLMSDGILDLLPEPTLKEKEAALPQRVRS AGGSLDGLRQVFGLATLGEMPDDIALLVLSRNL >198P_00128RNApolymerase-bindingtranscriptionfactorDksA MTEQDLLAQPLADYMNEAQQGFFRELLLAQRNELQVRIDAEFMVLREQEPNSDPADVGSA EEQRQWQLRLLEREKKLLDKIDEALEHLARGEYGWCRETGEPIGLKRLLLRPTATLCIEA KEREELRERHQRAI >198P_00997RNApolymerase-bindingtranscriptionfactorDksA MPTQAKQQSISGFQPYVESKGEEYMGKPMREHFSKILKQWKQDLMQEVDRTVDHMKDEAA NFPDPADRASQEEEFALELRARDRERKLIKKIDKTLQLIEDEEYGWCESCGVEIGIRRLE ARPTADLCVDCKTLAEIKEKQVGK >198P_03656RNApolymerase-bindingtranscriptionfactorDksA MTKEKLLAMPADDYMNAEQHAFFEQLLQDMKVEHHERIEQNRIAIESLDTPADPADAASV EEERTWLVNAIDRDQRMLPQLEQALGRIKEDSFGWCDDSGEPIGLKRLLISPTTKYCIEA QERHEQIDKHQRQA >198P_04298Coldshock-likeproteinCspD MASGKVKWFNNAKGYGFINEDGKEDDLFAHYSAIQMDGYKTLKAGQPVSFEIIQGPKGLH AVNIGAPVSLGTAKEDVAQKSEKQSA

[0053] It is important to mention that the GPI-1 isolate strain does not present IBP genes, which means that it does not produce ice-binding proteins, thus minimizing crystal formation.

[0054] Below are some PPI genes not present in the previously isolated, identified and characterized strain:

TABLE-US-00003 >sp|H7FWB6|IBP_FLAFPIce-bindingproteinOS=Flavobacteriumfrigoris (strainPS1)REV MKILKRIPVLAVLLVGLMTNCSNDSDSSSLSVANSTYETTALNSQKSSTDQPNSGSKSGQTL DLVNLGVAANFAILSKTGITDVYKSAITGDVGASPITGAAILLKCDEVTGTIFSVDAAGPACKIT DASRLTTAVGDMQIAYDNAAGRLNPDFLNLGAGTIGGKTLTPGLYKWTSTLNIPTDITISGSS TDVWIFQVAGNLNMSSAVRITLAGGAQAKNIFWQTAGAVTLGSTSHFEGNILSQTGINMKTA ASINGRMMAQTAVTLQMNTVTIPQ >sp|A5XB26|IBP_COLSXIce-bindingproteinOS=Colwelliasp.REV MKTLISNSKKVLIPLIMGSIFAGNVMAAGPYAVELGEAGTFTILSKSGITDVYPSTVTGNVGTS PITGAALLLNCDEVTGAMYTVDSAGPLPCSINSPYLLELAVSDMGIAYNDAAGRVPADHTELG TGEIGGLTLEPGVYKWSSDVNISTDVTFNGTMDDVWIMQISGNLNQANAKRVTLTGGALAK NIFWQVAGYTALGTYASFEGIVLSKTLISVNTGTTVNGRLLAQTAVTLQKNTINAPTEQYEEAP L >tr|B3GGB1|B3GGB1_FLAB3Ice-bindingproteinOS=Flavobacteriaceaebacterium (strain3519-10) MNKFLLLAASVAFMSFSGKAHAQAPTLGAAANFALFTTAGAVTNTGLSHITGDVGTNNAAST NFGNVDGVMQDSNGATSAAAADLLIAYNLLNAAIPTATLAPLLGNGTTLTAGNYFIGQGASLS GTLTLDGGGNSNSVFIFKIQGALSSAANTQVLLTNGALACNVFWKVEGLVDLATNTVMKGNV VANNAAIVLQSGVSLEGRALSTTGAITVTGVTVRKPILCGSAVLTGPVAPNLGTVVCYTIFSGN GALTNAGITYVTGDVGTNVGLTTGFQADNVNGTIHSNPDTSTAQAALDLNNAYTYLNTLPTDI ELLYPAAFGQNLVLTPHTYLLNAATVLNGKVTLDAQGNENAVFVIKINGALSTTVNASVELING AIAKNVFWKVDGAVDLNDYTKFKGSVIGNNGAVIINTGVEIEGRVLSTSGGISTFGINAQMTP GCELLGTGSNTVAIQAAKFYPNPFSSVLNVTMEDLNGGSTLTIYNAAGSQVFSKVLSTKTTSL SMKLPAGVYFYQMIGKNGAKQAGKLIAKP >tr|A0A654DWA0|A0A654DWA0_9BACTIce-bindingprotein(Modularprotein) OS=Marinoscillumsp.108 MKIIKSGLVLALLPILMFVGCDKDKDPVLVSPDVVSTAPADDATGIAVTAAVQFNFLADMNPET LNSTTVVLMEGTNKVATTVSYANKKLTMTPVANLKNSTVYTATVKTGAESELGAALENDFTITF TTVAEVDNEVPVISSTSPLANAVNITKGNSVSIVFNEPMNPATINVTTFTLVKGTTAVAGVVSY ADNTATFTATESFESNTAYTARITTGAQDLAGNGLAADTEWSFTTTDFAAPFINSTAPLSDAT GVARNKTVSVVFNEPMNPATISAATFQLKNGTTSVPGVVAYSGTTATFTSTTILEASTVYTAQI TTGAQDLSGNGLANNESWSFTTGEVTATLAMVNLGGASNYVILAKTAINNSSTSAITGHLGL SPAATSYITGLDLVDATGYATSSQVTGNVYAADMADPTPVNLTTAVNDMITAYNDAAGRPTPE FLELGTGNIGGMTLSAGLYKWTSTVTIPADVTLTGAADDVWIFQISGDLTQSSAINMTLNGGA QAKNIFWQVAGEATFGANSHFEGNILSMTGITFLTGASINGRALAQTAVILDANAVTKVQ >tr|K419X5|K419X5_PSYTTSecretedice-bindingcellsurfaceprotein OS=Psychroflexustorquis(strainATCC700755/ACAM623) MKNSLFTTAIISFLISFVSLEASVINSNTKGNIVNTTSISHMLVPEMMILPVFDTNPVLSNTRLS SVETSCANQLVADLIASHAELWGLTSTASHGAAFVNETLSPGVYDVITAATISGTLTLDAGGD PNALFVIRVVGALSTAVNTTVGLTGNARPENIFWVANGAISTGAGTTMKGTLIGGPPGDAAVS LGANTNHVGRMFTLGGAVTSGATGTILIIPTGTSVINLKSLSTFAMWSNLGAIATGADSNTTG DIGTFAGAISFGANSIHNGTVYSPGSDFCALIPTVWIGEVSTVAENISNWTNGFPDRDIDVLIN ITPNDPIFSENLEMKNLVIAIGASVSQTNESQIDIYGDLENNGTYNPGNSTLAFKGDGIQNFST DNTISVYNLTIDNDNSLNLLSGNVDIFNSLNLTTGDLITNYDHTIPDNNLVTFKSNATHTAIISE IKNSNTVHGEVMIERYITMQNRAFRFMTTSVNTTTSINENWQEGVNNTVNDYTQNKNPNPGY GTHITGSTTGVNGLDSTPTGNPSLFSWDSQNESWLTISNTNINTLVAGKAYGILIRGDRDTNI YSNNLVMGGDTRLRSLGTILTGDVNMDNDLNPNSEGFALIGNPYQAEVDMKATLANSSTHLD KRFYYAYKPSIGERGGYVTVDLDSEPVEHIPEVPLNNNMDSEKFRFLQVNQSVFVQTVSDLQP NEVPTLTFKEEFKTDDTSTSQVLRVNSNSKIDLNIFSNSNNELMDGVRFKFDATYDEEAGPDD ALKFWNDDETIGIHSDGNYLAIEKRPFPKDEDVFSFWIGNYRDLDYTMNVEVEDMPDYDIFLR DTYTEVDHQLNEGENDIAFSIDSSIPASVNSDRFKIRFEQITLGTSQNEMVASSQLYPNPSNS GFAYLKHNPDFNNELKVSVFNIIGQNIEIPKDLLSSSELKLNTSSLNSGIYLVKLTYQTQTTTHK LIIE >tr|K4I9B1|K4I9B1_PSYTTSecretedice-bindingcellsurfaceprotein OS=Psychroflexustorquis(strainATCC700755/ACAM623) MTIDNSHSLNLVSGNVDVFNSLNLTTGDLITNYDHSIPGNNLVTFKSNATNTAIISKIENGNTV HGEVMIERYITMQNRAFRFITTSVNTTTSINENWQEGVNNTVNDYTENKDPNPGYGTHITGST TGVNGLDATPTGNPSLFSWDSQNGSWLTISNTNTNTLEAGKAYGILIRGDRGTNMYLNNLAK GDDTRLRSLGTILTGDVNIDNDLNPNSEGFALIGNPYQAEVDMKATLKNSSTHLDKRFYYAYK SSIGDRGGYVTVDLDSEPVEHIPEVPLNNNMGPEKFRFLQVNQSVFVQTDENLOSNEVPSLTF KEEFKTDQTSTNEVLRVNSNSKIDLNIFSISNEKLMDGVRFKFDTAYDEEAGPDDALKFWND NETIGIQSDGKCLAIEKRPFPKDEDVFSFWIGNYRDADYTMNVEVEGMPDYNIFLRDTYTEVD HQLNEGENDIAFSIDSSIPASVNSDRFKIRFEQITLGTSKNEMTASSQLYPNPSNSGFAYLKHN PDFNNELKVSVFNMVGQNIEIPKDRMSSSELKLNTSSLNSGIYLVKLTYQTQTTTHKLIVE >tr|K4IDA2|K4IDA2_PSYTTIce-binding/adhesin-likeproteinOS=Psychroflexus torquis(strainATCC700755/ACAM623) MKTIILSIAIIAYSFSSIAQDEQPPIDIYLGTAANFILFTGAGAVANTGVSEITGDVGSHAGAIAG FGLPTVLNGTIQNTNSITAQALLDLAAACVQLQNIPATITDHSGIFGSLEGETIYPGVYSNAAAV SLTGTLTLDAQGDPDAMFIFKITGALNSVAGATVLLANGASSENVYWIAVGALALGANTTMKG TAIAYPGAVSLGAGASIDGSLYSTVGAIAINSTVGTKPTYNTPFGCDINAYLFQDNDVYTIDLA SGSSYEIATDITTGDINATGYNPVDGYIWGSLSSPEKTIVRVGKNFNTTSYYIDELPSSDTKIG DVSADGIYYLKGEDTTYYKIDLNPSSADFAQHQSTESLSQNISIDDWAFNAVDGNLYAIEKIS NILYRIDPSDGNVQTMGEVPILSGSTYTYDAVYFDVDGRFYISASEIGTIFVVQDVQDLDGSN AIDSNLFAFGPSSSNSDGARCPTALVAQEICDNGIDDDGDGLIDCEDPSCSGYGSCPIIESNT SGGNDGGLESNNRLSDKISQRNYNRAKINYRFDRGVARRVSKSSNYAKRSPNSNFQLQNFIP LTVIDEDYVIDSTPIDLLGITNAVDVYSV >tr|K4INU9|K4INU9_PSYTTSecretedice-bindingcellsurfaceprotein OS=Psychroflexustorquis(strainATCC700755/ACAM623) MNNLRFTTTIISFIISLASLEASVINSITEGDIVTNNPTHNVSVSEMTIFPVSGTDPVLSNAELSS SETFCATQAAADLITLYNELIAYPGGVTHPLVFGNGEILSPGVYDVGGAPSISGTLTMDGDGDP NSLFIIRGPGAFTTVAGTTVVLTGNAQPENIFWVSGAAMSTGASTIMKGTLVGGGGGAGAVS LGANTNHVGRMFTKLGAVSVGASSILAIPTGTPFFNLRSLSTFVMWSSGGALSDSASSDITG DVGTASGALAIAGTHNGRIYFPGVDYCALNPTVWTGDISTDAENVNNWTKGLPDRDIDVLINI SVNYPTFSEDVEMKNLSIATGAIVSQTNESQIDVYGDFQNNGTYNPGNSTLAFKGDEIQNFST NNTISVYNLTIDNDNSLNLLSGNVDVFNSLNLTTGDLITNYDHTIPGNNLVTFKSSATNTAIISE IKNSNTVVGEVMIERYIPMRNRAFRFMTTSVTTTTSIKDNWQEGVNNTVNDYEQNLNPNTGY GTHITGSTTGDNGFDATFTGNPSLFEWESQNGSWSTISNTNENTLEAGKAYGILLRGDRNTN IYSNNSVVGGDTRLRSLGTILTGDMNIDDDLNPNSEGFALIGNPYQAEVDMKATLANSSTHLG KRFYYAYKPNIGDRGGYVTVDLDSEPVEYIPEVSSNNNTNSEKFRFLQVNQAVFVKTDENLQP NEVPSLTFKEEFKTDQTSTNEVFRVNSNSKIDLNIFSISNNELMDGVRFKFDTTYDEEAGPDD ALKFWNDDETIGIQSDGKYLAIEKRPFPKDEDVFSFWIGNYRDADYIMNVEVENMPDYDVFLR DTYTEEDHQLNEGENDIAFSIDSSIPASVNSDRFKIRFEQITLGTSKNEMTASSQLYPNPSNSG FAYLKHNPDFNNELKVSVFNMVGQNIEIPKDRISSSELKLNTSSLNSGIYLVKLTYQTQTTTHK LIVE >tr|K419X0|K419X0_PSYTTSecretedice-bindingcellsurfaceprotein OS=Psychroflexustorquis(strainATCC700755/ACAM623) MKTIILSIAIIAYSFSSIARDEQSPIDIYLGTAADFILFTGAEAIANTGISDITGDVGSHVGAIAG YGPPTILDGTIQNTNSITAQALLVLASGSSYEIATDISTRNINAAGYNLVDGDIWGSFSSPEMK VLPVSDTNPVLSNIELSSSETFCATQATADLITLYNELIAYPGGVTHPLVFGNGEVLSPGVYDIG GAQSISGTLTMDGGGDPNSLFIIRGPGAFTTVAGSTVVLTGNAKPENIFWVSSAAMSTGASTI MKGTLVGGGGGAGAVSLGANTNHVGRMFTKLGAVSVGASSILAIPTGTPFFNLRSLSTFVMW SSGGALSDSASSDITGDVGTASGALAIAGTHNGAVYHPGIDYCALNPTIWIGEVSTVAENINN WTKGFPNRDIDVVINITPNDPIFSENIEIQNLSIATGASVSQANESQIDVYGDFQNNGTYNPG NSTLAFKGDEIQNFSTNNTISVYNLTIDNDNSLNLLSGNVDVFNSLNLTTGDLITNYDHTIPGN NLVTFKSSATNTAIISEIKNSNTVVGEVMIERYIPMRNRAFRFMTTSVTTTTSIKDNWQEGVN NTVNDYEQNLNPNTGYGTHITGSTTGDNGFDATFTGNPSLFEWESQNGSWSTILNTDTNTLE AGKAYGILIRGDRATNIYVNNNSRGGDTNLRSLGIILTGDVNIDADLNPNPDGFSLIGNPYQA EVDMKKTLANSSKHLDKKFYYAYRPNLGTRGGFVAVDLNANPVEGVPNDPTDENTIAAKFRYL QVNQSVYVQTDQNIQPTQVPLLTFKEKFKTDQSSTNVVFRDVPTSKVDLNIFSNSNNKLMDG VRFKFDATYDEEAGTDDALKFWNDDETIGIQSDGNYLAIEKRPFPKDEDIFSFWIGNYRDIDYI MNVEVESMSDYVIFLKDTYTEVDHQLNEGENDIAFSIDSSVPASINSDRFKIQFEKTTLGTSQ NEMAGSSQIYPNPSNSGFAYLLHNPDFNSELKVSVFNILGQSIAIPKDRLSSSELKLNTSSLNS GIYLIKLTYQTQTTTHKLIIE >sp|Q086E4|IBP1_SHEFNIce-bindingprotein1OS=Shewanellafrigidimarina (strainNCIMB400) MNHSIKKTYLVFTMLLGFILLAGCNGDNNNDNSNNDNNGVLLTSIAVTPATPSMPLGLKQQFT AMGTYSDGTSSDITNSATWSSDDSTVATINGSGLAMGVIPGSVAITASLIDSSSNEQSATTTL TITDATLTALAITPVNPSLAKGLTKQFMATGTYSDGTSPDVTTSVTWSSANTLVATVNASGLA SGVAIGSSIITASLGSDETTTELNITDAILSSIALTPVEPSIAKGITQQFTAIGTYSDGISVDITAS SNWSSADTLVATMNTSGAAKGVSIGSSIITADFQAQSATSLLTVTDASLTSIMLTPANPHIPK GNTLQLTATGIYSDGISVDITSSAIWSSADTLIATVNADGVVSGITSGSAIITATSAALSATTTV TVTDTTLTSIAVTPGNQTIVKGSNKQLTATGTYSDGSLANITASVTWSSADTLVATVNNSGLA SGIETGSSLISASSGALSGSTNLTITGAALNSIVVSPTNLSLVKGMNKQFAATATYSDGSVADI STSVTWSSADTLVATIDVNGLANGKAAGSSLITATSGAQSNSTNLTVTDATLNSIDVTPINPSI IKNSSQNFVATGHYSDGSTTNITSTVMWSSADTLVATLNPNEQLNSGRATAIEVGSSVIQASL SGVFADTTLNVTAALPNNPLAPELGEVARFAMLASQAITTTSGSAIVDGDLGILDQARSYYAGF TPGVNAGEFDELTNGLSYAGDDSTPPYVVPVPYASMVAFINQSRTDLGIAYNFLAADPNPNAA TQVCPIELGNLTLTRGVYKTAADVTLQTGTLTLDGEGDPDSVFIFTIGGNLTSGAPGGDIVLIN GAQAKNIYWRTAGKTVIGTNTNFSGNVFAWSEVNVRTGANVTGRLFAVTDQVTLDANAVTK AN

EXAMPLES

[0055] The present invention (microbial formulation) has been tested both in the laboratory and in the field. The laboratory test showed that it favors the growth of plants such as Arabidopsis thaliana, and also reduces the damage caused by low temperatures. On the other hand, field experiments demonstrated that the invention has an obvious protective effect against cold and water stress in avocados treated with the current invention.

[0056] As an example of the invention and its use in different environmental conditions, we describe below a set of examples with respect to different types of environmental conditions tested, as follows:

Example 1. Effect of the Invention on the Protection of Plants against Cold Stress

[0057] FIG. 1 shows the effect of the invention applied in avocado trees, subjected to frost for 10 days (with average temperatures of 2 C. at night). A) Avocados treated with the formulation of the invention and B) Untreated avocados, both subjected to the same conditions of soil, light, irrigation and temperature.

Example 2. Effect of the Invention on the Protection of Plants against Water Stress

[0058] FIG. 2, shows the effect of the invention on avocados subjected to water stress for 9 days. Untreated avocados (A) and treated with the formulation of the invention (Crioprotect) B) subjected to the same conditions of soil, light, temperature and absence of irrigation (drought).

Example 3. Pewman GPI-1 Promotes Plant Growth by Stimulating Lateral Root Growth

[0059] Trials in Arabidopsis thaliana have shown that the addition of GPI-1 to the substrate of the plant favors the formation and growth of lateral roots. This same result was obtained by exposing A. thaliana seedlings to volatile compounds generated by GPI-1. (FIG. 4) [0060] 1. Root growth stimulation assay by supplementing the substrate with GPI-1. [0061] 2. Root growth stimulation assay when exposing seedlings to volatile compounds generated by GPI-1.

Example 4. Example of Formulation of the Invention

[0062] Our formulation contains between 10.sup.5 to 10.sup.8 CFU/ml of total bacteria, where the formulation also contains: an aqueous solubilizer that contains amino acids, minerals, glycerol as a stabilizer and biopolymers, where at least one of these biopolymers is produced directly by one of the microorganisms of the formulation.

[0063] Under the growth conditions of the bacteria used in the formulation, these bacteria are able to form and secrete biopolymers at the nanometric level (FIG. 5), essential to generate better protection against cold stress.

Example 5. Protective Effect of the Formulation

[0064] A) A panoramic view of the division between the unprotected sector (left) and the protected sector (right) is shown where a clear discoloration is observed in the unprotected sector (FIG. 6). [0065] B) Plants in the unprotected sector have dry, damaged and yellow leaves, apart from completely dead plants being observed (FIG. 6). [0066] C) Plants in the protected sector visibly healthier, more vigorous and without dead plants (FIG. 6).

Example 6. Protection against a Cold Shock in Potato Cultivation

[0067] Se muestra el resultado correspondiente a plantas con y sin tratamiento de la formulacin de la invencin, que estaban en el campo. Las plantas fueron luego expuestas a un shock de fro a 15 C. por 5 min y posteriormente, se evalu el dao (FIGURA 7).

Ejemplo 7. Proteccin ante un shock de fro en Cerezos

[0068] Leaves extracted directly from plants in treated and untreated field trials with the formulation are shown and exposed to environmental frosts. A difference is observed in the size and number of necrosis points on the upper surface of the leaves (FIG. 8).

Example 8. The Biopolymer Produced by Pewman GPI-1 Promotes Tolerance to Freeze-Thaw Cycles

[0069] GPI-1 in the presence of the biopolymer survives a greater number of freezing cycles than when it is growing without the biopolymer. The effect of the biopolymer on the bacterium by producing and secreting the biopolymer was evaluated, and also by adding the purified biopolymer to GPI-1 cultures (two concentrations of biopolymer, 1 and 1.5 X) (FIG. 9).

Example 9. The Biopolymer Produced and Secreted by Pewman PGI-1 is a High Molecular Weight Sugar Polymer

[0070] El Biopolimero EPS sintetizado por GPI-1 fue analizado por espectroscopia de masas (MALDI-TOF). Los resultados coinciden mayormente con la mayora de los peaks encontrados en estndar de maltodextrina comercial, confirmando que corresponde a un polimero de dextroza con algunas modificaciones (cuya produccin y secrecin no ha sido descrita a la fecha en bacterias) (FIGURA 10).

Example 10Adding Biopolymer or CRIOPROTECT to Plants Decreases Damage from Leaf Freezing (FIG. 11)

Example 11Characterization of Bioactive Volatile Compounds Produced by Pewman GPI-1

TABLE-US-00004 TABLE 2 Organic volatile compounds produced by GPI-1 in LB media. Tiempo rea Retencin Compuesto Match R. Match Probabilidad (%) 3.37 Dimetildisulfuro 813 899 93.6 3.35 7.36 1-Noneno 915 918 26.2 0.21 13.74 1-Undecanol 898 903 6.64 89.78 18.88 2-Undecanona 922 929 90.6 6.66

Example 12Characterization of Volatile Compounds Generated by GPI-1, Performed by GC-MS

TABLE-US-00005 TABLA 3 Organic volatile compounds produced by GPI-1 in LB media supplemented. Tiempo rea Retencin Compuesto Match R. Match Probabilidad (%) 2.58 2-Pentano 856 865 72.6 0.31 3.44 3-Metil-Butanal 676 847 34.6 0.4 6.53 Metildiselenuro 844 846 97.3 1.16 6.90 2-Heptano 883 898 82.1 1.05 13.3 2-Nonanona 934 935 89.2 39.96 13.81 1-Undecanol 890 900 6.49 50.62 18.90 2-Undecanona 899 900 88.7 6.49

TABLE-US-00006 ListofSequences SeqIDNo.1 <210> 1 <211> 1532 <212> DNA <213> GPI-1(Cepaaislada) <220> <221> 16S <400> 1 TGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCG 60 AGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAATGCCTAGGAA 120 TCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACG 180 GGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTA 240 GTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAG 300 TCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGG 360 ACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTOGGATTGTA 420 AAGCACTTTAAGTTGGGAGGAAGGGCAGTAAATTAATACTTTGCTGTTTTGACGTTACCG 480 ACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCG 540 TTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAT 600 CCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACGAGCTAGAGTATGGTAGAGGGTG 660 GTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAG 720 GCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTA 780 GATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCT 840 TAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAAC 900 TCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAAC 960 GCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTT 1020 CGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT 1080 TAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTC 1140 TAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCC 1200 CTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGA 1260 GGTGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCG 1320 TGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCC 1380 TTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACC 1440 TTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGC 1500 CGTAGGGGAACCTGCGGCTGGATCACCTCCTT 1532