ES2336758B1 - PSEUDOMONAS FLUORESCENS N. 21.4 STIMULANT OF THE SECONDARY METABOLISM OF PHENOLIC COMPOUNDS. - Google Patents

Abstract

Pseudomonas fluorescens N21.4 stimulating the secondary metabolism of phenolic compounds.

Bacterial strain Pseudomonas fluorescens N21.4, microorganism of the Gram- group of bacteria, genus
Pseudomonas, stimulant of the secondary metabolism of phenolic compounds. This strain has been isolated from the rhizosphere of Nicotiana glauca, in nutritive agar (PCA), and has been characterized from the morphological, biochemical and genetic point of view by partial sequencing of the 16s gene. I can be used in order to increase the content of phenolic compounds in plant species of pharmacological and food interest, whose application would be to obtain more active ingredients and / or new foods with a standardized content of phenols, such as bean sprouts with higher content in isoflavones, or fruits of the forest with higher anthocyanin content, also having application as a phytosanitary, strengthening plants against the attack of pathogenic biological agents.

Description

Pseudomonas fluorescens N21.4 stimulating the secondary metabolism of phenolic compounds.

The present invention relates to a strain of Pseudomonas fluorescens (N21.4, internal laboratory code) for use in the treatment of plants in order to induce the synthesis of phenolic compounds. of secondary metabolism with pharmacological and nutritional interest, in addition to strengthening plants against external agents, so it is also of interest as a phytosanitary.

This strain has been deposited for the purpose of patent in the Spanish Type Culture Collection (CECT), dated October 29, 2009, where it has been assigned the number 7620. The CECT is headquartered in the University research building of Valencia, located on the Burjassot campus (DP 46100 - Valencia, Spain).

The invention falls within the fields of biotechnology, pharmacology and new foods- This strain bacterial can serve as a basis for the preparation of different types of stimulant products of secondary metabolism of plants. These products will improve the bioactive content of phenolic nature that may constitute active ingredients of various medications, improve the quality of certain foods and improve plant defense against agent attack Biological pathogens

State of the art

The mechanisms of action of bacteria promoters of plant growth, can be summarized in two types: Direct, when the metabolites produced alter the metabolism of the plant (hormonal activity, stimulation of the mechanisms defensive ..), and indirect, when they synthesize compounds that facilitate the uptake or mobilization of nutrients or prevent growth of pathogenic microorganisms on the plant, without alter the metabolism of the plant.

In this case, one of the direct mechanisms, that is to say that they alter the metabolism of the plant. The plant has a secondary metabolism, highly inducible, related to the defense of the plant and adaptations to adverse situations, which you have to face. Within this secondary metabolism is the metabolism of phenolic compounds., which in addition to being related to plant defense, are of interest to human health, both when you consume plant-based foods that contain them from natural form, such as plant extracts dedicated to supplements Nutritional They are also important as a source of principles assets for obtaining medicines.

Pseudomonas fluorescens belongs to the group of Gram - bacteria. The genus Pseudomonas is common among soil bacteria, and can be opportunistic pathogens in animals and plant pathogens. Following the taxonomy of the Bergeys Manual, March 2001 edition, this bacterium is framed within the Bacteria Domain, Phylum Proteobacteria, Gamma Proteobacteria Class, Pseudomonadal Order, Pseudomonaceae Family. The genus Pseudomonas is very common in the edaphic system, and has repeatedly been described as a protective bacterium against various plant diseases. Some bacteria in this group produce fluorescent pigments of yellow-green colors easily soluble in water. Among other functions, these pigments act as siderophores (molecules capable of capturing iron from the medium for the metabolism of the microorganism). In addition, they have great metabolic versatility due to a large number of plasmids containing inducible operons for the synthesis of specific enzymes that allow catabolizing the compounds present in the medium. The presence of Pseudomonas sp. in the rhizosphere of different plants it affects beneficially to its physiology indicating that its selection by the plant at the rhizospheric level is very possible. The effects of this strain on the physiology of different plants indicate that the plant selects them for their benefit.

Although there are references in the scientific literature that cite Pseudomonas fluorescens for its interest as a promoter of plant growth and protective against pathogens, its ability to stimulate the metabolism of phenolic compounds has not been cited. for therapeutic purposes, through human nutrition or as a source of active ingredients for nutritional supplements. (Evidence of disease resistance induced by rhizosphere pseudomonad against pseudomonas-syringae pv, phaseolicola Journal of general and applied microbiology Volume: 41 Issue: 4 Pages: 315-325 Published: AUG 1995. Alstrom, s .; Effect of plant growth-promoting rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cicer arietinum) Singh UP, Sarma BK, Singh DP CURRENT MICROBIOLOGY Volume: 46 Issue: 2 Pages: 131-140 Published: FEB 2003; Differential methods of inoculation of plant growth-promoting rhizobacteria induces synthesis of phenylalanine ammonialyase and phenolic compounds differentially in chickpeabasha SA (Basha, SA), Sarma BK (Sarma, BK), Singh DP (Singh, DP), Annapurna K (Annapurna, K.), Singh UP ( Singh, UP) MICROBIOLOGICAL FOLIA Volume: 51 Issue: 5 Pages: 463-468 Published: 2006).

Carrying out a retrospective search of patents worldwide in the Spanish production database Invenet (SPTO) and in the international Worlwide, through the Esp @ cenet system, the conclusion drawn from the scientific literature consultation is confirmed: the absence of Patent documents related to the bacterial species Pseudomonas fluorescens as inducer of the secondary metabolism of phenolic compounds. with pharmacological and nutritional interest, and also as a phytosanitary, since no patents have been found where the use of Pseudomonas fluorescens as a stimulant of phenolic compounds has been claimed. in relation to the defense of the plant.

After the aforementioned search, it has been found that there are 419 patents related to the Pseudomonas fluorescens bacteria published worldwide (seven with effects in Spain), of which only four (none Spanish) are related to phenolic compounds., But none of them Pseudomonas fluorescens is characterized by its stimulating capacity of the secondary metabolism of phenolic compounds. in plants, which is the object of the present invention.

Indeed, in the four patents found for Pseudomonas fluorescens they are related to the metabolism of phenolic compounds .; namely: GB2431926A "Charred biological material carrying microbes", KR2003008 6477A "Microbial agent sewage and watewater treatment", EP1537919A2 "Method for microbiologically decontaminating polluted materials resulting from road demolition" and US6406882B1 "Immobilized microbial consortium-for treatment of phenolic waste from petroleum refineries ". In all of them the phenolic compounds. they have the condition of contaminating agents, and the microorganism is used in order to decompose them in decontamination treatments of soils, sewage, biodegradable waste, etc., but not because of their stimulating capacity of the secondary metabolism of phenolic compounds. in plant species.

The invention

The object of the invention described herein and which, in view of the prior art, is understood to comply with the conditions of novelty and inventive activity necessary to be worthy of the patent right, is the isolation and characterization of the Bacterial strain Pseudomonas fluorescens N21.4 (CECT 7620), which is a microorganism of the Gram - bacteria group, genus Pseudomonas, capable of inducing the synthesis of phenolic compounds. of secondary metabolism in plant species that, due to the nature of such compounds, are of pharmacological and / or nutritional interest, in addition to being applied as a phytosanitary against the attack of pathogenic biological agents.

The physiological characteristics and the genetic analysis of this strain allow us to identify it unequivocally, differentiating it from other species of the genus Pseudomonas .

Once isolated and characterized, they were performed various tests to reveal biochemical activities Indicators of their potential defense induction capacity systemic and promotion of plant growth. These were, auxin production, degradation of 1-aminocyclopropane-l-carboxylate, phosphate solubilization and production of siderophores and chitinases, proving positive for the production of siderophores and chitinases and phosphate solubilization.

So far there have been consistent experiences in the inoculation of bacterial suspensions of Pseudomonas fluorescens on tomato seeds, a Solanacea of great agricultural interest. In these experiments a notable decrease in the biomass of tomato plants of 5 weeks has been detected. However, the plants subsequently treated with the pathogen Xanthomonas campestris pv tomato were better able to resist the attack of the pathogen reaching a 40% decrease in the pathology.

The direct inoculation of the strain in the model plant ( Arajbidopsis thaliana ) has also been tested, where it has induced an effect of systemic resistance against infections by the foliar pathogen P.syringae DC3000.

These two experiments performed in different plant species show that the bacteria is able to stimulate defensive metabolism, and yet there is no analysis of the compounds responsible for said defensive response.

On the other hand, elicitation experiments have been carried out with Pseudomonas fluorescens N21.4 in plants of the Leguminosae family, in particular in soybean seedlings ( Glycine max. Osumi), observing an increase in the production of isoflavones, which are both of pharmacological interest, by constituting active ingredients of various medications, such as nutritional, by having foods enriched in said phenolic compound. When N21.4 is applied to soybean plant seeds ( Glycine max. Osumi), it is possible to modify the isoflavone profile, with a higher concentration of aglycones, active forms both for the defense of the plant and for human health. These experiments have been carried out both by direct inoculation of Pseudomonas fluorescens in the biological material and by elicitation with fragments of a polysaccharide nature from its cell wall. Elicitation experiments have also been carried out with cell wall fragments on soybean cell cultures. In all cases the results have been similar.

Elicitation experiments have also been carried out with Pseudomonas fluorescens N21.4 in seedlings of Hypericum perforatum , family Hypericaceae, resulting in an increase in the content of phenolic compounds of pharmacological interest. Specifically, its application in Hypericum perforatum seedlings means an increase in the content of hypericin and pseudohypericine. These experiments have been performed by direct inoculation of Pseudomonas fluorescens in the biological material by applying several doses for 14 weeks.

Pseudomonas fluorescens N21.4 can also be applied to any species of plants that constitute red fruits or berries, such as strawberry, raspberry, blackberry, cranberry, or mirtillo, in order to increase their content in phenolic compounds of pharmacological interest , nutritional and / or phytosanitary. In particular, the application of the aforementioned bacterial strain in blackberry ( Rubus fruticosus ) is being tested.

The referred experiments on the use of Pseudomonas fluorescens N21.4 as elicitor of the secondary metabolism of phenolic compounds are presented at the end of the present specification, within the section embodiment.

The purpose that is pursued, in short, with this invention and that constitutes the technical advantage provided with the it is to have a bacterium that improves the content in secondary metabolites of phenolic nature in plants of interest pharmacological and nutritional, and at the same time have an effect such as phytosanitary, making contact by any means available to said bacterium or any of its parts with the plant.

Consequently, with the present patent application the use of the strain Pseudomonas fluorescens N21.4, or any fraction thereof, is claimed for application in any type of plant species, forming part of any preparation, either individually or in combination with other organisms, in order to increase the levels of secondary metabolites of phenolic nature that: 1) improve the quality of the natural product, either for direct consumption or for the preparation of nutritional supplements or plant extracts; 2) serve as active ingredients for the preparation of various medications; and 3) reinforce in any case the resistance of plants against external pathogens.

The use of this bacterium in plants, or in plant cell cultures, allows to stimulate the synthesis of secondary metabolic products of phenolic nature with Pharmacological, nutritional and / or phytosanitary interest.

Embodiment

The strain of the genus Pseudomonas described here was isolated by studying the rhizosphere of natural populations of Nicotiana glauca in a transect of the Mediterranean coast of Almeria. This plant species ( Nicotiana glauca ) was selected for being of the Solanaceae family and for having an active secondary metabolism.

The rhizosphere samples for the isolation of the bacterial strain were carried out in natural populations of Nicotiana glauca in three different soils on the coast of Almería, in the cold and warm seasons, during the years 1999 and 2000. As a result of said sampling they were collected 960 strains among which Pseudomonas fluorescens was found (N21.4, internal laboratory code). The isolation of said strain was performed in nutritive agar (PCA).

In the laboratory this microorganism is maintains a high 20% glycerol survival rate in nutritive broth (Pronadisa) at -80 ° C or in 15% glycerol in water at -20ºC and recover easily in the culture medium used for insulation in both solid phase and liquid phase a 28 ° C

For the characterization of the strains they considered different phenotypic characters that are detailed in this report: (i) colony morphology (ii) morphology of the cells, (iii) partial sequencing of the ribosomal DNA gene corresponding to the 16S subunit.

Morphological, biochemical and genetic characteristics of Pseudomonas fluorescens N21.4

Taxonomic characterization of Pseudomonas fluorescens was performed by identifying the strain by partial sequencing of 16S ribosomal DNA, its comparison with the sequences in the databases revealed a 98% homology with a strain of Pseudomonas fluorescens .

The morphology of colonies at 24 h of incubation at 28º on agar for methods standard (PCA).

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TABLE 1

one

Growing in liquid medium (nutritious broth Pronadisa) the color of the medium changes to yellow from the phase exponential growth to the stationary phase of increase.

The morphological characters of Pseudomonas fluorescens N21.4 at 24 h of incubation at 28 ° on agar for standard methods (PCA) correspond to a gram-negative bacillus.

Next, we proceeded to the genetic analysis of the strain for identification, for them the Next steps:

DNA extraction

For DNA extraction, colonies they grew for 24 hours in nutritive broth (Pronadisa) at 28ºC in agitation. After this time, genomic DNA was extracted from each bacterium with the Ultraclean ™ Microbial DNA isolation kit (MoBio, CA, USA), according to the manufacturer's instructions.

16S rDNA amplification

1500 bp were amplified by PCR corresponding to this region with the following primers: direct 5'-AGA GTT TGA TCC TGG CTC AG-3 'and reverse 5'-AAG GAG GTG ATC CAG CCG CA-3 '(Ulrike, 1989), in a 25 µL reaction with 1X of 10X buffer, 2.5 µM MgCl2, 250 µM of each DNTP, 2.5 µM forward primer, 2.5 µM reverse primer 1.25 DNA polymerase units (AmpliTaq Applied) and 100 ng of DNA bacterial. The amplification was performed in a GeneAmp thermocycler 2700 (Applied Biosystems) with the following conditions: 95ºC 5 minutes, followed by 25 cycles of 94 ° C 30 seconds, 65.5 ° C 30 seconds and 72ºC 30 seconds, ending with 7 minutes at 72ºC.

Display of the gels

The PCR product was resolved on agarose gel. 1% (w / v) in buffer Tris-Acetate-EDTA (TAE 1%) with ethidium bromide (0.5 mg / mL) and were visualized on an analyzer GelDoc2000TM 170-8126 image (Biorad, CA, USA).

DNA sequencing

Once the amplification is verified, the product PCR, was purified with the UltraClean ™ PCR kit Clean-up DNA purification (MoBio, CA, USA), is Sequence of GENOMICS SCIENTIFIC PARK OF MADRID-U.C.M. in an ABI PRIMS® 377 DNA sequencer Sequencer (Applied Biosystems, CA, USA).

Computer sequence analysis

The sequences were aligned with the Bioedit Sequence Aligment editor 5.0.3 program. ®, were manually reviewed and corrected and analyzed by BLASTN 2.2.6 (Altschul et al. , 1997) on the GeneBank EMBL and DDBJ (NCBI BLASTR website: http://www.ncbi.nlm.nih.gov /), resulting in the highest homology: Pseudomonas sp. DK4 EU158318, and the sequence being deposited in the GeneBank with the access number AY748893.

Pseudomonas fluorescens as elicitor of the secondary metabolism of phenolic compounds

1st. Experiment of elicitation in Hypericum perforatum plants by applying the bacteria in the root.- A bacterial suspension of strain N21.4 was inoculated into the root of Hypericum perforatum seedlings two weeks after germination and subsequently, inoculated every two weeks, for 12 weeks An induction of the synthesis of the active substances hypericin and pseudohypericine was observed, achieving an increase in the concentration of these active ingredients.

2nd. Elicitation experiment in soybean seedlings ( Glycine max var. Osumi) inoculating the bacteria at a radical level. Soybeans were pre-germinated and transplanted into pots filled with sterile vermiculite. The strain was inoculated and after 8 days of inoculation the seedlings were harvested and the concentration of total isoflavones was determined, analyzing in detail the different families of isoflavones present in soybeans, as well as the different chemical species and their distribution in the plant. It was found that this strain was capable of increasing the total isoflavone content, specifically increasing the concentration of isoflavones belonging to the families of Daizein and Genistein, especially in leaves and cotyledons. In addition, glycosylated and malon-constructed forms, which are the forms of transport, increased markedly, indicating that there is de novo synthesis and that there is transport to developing organs.

3rd. Elicitation experiment in soybeans ( Glycine max var. Osumi) applying the bacteria and polysaccharides of this bacterium. This experiment was performed by cutting the area of the soybean embryo and applying the bacteria and its two water-soluble polysaccharide fractions (greater than 10 kd and less than 10 kd), at a concentration of 0.01 mg / mL. The concentration of isoflavones was determined 4 days after treatment. Although in that short period of time the total isoflavone content was not increased, the relative concentration of these was altered, that is, the aglycone forms (active forms both for the defense of the plant and for human health) were increased. detriment of glycosylated and malontructivated forms - The results were similar both in the case of inoculation of the bacterium and when polysaccharide fractions are applied to the daizenia family; however, only the fraction with the highest molecular weight was able to significantly increase the isoflavone content of the genistein family.

4th. Elicitation experiment in soybean cell cultures ( Glycine max var. Osumi) applying surface polysaccharides of this bacterium. This experiment was performed by applying the two water-soluble polysaccharide fractions (greater than 10 kd and less than 10 kd), at a concentration of 0.01 mg / mL in the cell culture medium (cell calluses). Isoflavone concentration was determined 15 days after treatment. An increase in the total isoflavone content was detected, mainly due to the increase in the Daidzein family, especially increasing glycosylated and malonstituted forms to the detriment of aglycone forms; Only the fraction with the highest molecular weight was able to increase the isoflavone content of the genistein family.

Industrial application

Given the above-mentioned properties of Pseudomonas fluorescens N21.4 as a stimulator of the secondary metabolism of phenolic compounds, this bacterial strain has a specific application in the agri-food, chemical and pharmaceutical industry, being able to be used as part of any preparation (individually or in combination with other microorganisms) and bringing them into contact (to the strain or any part of it) with the seed, the radical or aerial system of the plants by any means available, in any plant species, or in any form of cultivation in vitro , to increase the concentration of secondary metabolites of phenolic nature with pharmacological and / or nutritional interest, or to strengthen plants against external pathogens.

Claims (11)

1. Pseudomonas fluorescens N21.4 (CECT 7620), a microorganism of the Gram genus Pseudomonas group of bacteria, characterized by its stimulating ability of the secondary metabolism of phenolic compounds in plant species, acting directly on plants of pharmacological and nutritional interest, and protecting them against the attack of pathogenic biological agents, also being of phytosanitary interest. 2. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived therefrom, according to claim 1, for application in any type of agricultural or forestry crop, in order to increase the production of phenolic compounds of the secondary metabolism of Pharmacological, nutritional and / or phytosanitary interest. 3. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived therefrom, according to claim 2, for application in any species of plants of the Leguminosae family in order to increase their production capacity of phenolic compounds of Pharmacological, nutritional and / or phytosanitary interest. 4. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived therefrom, according to claim 3, for application in soybean seedlings ( Glycine max. Osumi), in order to increase its isoflavone production capacity . 5. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived from it, according to claim 3, for application in soybean plant seeds ( Glycine max. Osumi), in order to modify the isoflavone profile , increasing the concentration of aglicones. 6. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived from it, according to claim 2, for application in any plant species of which constitute red fruits or berries, such as (strawberry, raspberry, blackberry, cranberry, or mirtillo, in order to increase its content in phenolic compounds of pharmacological, nutritional and / or phytosanitary interest. 7. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived therefrom, according to claim 6, for application in blackberry ( Rubus fruticosus ), in order to increase the content of anthocyanins and tannins. 8. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any of its parts, according to claim 2, for application in any species of plants of the Hypericaceae family, in order to increase the content of phenolic compounds of pharmacological interest . 9. Use of Pseudomonas fluorescens N21.4 (CECT 7620), according to claim 8, for application in Hypericum perforatum , in order to increase the content of hypericin and pseudohypericin. 10. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived from it, according to claims 1 to 9, either the individual strain or in combination with other organisms, forming part of any preparation, either individually or in combination with other organisms, and by any available means that puts the bacteria in contact with the seed, the radical or aerial system of the plants. 11. Use of Pseudomonas fluorescens N21.4 (CECT 7620), or any molecule derived therefrom, according to claims 1 to 9, either the individual strain or in combination with other organisms, or as part of any preparation, either individually or in combination with other organisms, and by any available means that puts the bacteria in contact with cells from any form of in vitro culture.