ES2388662B1 - NEW DUGANELLA VINTAGES ISOLATED FROM THE WILD AND CULTIVATED OLIVE RIZOSPHERE AND ITS USE IN VIOLACEIN PRODUCTION. - Google Patents

Abstract

New strains of duganella isolated from the rhizosphere of wild and cultivated olive and its use in the production of violacein. # The present invention relates to bacterial strains of Duganella spp. obtained from the rhizosphere of wild and cultivated olive trees. Preferably the strains are CECT 7779, CECT 7780 and CECT 7781 and more preferably the bacterial strain is CECT 7780. Furthermore, the present invention relates to its combinations with other microorganisms and to the compositions comprising the above products, as well as to a process for the production of violacein and the violacein produced for its biotechnological and agronomic application.

Description

  NEW DUGANELLA VINTAGES ISOLATED FROM THE OLIVE RIZOSPHERE IF LVESTRE and CUL TIV ADO and ITS USE IN THE PRODUCTION OF VIOLACEIN.

The present invention falls within the field of biotechnology and agronomy. Specifically, the present invention relates to bacterial strains of Duganella spp. obtained from the rhizosphere of wild and cultivated olive trees. Preferably the strains are CECT 7779. CECT 7780 and CECT 7781 and more preferably the bacterial strain is CECT 7780. Furthermore, the present invention relates to its combinations with other microorganisms and to the compositions comprising the above products, as well as to a process for the production of violacein and the violacein produced for its biotechnological and agronomic application.

STATE OF THE PREVIOUS TECHNIQUE

In recent decades, plant-microorganism interactions that occur in the rhizosphere have been subject to considerable studies since the rhizosphere is considered a potential reservoir for the discovery of microbial species of diverse biotechnological interest (Wen-Jun et al., 2004. International Journal of Systematic and Evolutionary Microbiology 54, 18111814; Ryan et al., 2009. Plant & Soil 321, 363-383). Soil and rhizosphere microorganisms have evolved functions that allow them to survive and be able to withstand the strong competition that characterizes most of those habitats. Rhizosphere bacteria can produce novel secondary metabolites with potential use as drugs and antibiotics which can be applied in humans, veterinary medicine, medicine, agriculture or industry.

The rhizosphere of wild woody trees is an unexplored environment that can be a good source of new bacteria producing bioactive compounds. Among the wild and cultivated trees, the olive tree is one of the species with the longest life and the most rich in genetic biodiversity. For thousands of years, the cultivated olive tree has been culturally and economically the main oil crop in the Mediterranean basin,

where there are about 9.5 million hectares of cultivation. The olive tree contains a high source of genetic variability, and this in the Mediterranean basin can be found mainly in two forms: wild olive

(Olea europaea subsp. Europaea var. Sylvestris (Miller) Hegi) and cultivated olive tree (Olea europaea subsp. Europaea varo europaea L.) (Green, 2002, Kew Bulletin 57, 91-140). The long history of olive cultivation (over 8,000 years) (Zohary and Spiegel-Roy, 1975, Science 187, 319-327) And the extensive hybridization between wild and cultivated olive trees make the western Mediterranean, and fundamentally southern the Iberian Peninsula (Besnard et al., 2002, Theoretical & Applied Genetics 104, 1353-1361) the region with the greatest genetic diversity for this tree (Lumaret and Ouazzani 2001, Nature 413, 700).

The longevity and high genetic diversity of the olive tree can favor selection

of specific microbial populations well adapted to the rhizosphere of this crop that may constitute unique reservoirs of microorganisms with

biotechnological interest

On the other hand, the pigmented molecules produced by some of these

microorganisms have been the subject of considerable research that has helped reveal how these molecules can provide certain microorganisms with an advantage to survive in their environment (Liu and

Nizet, 2009. Trends in Microbiology 17, 406-413).

Among these pigments is violacein, a secondary metabolite with blue-purple coloration produced by bacteria with important

Biological, biotechnological and industrial applications. Biological activities include: antibacterial, anti-viral, anti-trypanocidal, antiprotozoa and anti-ulcerogenic activity (Durán and Menck, 2001. Critical Reviews in Microbiology 27, 201-222; Durán et al., 2007. Biotechnology & Applied

Biochemistry 48, 127-133). In addition, it can have a promising clinical application in the treatment of cancer since it is effective against leukemia, cancer

Pulmonary and cell lymphoma (De Azevedo et aL, 2000. Phenomena and

Macrocyclic Chemistry 37, 93-101; Durán et al., 2007. Biotechnology & Applied Biochemistry 48, 127-133), can be used in dermatological therapies against ultraviolet (UV) radiation (Dessaux et al., 2004. La Revue de Medicine Interne 25, 659-662) , as a biological dye in the industry for staining natural and synthetic fibers (Shirata et al., 2000. Japan Agricultural Research Quarterly 34, 131-140) and can provide an advantage for survival

of certain microorganisms in their natural environment.

Violacein is produced by several Grarn-negative bacteria that inhabit

soils and seas in tropical, subtropical and glacial environments between

those that include Chromobacterium violaceum, the first and most studied

bacteria described as a producer of violacein (Durán and Menck, 2001. Critical Reviews in Microbiology 27, 201-222), Col / imanas sp. (Hakvag et al., 2009. Marine Drugs 7, 576-588), Duganel / a sp. B2 (Wang et al., 2009. Biochemical Engineering Journal 44, 119-124), Cordobacter Fluviatile (Logan, 1989. International Journal of Systematic Bacteriology 39, 450-456), Janthinobacterium Iividum (Shivaji et al., 1991. Polar Biology 11, 267-271; Lu et al., 2009. Biochemical Engineering Journal 43, 135-141), Microbulbifer sp. (Matz et al., 2008. PLoS ONE 3, e2744. Doi: 1 0.1371 / journal.pone.0002744),

Pseudoalteromonas luteoviolacea, Pseudoalteromonas tunicata and

Pseudoalteromonas ulvae (Matz et al., 2008. PLoS ONE 3, e2744. Doi: 1 0.1371 / journal.pone.0002744; Yang et al., 2007. Letters in Applied Microbiology 44, 625-630).

However, the inefficient production of violacein has restricted the industrial applications of this pigment, and therefore the problem of finding new sources of violacein producing in susceptible quantities persists.

of commercial exploitation.

EXPLANATION OF THE INVENTION

The present invention relates to bacterial strains that have an identity of between 98.6% and 99.3% with respect to the AON fragment of SEO ID NO: 1 of the AONr 16S gene, of between 93.7 % and 95.7% with respect to the SEO ID NO: 2 fragment of the gyrB gene and between 89.8% and 90.8% with respect to the SEO ID NO: 3 AON fragment of the gene saw. Preferably the bacterial strain is selected from the CECT 7779, CECT 7780 and CECT 7781 strains and more preferably the bacterial strain is CECT 7780. In addition, the present invention relates to its

combinations with other microorganisms and the compositions comprising the above products, as well as a procedure for the

Violacein production and the biolacein produced for its application

Biotechnological and agronomic.

The strains isolated in the present invention have been obtained from the rhizosphere

Olive, one of the species with the longest longevity and richness in genetic biodiversity, and have been characterized physiologically, biochemically and genetically as new strains belonging to the genus Duganefla spp. Violacein producers (example 3). These strains could be differentiated

based on phenotypic and genetic analyzes, in two groups according to

host of origin (wild olive versus cultivated olive) (example 2 and 3). The

wild olive trees represent a differentiated genetic cluster constituting a

Independent genetic pool of local olive cultivars (Belaj et al., 2010. Scientia Horticulturae 124, 323-330).

Thus, the strains isolated in the present invention, CICYT-60 (with deposit number CECT 7780), LO-22 (with deposit number CECT 7779), Mico-M (with deposit number CECT 7781), LOBA-24, Baetica-33, Mico-C and MicoM2, can be grouped according to their origin in strains of Duganefla spp. from wild olive trees (LOBA-24, Baetica-33 and LO-22) and strains of

Duganelfa spp. from cultivated olive trees (CICYT-60, Mico-M, Mico-C and Mico-M2).

All strains isolated and preferably strains CECT 7779, CECT 7780 and CECT 7781 were able to produce violacein at high levels of form

natural, that is, without trying any optimization of the process, a characteristic that makes them very attractive in biotechnological and agronomic applications because the low yields of violacein are considered one of the main limitations of wild strains for their production and

commercial exploitation. It is noteworthy that all isolated strains and, especially, the CECT 7779, CECT 7780 and CECT 7781 strains had higher levels of natural violacein production (of the order of 1.2 to 65 times more) than the levels previously cited for other bacteria producing

violacein when they grew in optimal conditions such as the strain

of the Amazon Chromobacterium violaceum (0.43 gil) (Mendes et al., 2001. Biotechnology Letters 23, 1963-1969), Janthinobacterium lividum (3.50 gil) (Lu et al., 2009. Biochemical Engineering Journal 43, 135 -141) or the first Duganelfa sp. B2 violacein producer (1.62 gil) (Wang, H. et al., 2009. Biochemical Engineering Journal 44, 119-124). However, strain CECT 7780 of Duganelfa sp. it was the one that showed the highest yields

producing almost 18 times more than Duganella sp. B2, which reached its maximum

Violacein production (1.62 gil) in the same optimal medium used in this

study and containing soluble starch as the sole source of carbon (Wang,

H. et al. , 2009. Biochemical Engineering Journal 44, 119-124) (example 4, figure 5).

In the present invention, the potential of the isolated strains as well as the crude violacein extract (bacterial extract comprising violacein) produced by them against Gram-positive bacteria is also revealed. The tests performed where the antimicrobial effect against Gram-positive bacteria Clavibacter michiganensis subsp.

michiganensis (phytopathogenic) and a strain of Bacilfus subtifis antagonist of Verticiflium dahliae isolated from the olive rhizosphere, highlights the antimicrobial activity of the isolated strains against these bacteria (example 5). In addition, according to a study by Barreta et al., 2008, Journal of Industrial Microbiology and Biotechnology 35, 783-790, which reveals the antifungal activity of violacein-producing strains and their supernatant, it is expected that strains isolated in the invention are able to act against pathogenic fungi.

On the other hand, violacein, as cited in the state of the art of the present document, has other biological activities, among which are: anti-viral, anti-trypanocidal, anti-protozoa, anti-ulcerogenic, anti-cancer activity, activity in dermatological radiation therapies

ultraviolet (UV) or activity as a biological dye in the staining industry

of natural and synthetic fibers.

Regardless of the production of violacein, it was also observed that the isolated strains of Duganefla spp. of wild and cultivated olive trees and, especially, the CECT 7779, CECT 7780 and CECT 7781 strains showed proteolytic, lipolytic and siderophores production. It is known that

Rhizobacteria producing siderophores improve the health status of plants at various levels, such as iron nutrition, and hinders the growth of pathogens, usually fungi, by limiting iron

available for pathogen growth (Kloepper et al., 1980. Nature 286, 885-886). Therefore, the fact that the strains of Duganella spp. olive oil will show lytic, lipolytic and siderophores production

it would imply a better competition capacity of the strains as inhabitants of the rhizosphere of the olive tree.

To date, the only strain of the genus Duganefla, producer of violacein, known in the state of the art is strain B2. The difference of said strain

82 with respect to any of the isolated strains of the present invention is that the strains of the invention produce between 2.5 to 17.5 times more violacein.

This fact constitutes an unexpected effect that represents a clear improvement.

of the state of the art.

Therefore, the present invention solves the technical problem posed by the difficulty of finding a bacterial strain capable of effectively producing violacein, a purple pigment with important agronomic and biotechnological applications. In summary, these isolated bacterial strains possess, between

other activities:

Antimicrobial, anti-viral, anti-trypanocidal, anti-protozoa, anti-ulcerogenic, anti-cancer activity, therapy activity

dermatological against ultraviolet (UV) radiation or activity as a biological dye in the industry for staining natural fibers and

Synthetic Violacein Extract (bacterial extract that

includes violacein), and proteolytic, lipolytic and siderophores production, which allow them to improve the health status of plants and hinder the growth of pathogens, usually fungi, in addition to increasing their ability to compete in the rhizosphere of

olives.

One aspect of the present invention relates to a bacterial strain with a

identity of between 98.6% and 99.3% with respect to the AON fragment of SEO ID NO: 1 of the AONr 16S gene, between 93.7% and 95.7% with respect to the fragment of SEO ID NO: 2 of the gyrB gene and between 89.8% and 90.8% with respect to the SEO ID NO: 3 fragment of the gene

saw. Preferably the bacterial strain comes from the rhizosphere of cultivated olive trees and / or wild olive trees. More preferably the bacterial strain is

CECT 7779, CECT 7780 or CECT 7781, And even more preferably the bacterial strain is CECT 7780.

The term "% identity" between two nucleatide sequences, as

understood in the present invention, refers to the number of positions

nucleotides over the total length of the sequence being compared, where all nucleotides in that position are identical.

A preferred embodiment of the present invention relates to a strain derived from any of the strains described in the preceding paragraph where said strain maintains or improves the capabilities described throughout the present invention. The derived microorganism can be produced naturally or intentionally, by methods of mutagenesis known in the state of the art such as, but not limited to, the growth of said original microorganism in the presence of mutagenic or stress-causing agents or by engineering. genetics aimed at the modification of specific genes. Preferably the derived strain is a genetically modified mutant. The terms mutant strain or derived strain can be used interchangeably.

Table 1 shows a comparative analysis of the 16S, gyr8 and vioA rDNA genes of the selected strains of the invention against the Duganella violaceinigra YIM 31327 strain, which according to phylogenetic analysis is considered the baseline group of all strains of Duganella spp. olive tree (example 3). Thus, strains that have a percentage of identity with respect to the SEO ID NO: 1 sequence (DNA sequence of the 16S rDNA gene of the Duganella violaceinigra strain), SEO ID NO: 2 (DNA sequence of the gyr8 gene of the strain Duganella violaceinigra) and SEO ID NO: 3 (DNA sequence of the vioA gene of the Duganella violaceinigra strain, sequenced by the researchers) of

between 98.6% and 99.3%, between 93.7% and 95.7% and between 89.8%

and 90.8%, respectively, are strains whose 16S rDNA, gyr8 and vioA genes are homologous to said genes of any of the strains selected in the present invention.

Table 1. Comparative study of the rDNA 168, gyr8 and ViDA genes of the isolated strains of the invention with respect to the Duganefla viDfaceinigra strain with strain no. YIM 31327.

DNA sequence analyzed

SEO ID NO. Duganeffa Violaceinígra Olive strains SEO ID NO. Strains % Identity regarding Duganeffa viofaceinigra

RDNA 168

one CICYT-60 4 98.6

MICO-C

19 99.1

MICO-M

10 99.0

MICO-M2

22 99, 1

Baeti ca-33

16 98.9

LO-22

7 98.9

LOBA-24

13 99.3

gyr8

2 CICYT-60 5 93.7

MICO-C

twenty 93.8

MICO-M

eleven 94.6

MICO-M2

2. 3 95.3

Baeti ca-33

17 95.1

LO-22

8 94.6

LOBA-24

14 95.7

saw

3 CICYT-60 6 90.5

MICO-C

twenty-one 90.7

MICO-M

12 90.5

MICO-M2

24 90.8

Baetica-33

18 89.8

LO-22

9 89.8

LOBA-24

fifteen 89.8

On the other hand, Table 2 shows a comparative analysis of the 168 rDNA, gyr8 and ViDA genes of different strains, selected for having a

higher degree of homology with respect to Duganella violaceinigra with nQ of

YIM 31327 strain. Following the results obtained in said table it is possible to

affirm that there are no other strains in the state of the art that

are within the range of percentages of identity, with respect to the SEO ID NO: 1, SEO ID NO: 2 and SEO ID NO: 3 sequences of Duganella

Violaceinigra, defined for the isolated strains of the invention.

Table 2. Comparative study of the AONr 16S, gyr8 and vioA genes of different strains with respect to the Duganel / a violaceinigra strain with strain YIM 31327.

% IDENTITY REGARDING DUGANELLA VIOLACEINIGRA

Strains

RDNA 165 GytS Saw

Duganel / a_sp._B2

96.2 86.6 77.9

Duganel / a zoogloeoides IAM 12670

95.3 87.5

Duganel / a nigrescens EF584756

98.7

Janthinobacterium lividum EU71441 O

95.8 86.9 77, 1

Janthinobacterium Iividum strain SKVTC8 EU732703

77, 1

Janthinobacterium Iividum DSM1522 00074977

77, 1

Janthinobacterium lividum EU330449

95.6 76.9

Janthinobacterium Iividum strain BP01 EF063591

76.9

Janthinobacterium sp. Marseil / e CPOO0269

83.9

Zoogloea ramigera AB014955

79, 1

Pseudoalteromonas luteoviolacea UST011230-013

61.7

Pseudoalteromonas luteoviolacea ATCC 33492

69.2

Col / imonas_sp. CT_MP11 E6 G0160909

77.3

Col / imonas sp. GU062792

77.5

Chromobacterium violaceum JCM 1249 AB032799

62, 1

Chromobacterium violaceum strain ATCC 12472 AE016825

62, 1

Chromobacterium violaceum AF17285 1

62.0

As shown in Table 2, the only two strains that have the genes

ADNr 16S, gyl13 and vioA are Duganefla sp. B2 and Janthinobacterium Iividum

EU714410. These strains have a homology with respect to the 16S rDNA gene not

greater than 96.2%, with respect to the gyl13 gene no greater than 86.1% and with respect to the vioA gene

not more than 77.9%.

From here on, to refer to any of the bacterial strains described in previous paragraphs that have between 98.6% and 99.3% identity with the SEO ID NO: 1 nucleotide sequence, between 93.7% And a 95.7% identity with the SEO ID NO: 2 nucleotide sequence and between 89.8% and a 90.8% identity with the SEO ID NO: 3 nucleotide sequence or any fragment thereof, you can use the term "bacterial cepals of the invention" or "cepals of the invention".

Preferably the bacterial strain of the invention is the strain with number of

tank CECT 7779, CECT 7780 or CECT 7781. More preferably the bacterial strain of the invention is CECT 7780.

Duganefla spp. LO-22 and CICYT-60 have been deposited in the Spanish type crop collection (CECT) on September 14, 2010 and were assigned the deposit numbers CECT 7779 and CECT 7780 respectively, and the strain of Duganefla spp. Mico-M was deposited in the Spanish collection of type crops (CECT) on October 13, 2010 and it was assigned the deposit number CECT 7781. The address of said Authority

International deposit is: University of Valencia / Research building

1 Burjassot Campus 1461 00 Burjassot (Valencia).

The scientific classification of the CECT 7779, CECT 7780 and CECT 7781 strains of the present invention is: Kingdom: Bacteria / Phylum: 3-Proteobacteria 1 Order: Burkholderiales / Family: Oxalobacteraceae / Genus: Duganefla / Species: two groups have been identified different genetics that can be separated from the Duganefla violaceinigra species depending on the host of origin (cultivated olive tree (CECT 7780 and CECT 7781) and wild (CECT 7779)). (Description

based on physiological and biochemical characterization (example 2), and the phylogenetic analysis of the 16S, 9yrB and vioA genes (example 3)).

5 the

The characteristics of the strains are: - OPTIMAL CULTURE CONDITIONS: Striated plate, R2A agar medium (Biolole, Milan, Italy); Incubation at 25 "on 48-72 h; Formula R2A (Biolife) agar (gil): Yeast extract: 0.5; Protein peptone: 0.5; Acid casein: 0.5; Glucose: 0.5; Soluble starch : 0.5; Dipotassium phosphate: 0.5; Magnesium sulfate: 0.024; Sodium pyruvate: 0.3; Agar: 12; final pH: 7.2 ± 0.2.

fifteen

-MAINTENANCE CONDITIONS: Cell suspension in TSB medium (Soy Broth-Triptone) + Glycerol 40% aqueous solution. (-80'C); Cell suspension in KMB medium (Kings B medium) + Glycerol 40% aqueous solution. (-80'C) Medium formula KMB (gil): Protein peptone: 20; Potassium Phosphate: 1.2; Dipotassium phosphate: 1.5; Magnesium sulfate: 1.5; Glycerol: 10; Agar: 20; pH 7.0 (sodium hydroxide).

twenty

-CONDITIONS FOR CHECKING THE VIABILITY: Striated plate, R2A agar medium or KMB medium; Incubation at 25 "48-72 h; No specific aeration conditions (aerobic).

25 30

Another aspect of the present invention relates to the combination of microorganisms comprising at least one of the strains of the invention. The combination of microorganisms is a set of cells of microorganisms of at least one of the strains of the invention, or at least one cell of at least one of the strains of the invention together with a set of cells of microorganisms of another of the strains of the invention or of a different strain of the same or different species. The cells of the combination of microorganisms can be viable or non-viable, in any

stage of the state of development and in any phase of growth, seasonal or

stationary, regardless of the morphology present.

A preferred embodiment of the present invention relates to the combination of microorganisms wherein said combination comprises at least one other microorganism different from the strains of the invention, for example, but not limited to, the microorganism that can be part of said combination is,

Without limiting this:

at least one other bacterium of the genus Duganel / a, where said bacterium is

select from the list that includes, but not limited to Duganella

violaceinigra (for example but not limited to the strain Duganel / a violaceinigra YIM 31327), Duganel / a nigrescens sp. Nov. (for example, but without limiting the strain Duganel / a nigrescens sp. nov. EF584756), Duganel / a sp. B2, Duganel / a zoogleoides or Duganel / a sp. tsz33;

at least one violacein producing bacterium, where said bacterium is selected from the list it comprises, but not limited to

Chromobacterium violaceum, Col / imanas sp., Lodobacter fluviatile, Janthinobacterium lividum, Microbulbifer sp., Pseudoalteromonas

luteoviolacea, Pseudoalteromonas tunicata or Pseudoalteromonas ulvae;

at least one strain of other phylogenetic groups, genera or species of prokaryotes such as but not limited to Archaea, Firmicutes,

Bacteroidetes,

Proteobacteria, Actinobacteria, Verrucomicrobia,

Fusobacteria,

Methanobacteria, Spirochaetes, Fibrobacters,

Deferribacteres,

Deinococcus, Thermus, Cyanobacteria,

Methanobrevibacterium,

Peptostreptococcus, Ruminococcus,

Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis,

Twin, Roseburia, Catenibacterium, Dialister, Anaerotruncus,

Staphylococcus, Micrococcus, Propionibacterium, Enterobacteriaceae, Faecalibacterium, Bacteroides, Parabacteroides, Prevotella,

Eubacterium, Akkermansia, Bacillus, Butyrivibrio or Clostridium.

Hereinafter reference may be made to any of the combinations of

microorganisms described in the previous paragraph as the "combination of

microorganisms of the present invention "or the" combination of microorganisms of the invention ".

Another aspect of the invention relates to the violacein produced by at least one of the strains of the invention or by the combination of microorganisms of the invention, hereinafter, violacein of the invention.

And another aspect refers to the bacterial extract comprising the violacein of the invention, hereinafter, bacterial extract of the invention. The term "bacterial extract" refers to both the extracts themselves.

as to the biomass obtained after the culture of the bacteria. Whether

If desired, these biomass can be, at least partially, dehydrated and / or

ground.

As demonstrated in the present invention, all strains of

Duganelfa sp. isolated from olive trees and specifically the strains with deposit number CECT 7779, CECT 7780 or CECT 7781, grew and produced violacein. As already mentioned, all isolated olive strains had natural levels of violacein production, without trying any optimization of the process, higher (from 1.2 to 65.8 times more) than the levels previously mentioned for other producing bacteria of violacein when they grew under optimal conditions (example 4).

And, considering that the yield of violacein obtained in pigment production is an important parameter for the production of violacein on an industrial scale, the present invention provides a set of strains that are highly suitable for future research and applications.

Biotechnology

Another aspect of the present invention relates to a composition, in

hereinafter composition of the invention, comprising the strain or strains of the

invention; or the combination of microorganisms of the invention; or the violacein of the invention; or the bacterial extract of the invention; or any of its combinations.

The composition, defined in general, is a set of components

which is formed by at least one strain of the invention; or at least for the

combination of microorganisms of the invention; or at least for the violacein of the invention; or at least by the bacterial extract of the invention; or any of its combinations. Therefore, said composition may be, but not limited to, a pharmaceutical composition or a product

phytosanitary or a product that can be the basis for the development of

The above products.

Violacein is a secondary metabolite that has been the subject of many

studies due to its important biological, biotechnological and

industrial (cited in the state of the art of this document), for its potential use as drugs and antibiotics which may have application

for example, but not limited to humans, veterinarians, medicine, agriculture or industry.

Thus, a preferred embodiment of the present invention relates to the composition of the invention, wherein said composition is a pharmaceutical composition. In an even more preferred embodiment, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier and / or excipient.

The term "pharmaceutical composition" refers to any product

intended for human or animal use presented in its pharmaceutical form, which

It has at least one application in improving physical, physiological or

psychological of a subject, which implies an improvement of the general state of his

Health, for example a cosmetic application, although it may not imply a physiological effect on the organism, implies an improvement in the well-being of the subject related to his psychology. Therefore, pharmaceutical compositions can be cosmetic, foods that have therapeutic action; pharmaceutical preparations based on natural resources, products generated by biotechnology, biological products, homeopathic products or a product that may constitute the basis for the preparation of the above products or the basis for the preparation of a medicine. Preferably the pharmaceutical composition is used for the preparation of a medicament.

The term "cosmetic" refers to a formulation of local application, based on scientific concepts, intended for the care and improvement of human skin and its annexes, without disturbing vital functions, without irritating, sensitizing, or causing undesirable side effects attributable to its systematic absorption or its local application. This substance or preparation is intended to be put in contact with the various surface parts of the human body (epidermis, hair and hair system, nails, lips and external genital organs) or with the teeth and oral mucous membranes, for the sole or main purpose to clean, perfume, modify their appearance, and / or correct body odors, and / or protect them or keep them in good condition.

The term "pharmaceutical preparation based on natural resources" refers to the packaged and labeled medicinal product, whose active ingredients are partially formed with natural resources for medicinal use or associations thereof, in the raw state or in pharmaceutical form and which is used for purposes therapeutic

The "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the composition. Therefore, the carrier is a substance that is used in the pharmaceutical composition to dilute any of the components of the composition of the

invention up to a certain volume or weight; or that still undiluted

said components is capable of allowing better dosing and

administration or give consistency and form to the pharmaceutical composition. When the form of presentation is liquid, the vehicle pharmaceutically

Acceptable is the diluent.

The term "excipient" refers to a substance that helps the

absorption of any of the components of the composition of the invention,

stabilizes these components or helps the preparation of the pharmaceutical composition in the sense of giving it consistency or providing flavors that make it more pleasant. Thus, the excipients could have the function of keeping the components together such as starches, sugars or

cellulose, sweetening function, coloring function, protection function

the pharmaceutical composition such as for example to isolate it from air and / or moisture, filling function of a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as that matter which, included in the galenic forms, is added to the active principles or to their associations to enable their preparation and stability, modify their organoleptic properties or determine the physicochemical properties of the Composition of the invention and its bioavailability. The excipient

"pharmaceutically acceptable" should allow the activity of the compounds of

the pharmaceutical composition, that is, that it is compatible with said components.

The "galenic form or pharmaceutical form" is the provision to which they adapt

the active ingredients and excipients to constitute a composition

Pharmaceutical It is defined by the combination of the way in which the

Composition is presented by the manufacturer and the way in which it is administered.

In addition, the excipient and the vehicle must be pharmacologically acceptable, that is, the excipient and the vehicle are allowed and evaluated so

that do not cause damage to the organisms to which it is administered.

5 The antimicrobial activity demonstrated by isolated olive strains and by the

bacterial extract of the invention (crude violacein extract) against

Gram-positive phytopathogenic bacteria (example 5), as well as the antifungal activity shown by several violacein-producing strains together

with other activities possessed by the strains of the invention such as

10 but not limited to, lytic, lipolytic and siderophores protein production, which can give these strains an important role in the biocontrol of plant diseases, a better ability to compete as inhabitants of the rhizosphere of the olive tree, and a potential antagonist against but without

limited, soil fungi, phytopathogenic bacteria, phytoparasite nematodes and 15 protozoa in the rhizosphere of the olive tree, make them especially useful for

development of phytosanitary products.

Therefore, another preferred embodiment of the present invention relates to the composition of the invention, wherein said composition is a product

20 phytosanitary.

The term "phytosanitary product" refers to that substance or mixture of substances intended to prevent the action of, or directly destroy,

insects (insecticides), mites (acaricides), molluscs (molluscicides), rodents

25 (rodenticides), fungi (fungicides), weeds (herbicides), bacteria (antibiotics and bactericides) and other forms of animal or plant life that are harmful to public health and also to agriculture (that is, considered as pests and therefore liable to be fought with

pesticides); during the production, storage, transport, distribution and processing of agricultural products and their derivatives. Among the products

Phytosanitary products also include pesticides, defoliants, desiccants and plant growth regulating substances or phytoregulators.

A more preferred embodiment of the present invention relates to the phytosanitary product wherein said product is a bactericide, which preferably acts against Gram-positive bacteria, or a fungicide.

In another even more preferred embodiment, the composition of the invention further comprises another active substance. In addition to the requirement of therapeutic efficacy, where said composition may require the use of other therapeutic agents, there may be additional fundamental reasons that compel or strongly recommend the use of a combination of at least one of the strains of the invention, the combination of microorganisms of the invention

or violacein, and another therapeutic agent.

The term "active substance" is any matter, whatever its origin, human, animal, plant, chemical or other, to which an appropriate activity is attributed to constitute a pharmaceutical composition or a phytosanitary product.

The form of presentation of the pharmaceutical composition or of the phytosanitary product in each case will be adapted to the type of administration used, therefore, the composition of the invention can be presented in the form of solutions or any other form of clinically permitted administration and in an amount therapeutically effective.

In the sense used in this description, the term "therapeutically effective amount" refers to that amount of the strain of the invention; or of the combination of microorganisms of the invention; or of the violacein produced by the strain or strains of the invention; or any of its combinations that when administered to a subject, preferably animal

or plant and more preferably a plant or a mammal, preferably a human, is sufficient to produce prevention and treatment, as defined below, of a disease or pathological condition of interest in said subject. The therapeutically effective amount will vary, for example,

according to the activity of the strain of the invention; or the combination of

microorganisms of the invention; or the violacein produced by the strain or strains

of the invention; or any of its combinations, in any form of presentation. The therapeutically effective amount will also vary according to the

metabolic stability and duration of action of the compound; and, in the case that the patient is a mammal, according to the age, body weight, general state of health, sex and diet of the patient; the mode and time of administration; the rate of excretion, the combination of drugs; the severity of the particular disorder or pathological condition; and the subject that

undergoes therapy, but can be determined by an expert in the field according to his own knowledge and this description.

The term "treatment" as understood in the present invention is

refers to combating the effects caused as a result of a disease or pathological condition of interest in a subject that includes:

(i)

inhibit the disease or pathological condition, that is, stop its development;

(ii)

relieve disease or pathological condition, that is, cause the

regression of the disease or the pathological condition or its symptomatology;

(iii) stabilize the disease or pathological condition.

The term "prevention" as understood in the present invention is to prevent the onset of the disease, that is, to prevent the occurrence of

disease or pathological condition in the subject, in particular, when said

subject has a predisposition for the pathological condition, but has not yet

diagnosed to have it.

Preferably, the pharmaceutical composition is presented in a form

adapted to oral administration. Another possibility is that the composition

Pharmaceutical is present in a form adapted to sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled administration.

Preferably the phytosanitary product is presented, in a form adapted to

Spray application, the most frequent method of administration of the phytosanitary product, since most of these products are formulated for dispersion in water, that is, they can be formulated in the form of powder, granules, capsules, soluble powder, wettable powder, soluble liquid, emulsifiable liquid or colloidal suspension.

The strain of the invention; the combination of microorganisms of the invention; the

Violacein or any combination thereof, obtained from the strain of the invention, or from the combination of microorganisms of the invention; they can be associated, for example, but not limited to liposomes or micelles. A liposome is a spherical vesicle with a phospholipid membrane. He

Liposome contains a core of aqueous solution. The micelle is an acid

spherical containing non-aqueous material. Both liposomes and micelles can be used as carriers of various substances between the

outside and inside the cell.

Another aspect of the present invention relates to the use of the composition of the invention where the phytosanitary product prevents the action of at least one

Gram-positive bacteria or causes the death of said bacteria.

Another aspect of the present invention relates to the use of the strain of the invention; or of the combination of microorganisms of the invention; or of the violacein of the invention; or of the bacterial extract of the invention; or of the composition of the invention; for the manufacture of a medicine. The medicament referred to in the present invention may be for human use.

or veterinarian

The term "medicament" has a more limited meaning than the meaning of "pharmaceutical composition", as defined in the present invention, since the medicament necessarily implies a therapeutic effect, that is, a physiological effect on the subject's metabolism.

The term "medication", as used herein, refers to

to any substance obtained from active ingredients, with or without

auxiliary substances, used for prevention, diagnosis, relief, treatment, cure or rehabilitation of diseases in man and animals. Also included in this definition are foods that have an action or are administered for therapeutic purposes or advertised with medicinal properties.

Therefore, the medicament referred to in the present invention can be for human or veterinary use. The "medicine for human use" is any substance

or combination of substances that are presented as having properties for the treatment or prevention of diseases in humans or that

can be used in humans or administered to humans in order to

restore, correct or modify physiological functions by exercising a pharmacological, immunological or metabolic action, or establishing a medical diagnosis. The "veterinary medicinal product" is any substance or combination of substances that is presented as having curative properties or

preventive regarding animal diseases or that can be administered to the animal in order to restore, correct or modify its

physiological functions exerting a pharmacological action. immunological or metabolic, or to establish a veterinary diagnosis. "Premixes for medicated feed" prepared to be incorporated into a feed will also be considered "veterinary medicinal products".

The form of presentation of the medication will be adapted to the type of

Administration used, therefore, the medicament may be presented in the form of solutions or any other form of clinically permitted administration and in a therapeutically effective amount.

Preferably the medicament is presented in a form adapted to the

parenteral, cutaneous, oral, epidural, sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled administration.

The term "adapted form" refers to how to adapt the

medicine, pharmaceutical composition or phytosanitary product of the

present invention so that they can be administered.

The form adapted to parenteral administration refers to a physical state that can allow its injectable administration, that is, preferably in

liquid state. Parenteral administration can be carried out via

intramuscular, intraarterial, intravenous, intradermal, subcutaneous or intraosseous administration but not limited to these types of parenteral administration routes. The form adapted to oral administration refers to a physical state that can allow oral administration, that is, it can be

formulate in solid, semi-solid, liquid or gaseous form. Said form adapted to oral administration is selected from the list it comprises, but without

limited, drops, syrup, tea, elixir, suspension, extemporaneous suspension,

solution, drinkable vial, tablet, capsule, powder, granulate, seal, pill, tablet, tablet, troches or lyophilisate. The form adapted to rectal administration is

select from the list comprising, but not limited to, suppository, capsule

rectal, rectal dispersion or rectal ointment. The form adapted to transdermal administration is selected from the list comprising, but not limited to, transdermal patch or iontophoresis.

The medicament can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art. Thus, they can be, without limitation, in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, and the like, and

Nutrients including glucose, dextrose, vitamins and minerals.

Alternatively, the medicament can be prepared for solid administration. The medicine may be combined with several inert vehicles or excipients, including but not limited to: binders such as

microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as

starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate; sliders such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.

On the other hand, previous research has shown that the production of

Violacein for violacein producing bacteria including

Chromobacterium violaceum, Duganella sp. B2, Janthinobacterium Iividum and

Pseudoalteromonas luteoviolacea is affected by various growth factors and culture conditions such as temperature, agitation,

culture volume, media components and pH (Inniss and Mayfield, 1979. Microbial Ecology 5, 51-56; Stephens, 2004. Current Biology 14, R65-R66; Pantanella et al., 2007. Journal of Applied Microbiology 102, 992 -999; Yang et al., 2007. Letters in Applied Microbiology 44, 625-630; Lu et al., 2009. Biochemical Engineering Journal 43, 135-141; Wang et al., 2009. Biochemical Engineering Journal 44, 119- 124).

Thus, another aspect of the present invention relates to the method, hereinafter the method of the invention, for the production of violacein or for the production of the bacterial extract containing vioalacein, wherein said method comprises the steps of:

to.

Select a bacterial strain of the invention or combination

of microorganisms of the invention,

b.

cultivate the strain or the combination of microorganisms from step (a)

in

terms from culture adequate for he increase Y

microorganism development, and

C.

extract the violacein or bacterial extract from the step culture

(b).

In a preferred embodiment of the method of the invention, the strain or combination

of microorganisms according to step (a) is selected from a sample

from the rhizosphere of cultivated olive trees and / or wild olive trees.

The in vitro evaluation for the production of violacein (example 1) determined that all bacterial strains isolated from olive trees grew better and produced purple pigment at 25 ° C compared to other temperatures

assayed (including 4, 10, 25, 28, 30, 37, and 40 ± l'C) after five days of incubation in R2A medium (example 1, Table 4). When different culture media were evaluated at 25'C (R2A agar, tryptone soybean agar (TSA), 1/10 tryptone soy broth (TSB) supplemented with antibiotics, yeast extract malt agar (YMA), and nutritive agar (NA) ) The best growth occurred in the R2A medium where all strains showed a strong production of the purple pigment after 48h of incubation, followed by TSA media, and 1 / 3x KMB with and without antibiotics (example 1, Table 5). Other conditions

used specifically for the production of violacein were in between

Liquid: pH 6.7, potassium nitrate, ferrous ammonium sulfate, dipotassium phosphate, magnesium sulfate, meat extract, L-tryptophan and soluble starch as described in (Wang et al., 2009. Biochemical Engineering Journal 44, 119-124).

The extraction of violacein or the bacterial extract from the culture of step (b) is

performed by any technique described in the state of the art and known to the person skilled in the art. Example 4 of the present invention describes one of the techniques that can be used for extraction.

In addition, the raw violacein can be separated and, if desired, purified by conventional methods, for example, by high performance liquid chromatography.

resolution (see example 4).

The present invention also contemplates violacein or bacterial extract.

comprising violacein, obtained by the method described in previous paragraphs.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Shows the phylogenetic relationship (Neighbor-Joining) of the 16S rDNA sequences of the Duganella spp. of wild olive and cultivated with other violacein producing bacteria.

The closest DNA sequences of the 16S rDNA gene in the GenBank were used for comparison. Strains marked in bold are type strains or specifically referred to throughout this document. The sequence AJ496445 of Col / imonas fungivorans CTE227 was used as an external group. The deposit numbers obtained from the GenBank database are shown in front of the name of each of the strains. Only Bootstrap values greater than 50% are indicated on the nodes of the main clusters.

Fig. 2. Shows the phylogenetic relationship (Neighbor-Joining) of the sequences of the gyr8 genes (Fig. 2A) and vioA (Fig. 28) of the Duganella spp. of wild olive and cultivated with other bacteria

Violacein producers.

The DNA sequences of both genes available in the GenBank were used for comparison. The sequences of the gyrB gene of Pseudoafteromonas futeoviofacea (Fig. 2A) and vioA of Coffimonas sp. (Fig. 2B) were used as

external group Only Bootstrap values greater than 50% are indicated in the

nodes of the main clusters.

Fig. 3. It shows the Cluster analysis of the physiological data in Table 6 (example 2), and APIZYM and Biolog GN2 results treated independently of Duganella spp. of wild and cultivated olive trees.

The UPGMA algorithm was applied to the similarity matrix generated for each experiment independently using the Dice (binary) coefficient (Fig. 3A) or the Pearson correlation coefficient (APIZYM and Biolog data) (Fig. 3B and 3C, respectively ). Values in the nodes indicates the support of

Bootstrap.

The white-black color in the data corresponding to table 6 in Figure 3 indicates values of O (no activity) and 1 (activity), respectively, and the greater gray intensity denotes higher activity values (APIZYM and Biolog data ).

Fig. 4. It shows the Cluster analysis combining the physiological data of Table 6 (example 2), APIZYM results and Biolog GN2 of Duganella spp.

of wild and cultivated olive trees.

The UPGMA algorithm was applied to the consensus of the similarity matrix

generated by combining the matrix obtained in each experiment so

independent using the Dice (binary) coefficient (Fig. 3A) and the Pearson correlation coefficient (APIZYM and Biolog data) (Fig. 3B and 3C, respectively). Values in the nodes indicates Bootstrap support.

The white-black color in the data corresponding to table 6 in Figure 4 indicates values of O (no activity) and 1 (activity), respectively, and the greater gray intensity denotes higher activity values (APIZYM and Biolog data ).

Fig. 5. Shows the production of violacein by the strains of Duganella spp.

Fig. 5A. It shows the production of violacein by Duganelfa spp. growing in the medium described by Wang et al., 2009, Biochemical Engineering Journal 44, 119-124, using 10% or 20% v / v onset of culture.

Bars with different letters on them in the same color and shape or with an asterisk n indicated significant differences (PS; 0.05) between strains or between initial cultures, respectively, according to Fisher's protected contrast of the minimum significant differences. The results are mean values ± standard deviation of three repetitions.

Fig. 5B. It shows the visible-UV spectrum of commercial violacein and crude ethanol extract from Duganelfa spp. of wild olive (LO-22) and cultivated (CICYT-60 and MICO-M).

To avoid repetition, only three UV-vis HPLC spectra of the selected strains are shown.

Fig. 6. Shows the antibacterial activity of the strains of Duganella spp. against a strain of Bacillus subtilis isolated from the olive rhizosphere.

The antibacterial activity was determined by in vitro dual culture assays using the Waskman agar medium.

commercial, commercial violacein.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of different

strains from the rhizosphere of wild and cultivated olive trees for

produce violacein, a purple pigment that has shown important

Biotechnological and agronomic applications.

Throughout the different examples it is demonstrated how bacterial strains isolated from the rhizosphere of wild and cultivated olive (CICYT-60 (with deposit number CECT 7780), LO-22 (with deposit number CECT 7779), Mico-M (with deposit number CECT 7781), LOBA-24, Baetica-33, Mico-C and MicoM2) and which were characterized physiologically, biochemically and genetically (by phylogenetic analyzes of the 16S rDNA, gyrB and vioA genes) as

new strains of Duganella spp. produce violacein with a higher yield compared to other violacein producing bacteria previously

documented when they grew in optimal conditions. In addition, you are

strains could be differentiated based on phenotypic and genetic analyzes

in two groups according to the host of origin (wild olive versus cultivated olive).

On the other hand, the antimicrobial activity of the bacteria and the crude extract of violacein obtained from them has also been evaluated.

Phytopathogenic bacteria, finding antimicrobial effect against the two Gram-positive bacteria evaluated, the phytopathogenic Clavibacter michiganensis

subsp. michiganensis and a strain of Bacillus subtilis antagonist of Verticillium

Dahliae isolated from the olive rhizosphere.

Regardless of the production of violacein, it has been observed that strains isolated from Duganella spp. of wild and cultivated olive trees showed proteolytic and lipolytic activity and production of siderophores, which would imply that these strains would have a better ability to compete as an inhabitant of the rhizosphere of the olive tree, because they would be able to improve the sanitary state of the plants to several levels, such as iron nutrition, and hinder the growth of pathogens, usually fungi, by limiting the iron available for the growth of the pathogen.

The following specific examples provided in this document

The patent serves to illustrate the nature of the present invention. These

Examples are included for illustrative purposes only and should not be construed as limitations of the invention claimed herein. Therefore, the examples described illustrate the invention without limiting its scope of application.

EXAMPLE 1. ISOLATION AND CONDITIONS OF CULTURE OF THE VINTAGES WITH PURMENTS. 10

The olive roots of 92 commercial farms located in the main areas

olive groves in the provinces of Córdoba, Jaén, Sevilla and Granada and 11 areas with wild olive trees located in the potential area where you can find both

true olive trees (for example, wild forms in natural areas) such as 15 feral forms (for example, secondary sexual derivatives of clones

cultivated or hybridization products between cultivated trees and nearby olive trees) (Lumaret and Ouazzani, 2001, Nature 413, 700), located in the provinces of Córdoba and Cádiz, were sampled in Andalusia, southern Spain in the spring of 2009 (Table 3 ). Bacterial suspensions of the rhizosphere

20 were serially diluted and seeded in R2A agar medium (Bio / ile, Milan, Italy) and incubated at 28 ± 1 'c for 72h in the dark.

Table 3. Location, altitude and climatic characteristics of the sites of origin of

the isolated strains of Duganella spp.

Strain

Olive plants Location Coordinates Alt ' Climatic characteristicsb

Classification

'C mm

LO-22

Wild> 200 years 8.0 Km SW Benalup-Casas Viejas, Cádiz 36 '18'43.65 "N 5'54'1.14" O 53 Mediterranean Maritime 1719 600-800

LOBA-24

Wild> 200 years 7.4 Km SW Benalup-Casas Viejas, Cádiz 36'18'26.64 "N 5'52'56.0 1" O 47 Mediterranean Maritime 1719 600-800

Baetica-33

Wild> 200 years 1,5 km E Vejer, Cádiz 36'14'32.64 "N 5'57'1.63" O 14 Mediterranean Maritime 1719 12001600

MICO-C MICO-M

Cultivated cv. Arbequina 5 years 6.03 Km S Villafranca, Córdoba 37 '54'27.53 "N 4" 34'3.52 "0 160 Subtropical Mediterranean 1517 400-600

MICO-M2

Cultivated cv. Arbequina 4 years 3.05 Km SW, Cordoba 37 "51'24.54" N 4 '48'5.32 "0 175 Subtropical Mediterranean 1517 400-600

CICYT-60

Cultivated ev. Picual> 100 years 3.2 Km SE Alcaudete, Jaén 37 "36'17.65" N 4 '7'8.95 "0 530 Subtropical Mediterranean 1517 400-600

at Altitude above sea level (m). b Climate classification (J. Papadakis, 1966), average annual temperature in 'C, and

annual precipitation in mm. were obtained from SigMapa, Information System

Geographic of the Spanish Ministry of "Environment, and Rural and Marine Environment" (httpJ / sig.mapa.es / gecportal /). CV., Cultivate.

After the incubation period, the appearance of colonies was observed

bacteria with purple pigmentation in about 10% of the samples that

They were purified for later physiological, biochemical and molecular characterization.

Bacteria with purple pigmentation were identified in 27.3% of the root samples taken from wild olive trees in the province of Cádiz, and

Only in 4.4% of the root samples of 92 commercial olive farms sampled throughout Andalusia. It is noteworthy that although eight

different varieties grown without pattern were sampled on commercial farms, bacteria with purple pigmentation were isolated only

of cvs. (cultivars) Arbequina and Picual (Table 3).

Seven strains of pigmented bacteria were selected for studies

later. Of these, three strains (Baetica-33, L022 and LOBA-24) came from the rhizosphere of centennial olive trees (age over 200-years based on the diameter of the trunk; Michelakis, 2002. Proceedings of International Sysmposium, Sitia, Crete, Greece) located in different geographical areas; two strains (MICO-C, MICO-M) came from the same commercial estate from the rhizosphere of olive trees of cv. (cultivate) Arbequina with an inoculated age of 5 years (MICO-M)

or not (MICO-C) with a commercial product based on mycorrhizae (Mycosim-TR / TON, MYCOS / M / nternationa / AG, Base / Switzer / and) during its phase of

nursery production; the strain MICO-M2 came from a different commercial field than

from the rhizosphere of olive trees of cv. Arbequina 4 years old inoculated with

the same mycorrhizal inoculum; and strain CICYT-60 came from a commercial estate

with centuries-old olive trees from cv. Picual (> 100 years old).

1.1. Optimal culture conditions.

The growth and production of the purple pigment of all bacterial strains was evaluated in different solid and liquid media at 25 ± 1 "C for 48 hours. In the dark. The media evaluated were: R2A agar, tryptone soybean agar (TSA), 1 / 10 lriptone soy broth (TSB) supplemented with antibiotics (Landa et al., 2003. Phytopatology 93, 982-994), yeast extract malt agar (YMA), and nutritive agar (NA). All media were purchased from O / I read Laboratories (Detroit, Michigan,

USES). Additionally, the growth and production of violacein in the

5 seven bacterial strains in R2A medium at different temperatures including 4, 10, 25, 28, 30, 37 Y40 ± 1 'C and in dark conditions.

All bacterial strains grew better and produced purple pigment at 25 'C compared to temperatures of 15, 20, 28, 30, Y34'C; but nevertheless,

10 none of the strains grew at 40QC after five days of incubation in between

R2A The strains MICO-M, MICO-M2, and CICYT-60 were able to grow at 4 and 37 'C, but the production of the purple pigment was not evident (Table 4).

Table 4. Effect of temperature on the growth and production of purple pigment in R2A medium of strains isolated from the rhizosphere of wild and cultivated olive

Wild olive

Cultivated olive

T '

(' C)

LO-22 LOBA-24 Baetica-33 Mico-C Mico-M Mico-M2 CICYT-60

4

.j.a ± I - / - / ± I ± I ± I

fifteen

+ / + + / + + / + + / + / ± + / ± + / ±

twenty

++ J ++ ++ / ± + / + + / ++ J ± ++ J ± ++ 1 +

25

++; ++ ++; ++ ++ / ++ ++ / ± ++ / ++ ++ / ++ ++ / ++

28

++ / + ++ 1 + ++ / + ++ / ± ++ / + ++ 1 + ++ 1 +

30

++ / + H / ± ++ / ± ++ /. ++ /. ++ /. ++ 1 +

3. 4

+ / + / ± I + / + / + / + /

37

- / - / - - / - / +/- + / +/-

40

- / - / - / - / - / - / - /

a Growth / production of purple pigment (++) ~ strong, (+) ~ positive, (±) ~ weak 20 or (-) ~ negative.

When different culture media were evaluated at 25! I! C, the best growth occurred in R2A medium where all strains showed a strong production of the purple pigment after 48h incubation, followed by TSA media, and 1 / 3x KMB with and without antibiotics (Table 5). Additionally, all strains were

5 capable of growing in liquid medium 1 / 3x KMB +++ (supplemented with the antibiotics ampicillin, chloramphenicol and cycloheximide) and in 1 / 10x TSB + (supplemented with cycloheximide).

Table 5. Purple growth / production in culture medium (25'C) of the

10 strains of Duganella spp. isolated from the rhizosphere of wild and cultivated olive trees and two Duganella spp. described to date

Culture media "

Wild olive trees Olives cullivated Reference strains

LOBA-BaellcaLO-22 24 33

Mico-Mlco-Mlco-CICYT-e M M2 60 D. D. vlo / acelnlgra zoogloeoldes YIM 31327 IAM 12670

R2A 1 / 3x King "s B agar (1 / 3x KMB) 1J3x KMB + H agar Triptone soybean agar (TSA) Yeast mesh agar (YMA) Nutritive agar (NA)

++ 1 ++ ++ 1 ++ -H / ++ ++ / ++ ++ / ++ - ++ / ++ ++ / + ++ / + ++ J + ++ / + ++ / + ++ J + + / ± + / ± + / ± + 1 · + 1 · + 1 · H / ± ++ 1 ++ -H./++ ++ /. H ++ / ± ++ / ++ ++ / ++ ++ / ++ ++ / ± ++ J + ++ J + ++ / + ++ / ± ++ J + ++ J + ++ / + + / ± + / ± + / ± + / ± + 1 · + 1 · + 1 · + 1 · nr nr nr nr nr nr nr nr +1+ nr · 1 · + 1 ·

a Tests of growth, assimilation, ° production (++) = strong, (+) = positive, 15 (±) = weak, (-) = negative.

b Data from Duganefla violaceinigra YIM 31327T were taken from Li et al. 2004, International Journal of Systematic and Evolutionary Microbiology 54, 1811-1814 and of Duganefla zoogloeoides IAM 12670T of Hiraishi et al. 1997, International Journal of Systematic Bacteriology 47: 1249-1252. nr = not cited. (+ ') Referred to as use of carbon sources in Li et al. 2004, International Journal of Systematic and Evolutionary Microbiology 54, 18111814.

Medium formula R2A (Biolife) agar (gil): yeast extract: 0.5; peptone protein: 0.5; acid casein: 0.5; glucose: 0.5; soluble starch: 0.5; dipotassium phosphate: 0.5; magnesium sulfate: 0.024; sodium pyruvate: 0.3; agar: 12; Final pH: 7.2 ± 0.2. Medium formula 1/3 KMB (gil): peptone protein: 6.7; potassium phosphate: 0.4; dipotassium phosphate: 0.5; magnesium sulfate: 0.5; glycerol: 3.3; agar: 20; pH 7.0 (sodium hydroxide). Medium formula TSA (gil): casein peptone: 15; soy peptone: 5; sodium chloride: 15; agar: 15; Final pH: 7.3. TSB (gil) medium formula: casein pancreatic digestive: 17; enzymatic digestive of soy: 3; sodium chloride: 5; dipotassium phosphate: 2.5; dextrose: 2.5 g; agar: 15; Final pH: 7.3. Medium formula YMA (gil): yeast extract: 3; malt extract: 3; peptone: 5; glucose: 10; agar: 20, final pH: 7.3. Medium formula NA (gil): gelatin peptone: 5.0; meat extract: 3 g; agar: 15; Final pH: 7.2.

EXAMPLE 2. PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION.

The glucose oxidation-fermentation test (O-F) and the ability to produce

acid from carbohydrates and related compounds was monitored by the

Method of Hugh, R. and Leifson, E., 1953, Journal of Bacteriology 66, 24-26. The

Catalase and oxidase activity was determined following the procedures

described by Bergey, D.H., 1994, Baltimore: Williams & Wilkins, 636-642. Gram staining was performed using 48h pure bacterial cultures. old. Proteolytic activity was evaluated using the plate assay with a medium

semi-quantitative using skim milk-agar, while lipolytic activity was evaluated using Tween 80-agar medium; both activities are

detected by the presence of clear halos and halos halos respectively

around the growth of bacterial colonies. Siderophores production

was evaluated under conditions of iron limitation using the universal test

for the production of siderophores (Schwyn, B., Neilands, J.B., 1987. Analytical Biochemistry 160, 47-56). All physiological and biochemical tests were

made at 28 "C in dark conditions.

To test the use of carbon sources and enzymes, using the Biolog GN2 microplate system (Biolog, Barcelona, Spain) and the APIZYM system

(Biomerieux, Madrid, Spain) Aqueous bacterial suspensions were used in

0.85% NaCI.

For APIZYM plates, the fluids were incubated at 28 ± 1 'C for 24 hours.

Subsequently, the galleries were activated by adding 30 111 of each reagent

(ZYM A YZYM B; BioMerieux), and after a period of 5 min at room temperature a semi-quantitative evaluation of the enzymatic activity was carried out using as a reference a standard calorimetric table assigning a numerical value of O

to 5 (equivalent of O to 40 nmol) (Sciancalepore et al., 1996. Toxicological & Environmental Chemistry 55, 145-158), depending on the color intensity of the

chromogenic substrate produced by the hydrolysis reaction.

Biolog plates were incubated for 5 days at 28 ± 1'C measuring

periodically absorbance at 585 nm. using a spectrophotometer of

Tecan Safire fluorescence (Tecan Spain, Barcelona, Spain). The average color development of all wells (Average Weff Color DevelopmenQ (AWCD) of each plate was calculated as the average of the absorbance values for all well responses for each reading time.

AWCD was used to determine the speed and asymptotic level of assimilation

of the substrates. A cluster or aggregation analysis was performed in which the dendrograms were constructed using the similarity matrix from the

Phenotypic characterization data using the Dice coefficient (binary data)

or the Pearson correlation coefficient (quantitative data). Analysis was used

UPGMA (Unweighted Pair Group Method with Arithmetic Averages) for aggregation analysis with the Bionumerics 6.1 package (Applied Maths, Sint-Martens

Latem, Belgium). The significance of the nodes was evaluated by Bootstrap analysis

or support over 1,000 randomizations.

The isolated strains of wild and cultivated olive were Gram-negative, not

fermentative (they catabolized glucose in oxidative form in the O-F test), catalase +, oxidase +, and most of them showed proteolytic and lipolytic activity. In addition, all strains showed siderochlor production. The majority of the strains produced acid from galactose, but in general, they did not

they did when other carbon sources were used. The strains differed in

many characteristics with respect to the type species of Ouganella (D. zoogleoides) (Hiraishi, A., et al., 1997. Intemational Joumal 01 Systematic Bacteriology 47, 1249-1252) and in some characteristics with respect to Ouganella 15 violaceinigra (Wen- Jun et al., 2004. International Journal 01 Systematic and Evolutionary Microbiology 54, 1811-1814), mainly in the production of

acid from glucose in oxidative form and in oxidase activity. Too

all strains diluted Ouganella violaceinigra in at least two of the five enzymatic activities determined by the API system compared to the

20 cited in Wen-Jun et al., 2004, International Journal 01 Systematic and Evolutionary Microbiology 54, 1811-1814 (Table 6).

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a Tests for growth, assimilation, or production (++) = strong, (+) = positive, (±) = weak, (-) = negative. b Data from Duganella violaceinigra YIM 31327T were taken from Li et al. 2004,

International Journal 01 Systematic and Evolutionary Microbiology 54, 1811-1814, Y of D. zoogloeoides IAM 12670T from Hiraishi et al. 1997, International Journal 01 Systematic Bacteriology 47: 1249-1252. nr = not cited. (+ ') Referred to the use of carbon sources in Li et al. 2004, International Journal of Systematic and Evolutionary Microbiology 54, 1811-1814.

Based on the cluster analysis of phenotypic characteristics (Table 6), in the APIZYM and 8iolog GN2 tests, the seven strains could be clearly differentiated and were consistently grouped into two main clusters according to the origin of the strains, well when the data of each one of them were independently processed (Fig. 3) or when the data of the three Experiments were combined (Fig. 4).

EXAMPLE 3. MOLECULAR CHARACTERIZATION THROUGH PHILOGENETIC ANALYSIS OF 16S rDNA genes, gyrB and vioA.

The DNA of the bacterial strains was isolated using the UltraCleanTM microbial DNA kit (MoBio Laboratories, Inc., CA, USA) according to the manufacturer's instructions. Additionally, DNA from the Duganella violaceinigra type strain CCUG 50881T (equal to Duganella violaceinigra YIM 31327T) was used in molecular studies.

The strains were identified at the genus / species level by

sequencing of the 16S rDNA region using the 8f primers (SEO ID NO: 25) and 1492r (SEO ID NO: 26) according to Landa et al., 2003, Phytopatology 93, 982-994. The gyrB gene was amplified with the Up-1 G- (SEO ID NO: 27) and Up-2G (SEO ID NO: 28) primers described by Mavrodi et al. 2010, Applied & Environmental Microbiology 76, 866-879. In addition, the pair of degenerate primers VPA3 (SEO ID NO: 29) NPA4 (SEO ID NO: 30) (Hakvag et al., 2009. Marine was used.

Drugs 7, 576-588) to amplify an approximately 1.0 kb segment of the vioA gene encoding the flavoenzyme vioA, which was selected due to the higher

number of sequences available in the GenBank database for this gene compared to other genes for the synthesis of violacein in different genera of bacteria.

The amplified sequences were purified using the PureLink PCR kit (Invitrogen, Barcelona, Spain). Nearly the entire length of the amplified genes rDNA 168 (approximately 1.5 kb), gyrB (approximately 1.0 kb) and vioA (approximately 1.0 kb) was directly sequenced using

the same initiators that were used for the amplification in the facilities

from STABVIDA (Caparica, Portugal). All 168 sequences were analyzed using the NCBI-BLA8T program, and the sequences closest to the

strain sequences were extracted and aligned using the software

Bionumerics 6.1 for phylogenetic analysis. Phylogenetic trees were constructed using analysis of Neighbor Joining (NJ) or the nearest neighbor. The robustness of the originated groups was tested by Bootstrap or support analysis on 1,000 randomizations.

The analysis of almost the entire length of t 68 rDNA sequences of

all strains showed that they are closely related to each other

(> 99.3% similarity of the sequences) and could be assigned to the class f3Protecbacteria, order Burkholderiales, family Oxalobacteraceae, with the closest genus Duganella. Based on the homology of the 168 rDNA genes of the bacteria, the phylogenetic tree was constructed by analysis of Neighbor Joining (NJ) containing the sequence of gene 168 of the seven bacterial strains obtained from olive trees, the reference strains of Duganella spp. and Janthinobacterium

Iividum and several related sequences of non-cultivable organisms contained in clones from the GenBank database (Fig. T). Phylogenetic analysis of the rDNA gene 168 indicated that the seven strains with purple pigmentation were identified as Duganella spp. This is the first date of Duganella spp. in the rhizosphere of woody plants and as a specific inhabitant of the rhizosphere of wild and cultivated olive trees. All 168 rDNA sequences of Ouganella spp. of wild and cultivated olive trees showed a lower homology (95.5-96.0%) compared to that of the type species of Ouganella (D. zoogleoides) and the isolate of Ouganella sp. 82, the first Ouganella sp. Violacein producer cited so far (Wang et al., 2009. Biochemical Engineering Journal44, 119-124). It is noteworthy that although strain B2 has been cited as an Ouganella sp., In

our study, phylogenetic analyzes showed that it is more closely

related to Janthinobacterium lividum because its 168 rDNA sequence grouped with Ouganella sp. tsz33 (an isolated strain of a Glacier in China) as a sister group of all the Janthinobacterium lividum sequences of Gen8ank (58% support). On the contrary, the sequence of the rDNA 168 genes of wild and cultivated olive eepas showed a hamo loggia of 98.6-99.3% with the Ouganella violaceinigra YIM 31327 lipo strain (Table 1) and were grouped

with other strains and sequences of organisms not cultured in a sister cluster

different from that of the lipa species (58% support). Although the seven olive strains showed high homology in the 168 rDNA sequence (99.4-100%), two subgroups could be differentiated between them according to their host of origin (wild olive versus cultivated olive).

Phylogenetic analysis of the gyr8 gene using a smaller data set showed similar results to those obtained with the ADNr 168 gene, with the strain Ouganella sp. B2 being genetically different from the lipa strain of Ouganella violaceinigra YIM 31327 And from the olive strains (99% support), which were grouped again according to their origin (Fig. 2A). Thus, the three strains of wild olive trees were grouped as a basal group of the Ouganella violaceinigra YIM 31327 type strain (54% support; 93.7-95.7% similarity, Table 1) and all these oepas formed a sister group of which come from cultivated olive (100% support).

The phylogenetic analysis of the partial gene vioA grouped Ouganella sp. B2 as a basal group of the remaining sequences of Ouganella spp. (62% support)

showing a higher similarity in the sequence of the vioA gene with all the

Janthinobacterium lividum sequences (80.4-81.2%) than with all

Duganella spp. including olive strains and the Duganella violaceinigra type strain (77.3-77.9%). Duganella violaceinigra was a basal group of all strains isolated from Duganella spp. of olive tree and a soil sample from Ithaca, New York, USA. (89% support; 89.8-90.8% similarity, Table 1) (Brady et al., 2001. Organic letters 3, 1981-1984.). Within this cluster, the seven Duganella spp. they were grouped according to their host of origin, wild olive (lOBA-24, 10-22, Baetica-33) or cultivated (CICYT-60, MICO-C, MICOM, MICO-M2) with homology in the sequences in a range from 91, 6 to 91, 8% between both groups (Fig. 2B).

EXAMPLE 4_ DETECTION, PRODUCTION AND EXTRACTION OF VIOlACEIN.

The in vitro evaluation for the production of violacein was carried out using the optimal conditions and culture medium described above for a

Duganella sp. (Wang et al., 2009. Biochemical Engineering Journal 44, 119-124) (pH 6.7, potassium nitrate 1.18 gil, ferrous ammonium sulfate 0.08 gIL, dipotassium phosphate 0.25 gIL, magnesium sulfate 0.75 gil, meat extract 1.53 gi l, lipriphan 0.74 gil, soluble starch 13 gil, 25 ml of the medium in 250 ml flasks) with an initial inoculum size of 10% (viv) or 20% (viv) of a crop

bacterial in the same medium in a stationary growth phase. The

cultures were incubated on a rotary shaker at 60 rpm and 25 ± I 'C for 5h (when the cultures reached the stationary phase).

Raw violacein was extracted from the cells using the ethanol extraction method according to Wang et al., 2009, Biochemical Engineering Journal44, 119-124. Raw violacein production was measured based on the absorbance of the ethanol solution at 585 nm using an ultraviolet-visible spectrophotometer (Metertek SP-850; Metertech Inc., Taipei, Taiwan) (Mendes et al., 2001. Biotechnology letters 23 , 1963-1969). Further,

the raw violacein obtained by the extraction process previously

described was separated and purified by high performance liquid chromatography, in an Agi / ent 1200 HPLC system equipped with a diode detector (DAD) (Agitent Teehn% gies, Wa / dbronn, Germany) with an octadecylsilane column (ODS) ( Bring Exee / 120 ODS-B, 5 mm, 250 x 46 mm), for

measure the composition and compare it with commercial violacein obtained from

Janthinobaeterium. lividum (Sigma-A / drieh, Madrid, Spain) and with previously published data (Retori and Durán, 1998. World Journal of Microbiology & Biotechnology 14, 685-688 .; Sánchez et al., 2006. ChemBioChem 7, 12311240; Wang et al., 2009. Biochemical Engineering Journal 44, 119-124; Jiang et al., 2010. Applied Microbiology & Biotechnology 86,1077-1088).

The measurement wavelength was 570 nm and the mobile phase consisted of 70% methanol at a flow rate of 1 ml / min at a temperature of 30'C (Jiang et al., 2010. Applied Microbiology & Biotechnology 86, 1077-1088). The

Differences in the production of raw violacein were analyzed by standard analysis of variance and the comparison of means between treatments using

Fisher's protected contrast of the minimum differences (LSD) (P,; 0.05) using the STATlSTlX9.0 program (Ana / ytiea / Software, St. Pau /, MN, USA).

All isolated olive strains and Duganella strain saw / aeeinigra CCUG 5088n (same as Duganella saw / aeeinigra YIM 31327) possess the vioA gene for the production of violacein (determined by the amplification of a fragment of approximately 1.0 kb using the pair of initiators VPA3 (SEO ID NO: 29) NPA4 (SEO ID NO: 30)). As far as we know, this is the first

demonstration that Duganella violaceinigra has genes for the production of

Violacein Also all isolated olive strains produced violacein,

evidenced by quantitative determination by spectrophotometry and

liquid chromatography (LC) (Fig. 5 A, B). When raw extracts of

Violacein were examined by HPLC, the fraction collected consisted of a

main peak (6.5 min) for all bacterial strains.

The visible UV spectrum of raw violacein extracts was similar to that observed for commercial violacein obtained from Janthinobaeterium lividum (Sigma, Madrid, Spain) and for that cited for other violacein producing bacteria including Duganelfa sp. B2, and Chromobacterium viofaceum (Retori and Ourán, 1998 World Journal of Microbiology & Biotechnology 14, 685-688; Sánchez et al., 2006. ChemBioChem 7, 1231-1240; Wang et al., 2009. Biochemical Engineering Journal 44, 119 -124; Jiang et al., 2010. Applied Microbiology & Biotechnology 86, 1077-1088) with a strong absorption in the visible region due to the resonance of violacein (Riveros et al., 1988. Arquivos de Biologia e Tecnologia 31, 475-487) (Figure 5B). In addition to the above, the addition of 10% sulfuric acid to the extracts of crude violacein in ethanol changed the violet color to green, and when NaOH was added, it changed to a reddish brown color, as described for the solution of ethanol violacein from Janthinobacterium fividum (Shivaji et al., 1991. Polar Biology 11, 267271).

Bacterial strains differed significantly (P <0.0001) in their ability

to produce violacein when it was determined for a similar number

of cells / mi (00600 1,280 ± 0.1879) in the stationary phase of growth after 48h incubation at 25 ° C (Fig. 5A). Baetica-33, CICYT60, and LO-22 strains produced significantly higher amounts (P <0.0001) of violacein when the culture was started with 10% (v / v) than with 20% (v / v) of a

bacterial culture in the stationary phase. When the bacterial culture started

With 10% (v / v) for some strains (Baetica-33, LO-22), the production of violacein was evident however not abundant (00 585 = 0.230-0.405; equivalent to 4.10-7.22 mg / L). In contrast, when the culture started with 20% (v / v), the CICYT 60 strain produced significantly higher (P <0.0001) amounts of raw violacein (00585 = 1.585; equivalent to 20.30

mg / L) compared to the other six strains.

EXAMPLE 5. ANTIBACTERIAL ACTIVITY IN VITRO

The antibacterial activity of the isolated olive strains was determined by in vitro dual culture assays using the Waskman agar medium

(Berg et al., 2002. Applied & Environmental Microbiology 68, 3328-3338) against a strain of phytopathogenic bacteria (C / avibacter michiganensis subsp.

michiganensis), and a strain of Bacillus subtilis isolated from the olive rhizosphere.

Bacterial suspensions were adjusted to an approximate concentration of t x 1 O 'colony forming units (cfu) and were used to completely inoculate the surface of the Waskman agar medium contained in 9 cm diameter petri dishes. Filter paper discs were inoculated with 30 111 of the bacterial strains Bacillus subtilis and Clavibacter michiganensis growing in the optimal culture medium for violacein production

(Wang et al., 2009. Biochemical Engineering Journal 44, 119-124) And they were placed on the plates 1 cm away from the edge of the plate.

On the other hand, the crude violacein filtrates (10 111) from the six isolated bacterial strains (MICO-M, MICO-M2, LOBA-24, CICYT 60, LO-22 and Baelica-33) and a commercial violacein solution (50 11M) in elanol were inoculated on the Waskman agar medium that had previously been inoculated with the bacteria Bacillus subtilis and Clavibacter michiganensis in the manner described above. Ethanol was used as a control. Three repetitions were performed per bacterial strain. The plates were incubated at 25'C and were

performed a qualitative evaluation of inhibition based on

presence / absence of inhibition halos around the point of inoculation (Figure 6).

Bacterial strains producing olive purple pigment and filtrates of

Raw violacein showed moderate (2 mm halo inhibition of

growth) to strong inhibition (1 cm diameter of halo at the point of inoculation) respectively, against the Gram-positive bacteria (Baciflus subtilis) evaluated in our study.

In addition, crude violacein filtrates from strains LOBA-24, LO-22, Baetica-33 and CICYT-60 inhibited the growth of Clavibacter michiganensis

subsp. michiganensis. The halos of inhibition of the crude violacein filtrates were similar for all bacterial strains and equivalent to that observed

when commercial violacein was used (50 ~ M).

Claims (17)

1. Bacterial strain of the genus Duganella spp. Violacein producer isolated from the rhizosphere of cultivated and / or wild olive trees with a identity:

to.

from 98.6% Yun 99.3% with respect to the DNA fragment of SEQ ID NO: 1 of the 16S rDNA gene,

b.

from 93.7% Yun 95.7% with respect to the DNA fragment of SEQ ID NO: 2 of the gyrB gene, and

C.

between 89.8% and 90.8% with respect to the DNA fragment of SEQ ID NO: 3 of the vioA gene.

2. Bacterial strain according to claim 1, wherein said strain is Select from the CECT 7779, CECT 7780 or CECT 7781 strains. 3. Bacterial strain according to any of claims 1 or 2, wherein The strain is CECT 7780.

Four.

Combination of microorganisms comprising at least one strain according to any one of claims 1 to 3.

5.

Combination of microorganisms according to claim 4. further comprising at least one other microorganism.

6. Violacein produced by the strain according to any of claims 1 to 3 or by the combination of microorganisms according to any of claims 4 or 5. 7. Bacterial extract comprising the violacein according to claim 6.

8.

Composition comprising the strain according to any one of claims 1 to 3; the combination of microorganisms according to any of claims 4 or 5; the violacein according to claim 6; or the bacterial extract according to claim 7.

9.

Composition according to claim 8, wherein said composition is a pharmaceutical composition.

10, Pharmaceutical composition according to claim 9, wherein it further comprises a vehicle and / or a pharmacologically excipient acceptable. 11. Composition according to claim 8, wherein said composition is a phytosanitary product 12. Composition according to claim 11, wherein the phytosanitary product It is a bactericidal. 13. Composition according to claim 11, wherein the phytosanitary product is a fungicide. 14. Composition according to any of claims 8 to 13, further comprising another active substance. 15. Use of the composition according to claim 12, to prevent the action of at least one Gram-positive bacterium or to cause death of said bacteria. 16. Use of the strain according to any of claims 1 to 3; of the combination of microorganisms according to any of the claims 4 or 5; of the violacein according to claim 6; of the bacterial extract according to claim 7; or of the composition according claim 8, for the manufacture of a medicament. 17. Method for the production of violacein or for the production of 5 bacterial extract containing violacein, comprising: to. Select a bacterial strain according to any one of claims 1 to 3, or the combination of microorganisms according to any of claims 4 or S, 10 b. cultivate the strain or the combination of microorganisms from step (a) under culture conditions suitable for the growth and development of the microorganism, and C. extract the violacein or the bacterial extract from the culture of step (b).