ES2575752B2 - Obtaining and using liquid extracts to improve the recovery of microorganisms associated with lichen - Google Patents

Abstract

Obtaining and using liquid extracts to improve the recovery of microorganisms associated with lichens. # The present invention relates to a liquid extract and the use thereof to improve the recovery of microorganisms associated with lichens. Thus, the invention is directed to a method for improving or optimizing the isolation and / or cultivation of microorganisms associated with lichens which comprises the processing of washed or washed and crushed liquids in a culture medium comprising the liquid extract of a population of the lichen species from which microorganisms are to be isolated. The method of obtaining the liquid extract and the composition comprising the liquid extract thus obtained are also described.

Description

Obtaining and using liquid extracts to improve the recovery of microorganisms associated with lichen

The present invention relates to obtaining liquid extracts and using them to improve the recovery or isolation of microorganisms associated with liquid talus. Thus, the invention provides a new method for obtaining liquid extracts and preparing new culture media enriched with

said liquid extracts that mimic the nutrients present in the liquid tali. Therefore, the present invention belongs to the area of Microbiology, specifically, to the field of microbiological techniques for the isolation of microorganisms.

STATE OF THE TECHNIQUE Lichen is a symbiotic organism formed by at least one fungus (mycobiont) and a green microalgae or a cyanobacterium (phycobiont). While fungi are responsible for the supply of water and mineral salts, microalgae or cyanobacteria provide photosynthetic products, such as carbohydrates. It is estimated that there are approximately 14,000 species grouped into 600 genera. Additionally, there are microorganisms associated with liquid symbiosis, such as bacteria and viruses, as well as other fungi and microalgae, whose isolation and identification is desirable both from the ecological or environmental point of view, and from the biotechnological point of view due to the large number and diversity of applications that said microorganisms can have. However, there are currently many difficulties to isolate these microorganisms associated with liquid symbiosis as well as to recover them in large numbers. This is due, among other causes, to the use of synthetic culture media that do not reproduce the nutritional conditions of the liquid tali, mostly frozen samples, or adequate microbiological analysis of the samples of the liquid talus

Cardinale al. 2006 (FEMS Microbiol. Ecol., 57: 484-495) carried out a molecular analysis of the heterotrophic bacterial communities associated with several species of lichens by methods independent and independent of the culture. In the bacteriological analysis they used eleven samples of frozen liquid talus from eight different species collected in Austria, one of them being Pseudevernia furfuracea, and two synthetic culture media for the culture of the bacteria

liquid. As a result, they obtained only a few dozen bacterial colonies from nine of the eleven liquid tali analyzed, evidencing the

difficulty in recovering liquid bacteria, both from the surface (ectoliquénicas) and from the interior (endoliquénicas) of the liquid tali. These authors did not provide quantitative data on isolated cultivable bacteria in colony forming units (CFU) per gram (CFU / g).

Liba al. , 2006 (J. Appl. Microbiol., 101: 1076-1086) also made isolates of heterotrophic bacteria (mainly nitrogen fixing bacteria) associated with different species of lichens, but recovered them by means of a previous enrichment step in liquid medium. Despite the enrichment, they isolated few bacteria with this capacity and when using a liquid culture medium they also did not give quantitative data on the CFU / g.

Selbmann al., 2010 (Polar Biol., 33: 71-83) conducted a study of arable bacteria associated with Antarctic lichens with samples of freeze-preserved liquid talus. Freezing of the liquid tali, especially in the absence of cryoprotective agents, decreases both the viability and the cultivability of the microorganisms in the culture media. Therefore, of the sixteen liquid tali of the different species and genera analyzed, they only managed to isolate bacteria in five of them and in very low numbers compared to those obtained with the present invention. As in the two previous studies, the authors did not provide specific data of CFU / gr.

Grube the al. 2009 (ISME J., 3: 1105-1115) described the isolation of considerable bacterial densities (107_104 UFCfg) in some species of lichens through the use of a nutrient-poor medium developed for the count of bacteria from oligotrophic waters. The liquid species that grow on soils (earthlings) had higher bacterial population levels (107 CFU / g, since they inhabit environments in which microbial populations tend to be much higher (108_ 109 bacterial cells / g) than those that grow on rocks (104 CFU / g) However, Grube al. 2009 did not employ specific culture media that simulated the nutritional conditions of the analyzed liquid talus. More recently, the Grube group and collaborators (Grube el al., 2014; ISME J .; doi: 10.1038 / ismej. 2014.138) have described more than 800 bacterial species in the lichen model Lobada pulmonaria by means of independent culture techniques, these authors have proposed that these bacteria have the ability to contribute in multiple aspects of the liquid symbiosis , including different essential functions, such as: i) provide nutrients, especially nitrogen, phosphorus and sulfur, ii) confer protection n against biotic (such as pathogens) and abiotic stressors, jii) contribute to photosynthesis by providing vitamin 812, IV) contribute to the growth of fungi and liquid algae by providing hormones, v) detoxification of toxic metabolites, and vi) degradation of the oldest parts of the liquid thallus (nutrient recycling). However, to confirm all these potential functions, as well as to study their possible biotechnological applications, it is necessary to have arable bacteria and for this the method of the present invention is required.

Lasken & McLean, 2014 (Nat. Rev. Genet. 15: 577-584) describe a new method based on the new generation sequences or NGS (from its initials in English New Generation Sequences) for the identification of microbial species that have not yet they have been able to be cultivated by routine microbiological culture techniques, followed by partial or complete sequencing of the 16S ribosomal gene. However, this study technique based only on the analysis of DNA sequences is independent of the culture of the liquid microorganisms, and although it is useful for diversity studies, it does not allow to obtain pure cultures thereof, which is a necessary requirement both for the description of new microbial taxa as for their biochemical, physiological and metabolic characterization for their possible biotechnological applications. Especially complex and difficult is the isolation and characterization of microorganisms that are associated with cyclic mutualistic symbiosis, as is the case with liquid tali.

Therefore, there is still a need in the state of the art to provide a solution to this problem that has not yet been resolved, such as the provision of a culture method that can not only be used in the isolation and identification of microorganisms associated with liquid talus. whose cultivation has not been possible until now or presents great difficulties with the usual methods, but also serves to significantly increase the recovery and isolation of liquid microorganisms, both in quantity and diversity.

DETAILED DESCRIPTION OF THE INVENTION The inventors of the present invention have discovered that the addition of a liquid extract derived from talos of the same population of a liquid species in a culture medium allows, surprisingly, both the isolation and the cultivation of microorganisms associated with the population of the lichen species from which said liquid extract has been obtained more effectively than if isolation and cultivation are carried out in a standard culture medium without liquid extract.

The advantages of the present invention over the various methods that exist in the state of the art for isolating microorganisms associated with a population of a lichen species include:

-

It allows to increase the number of microorganisms recovered after cultivation, both oligotrophs and copiotrophs and in both quantity and diversity, thanks to the fact that a culture medium is used that simulates the nutritional conditions existing in the liquid thallus from which you want to isolate the microorganisms of interest -It allows to increase the recovery of microorganisms associated with lichens, since microbiological analyzes are carried out from fresh liquid talus samples. -It allows to increase the number of microorganisms recovered after cultivation, both ecto-as endoliquénicos, thanks to an extensive washing of liquid talols / s that allows, in turn, the elimination of the disinfection stage of said liquid talus that negatively affects the recovery of said microorganisms.

Based on this discovery, a series of inventive aspects have been developed that will be described in detail below.

Method of obtaining the lumen extract of the invention

As explained previously, the present invention is based on the use of an extract derived from talus from the same population of a liquid species in a culture medium to isolate or cultivate more effectively the microorganisms associated with said liquid species. Therefore, the implementation of the invention comprises obtaining said liquid extract.

Thus, in one aspect, the invention relates to a method for obtaining a liquid extract, hereinafter "method for obtaining the liquid extract of the invention", which comprises:

(to)

collect at least one lichen talus from a liquid species of interest,

(b)

wash and proceed to the disruption of the liquid thallus with a sterile isotonic solution, and

(and)

sterilize by filtration the product obtained after carrying out step b).

The different stages that comprise this method allow to obtain a liquid extract that preserves, unchanged, all the natural components and nutrients that are present in the liquid species, which differentiates it from other methods described in the state of the art.

Thus, in a first stage, the method of obtaining the liquid extract of the invention comprises collecting at least one liquid talus from a liquid species of interest.

In the present description, "liquid species" or "lichen" means the organism (talus, holobiont) that arises from the symbiosis between at least one heterotrophic fungus (mycobiont) and one or more photosynthetic organisms that can be green microalgae

or cyanobacteria (photobionts). The basis of mutualistic symbiosis is the morphological, metabolic and genetic integration between symbionts (talos), where the fungus acquires the carbohydrates necessary for its metabolism from photobionts, while the fungus provides water, mineral elements and signal molecules ( eg NO) for the protection of algae in the aggressive air environment. The nomenclature of the liquid species is made by the name of the symbiote fungus.

In the present invention, "liquid thallus" or "thallus" means the vegetative body of lichen, which is composed of at least one fungus (mycobiont) and one or several photobionts (microalgae and / or cyanobacteria) and other associated microorganisms (bacteria , viruses and other fungi or algae or other organisms), not differentiated in a caulinar axis with leaves and roots, and that does not present true tissues.

As the person skilled in the art understands, the collected liquid species may be part of a population of a liquid species. Thus, in a particular embodiment of step (a), the collected liquid tali belong to the same population of the liquid species. In the present invention, it is understood by

"population" or "biological population" to all organisms or individuals of the same species that coexist in the same space and time, and that share certain biological properties, which produce a high reproductive and ecological cohesion of the group. Reproductive cohesion implies the exchange of genetic material between individuals. Ecological cohesion refers to the presence of interactions between them, resulting from having similar requirements for survival and reproduction, by occupying a generally heterogeneous space in terms of resource availability.

Methods for collecting at least one liquid thallus of the liquid species of interest are widely known in the state of the art and are routine practice for the person skilled in the art. In the collection, scalpels, knives, knives, scissors, gloves, tweezers, etc. can be used. And, once collected, it can be stored in bags, Petri dishes or plastic tubes for transfer to the laboratory.

The processing of the liquid species can be carried out from fresh or frozen talus. Thus, in a particular embodiment, the liquid thallus is preferably fresh and, failing that, may be frozen. In the case of using fresh talos, they can be processed directly after collection or they can be kept refrigerated (for example, around 4 ° C) until they are processed, within a few days. The term "fresh" includes both freshly collected talos and chilled talos. Alternatively, after the collection of the talus it can be frozen in case the storage until its processing is going to be more prolonged. In this case, the person skilled in the art understands that before using the frozen talus to obtain the liquid extract it has to be thawed. Techniques for freezing and thawing liquid tali are widely known in the state of the art and are routine practice for the person skilled in the art.

After the collection of the liquid thallus, the method comprises washing and subsequent disruption of the liquid thallus with a sterile isotonic solution [step b)].

Both washing and thallus disruption are carried out with a sterile isotonic solution. Any sterile isotonic solution can be used in the washing and disruption of the liquid thallus. In the present invention, "isotonic solution" is understood as the composition comprising several salts dissolved in water for the purpose of creating

a saline solution with a concentration of salts equivalent to biological tissue. In a particular embodiment, said sterile isotonic solution is the Ringer solution. The typical Ringer solution generally comprises sodium chloride, potassium chloride, calcium chloride and sodium bicarbonate (the latter used to balance the pH). For example, a typical standard isotonic solution comprises 5 9 of NaCl, 0.42 9 of KCI, 0.25 9 of CaCI2 and 0.2 9 of sodium bicarbonate dissolved in one liter of distilled water.

Additionally, the sterile isotonic solution employed in the present method may be supplemented with a surfactant. In the present invention, "surfaclanle agent" or "surfactant" or "detergent" is understood as the substance that influences by means of super-tension on the contact surface between two phases. The classification is based on the power of dissociation of the surfactant in the presence of an electrolyte and its physicochemical properties. The surfactants can be ionic or non-ionic; and within the ionic ones according to the load that the part that presents the surface activity possesses will be: anionic, cationic and amphoteric. Any surfactant can be used in the context of the present invention. However, in a particular embodiment, the surfactant is polysorbate 20 or polyoxyethylene sorbitan monooleate, commonly known as Tween 20®.

To obtain the liquid extract first, a brief washing, with sterile distilled water, of the liquid talus collected to remove the thicker impurities (dust, soil, etc.) is performed. The talus is then subjected to a longer wash with a sterile isotonic solution, for example, for 30 minutes at 200 rpm at room temperature. After said washing of the thallus, it is disrupted. In the present invention, "disruption" is understood as the breaking of the liquid thallus into smaller fragments. The disruption can be carried out by dilating, crushing, crushing, etc. However, in a particular embodiment, the disruption of the liquid thallus is carried out by crushing.

In the present invention, "dilated" is understood as the technique comprising fragmenting the liquid thallus into smaller pieces. The dilatation of the liquid talus can be done, for example, by the use of blades, scalpels, etc.

In the present invention, "crushed" is understood as the technique that involves hitting something to deform it, crush it or reduce it to small fragments without crushing it. Crushing of the liquid talus can be done, for example, by using a mortar or with a mace in a plastic bag.

In the present invention, "crushing" is understood as the technique comprising grinding

or shred the liquid talus without reducing it entirely to dust. The crushing of the liquid talus can be done, for example, by a crushing machine.

Once step (b) has been carried out, a product is obtained which comprises the washed and fragmented liquid thallus in the isotonic solution, together with the bioactive compounds and substances released and dissolved in said solution. Said liquid extract will be subjected to a sterilization by filtration [step c) of the method to obtain the liquid extract of the invention).

The use in the present method of a physical filtration process without the use of heat or chemical compounds (for example, solvents), allows the properties of the liquid substances that have been extracted in step b) of the method not to be altered. Thus obtaining a liquid extract that preserves intact all the compounds and bioactive substances present in the liquid species in its fresh state. For this reason, the use of the liquid extract obtained by this method is optimal for cultivating and isolating microorganisms associated with a population of a liquid species of interest. This aspect will be explained in detail later in this description.

Filtration sterilization is achieved by the passage of a liquid or a gas through a material capable of retaining the microorganisms present. There are several types of filters and any of them can be used in the present invention. Examples of filters include, without limitation, depth filters and surface filters or membrane filters.

The depth filters are made of a fibrous material (paper, asbestos or glass fiber) arranged randomly, so that tortuous pathways are created within the filter structure where most of the contaminants present can be retained. Given their characteristics, depth filters are mainly used as prefilters, since they allow the removal of large particles but not the total elimination of microorganisms. Thus, in a particular embodiment, filtration sterilization comprises the use of a pre-filter.

Surface filters are filters generally made from cellulose acetate or cellulose nitrate and contain pores of uniform size. By knowing the pore size they present, filters capable of retaining the microorganisms of the solution can be selected. In a particular embodiment, the pore size of the filter is between 0.22 and 0.45 µm. For optimal sterilizing filtration combinations of a depth filter (pre-filter), with a pore size of, for example, 0.8 I-Im, can be used with a surface filter having a pore size, for example, of 0.22 IJm. In another even more particular embodiment, the sterilization by filtration of step e) is carried out with a prefilter (with a pore size of 0.8 IJm) and a filter, where the pore size of the filter is between 0 , 22 and 0.45 IJm.

Any liquid species can be used to obtain the liquid extract of the invention. The liquid thallus consists of lichenizing fungi along with the photobionts. Examples of lichenizing fungi that make up a liquid species include, but are not limited to, Lecanoromycetes that contain 14,000 known species of which 95% are forming lichens (ascolyquens), establishing mutualistic symbiosis with green microalgae (chlorolkenes) or cyanobacteria (cyanolkenes); Only a few establish internal symbiosis with algae and cyanobacteria at the same time (cephalodiums). In Miadlikowska etal's work., 2014 (Mol Phylogenet Evol 79: 132-168) the genera of fungi of this class that are lichenized appear, in short 1139 taxa, 317 genera, 66 families, 17 orders and 5 subclasses: Acarosporomycetidae , Lecanoromycetidae, Ostropomycetidae, Umbilicariomycetidae and "Candelariomycetidae". Only a small percentage of Basidiomycetes are capable of lichenizing (basisioliquens) and the most common genera (Omphalina, Dyctionema, etc.) are listed in Lutzoni, 1997 (Syst Biol. 46 (3): 373-406) and from Hodkinson et al., 2014 (Fungal Divers. 64 (1): 165-179).

Some more detailed examples of liquid species can be

Acarospora spp. Gladonia spp., Evernía spp., Flavoparmelia spp., MeJanelia spp., Pamotrema spp., Platismatia spp., Pseudevernia spp. , Ramalina spp., Tephromela spp., Usnea spp. or Xanthoparmelia spp. However, in a particular embodiment, the liquid species is selected from the group consisting of Pamotrema spp., Pseudevernia spp. and Ramalina spp.

Examples of Ramalina spp. include, but are not limited to, R. asahinae, R. bourgaeana

R. canariensis, R. farinacea, R. fasligiala, R. fraxinea "R. impleclens, R. subgeniculata,

Examples of Parmotrema spp. include, but are not limited to, P. arnold; ¡P. austrosinense, P. crinilum ,. P. hypoleucinum, P. perlatum, P. pseudotinctorum.

Examples of Pseudevernia spp. include, but are not limited to, P. ceralea, P. cirrhata,

P. consocians, P. furfuracea, P. furfuracea varo ceralea, P. furfuracea varo furfuracea,

P. isidiophora, P. kamerunensis, P. mauritiana.

Examples of Evernia spp. include, but are not limited to, E. divaricata, E. iIIyrica, E. furfuracea, E. mesomorpha, E. prunastri

Examples of Parmelia spp. they include, but are not limited to, P. barrenaae, P. saxatilis, P. serrana, P. sulcata.

Examples of Usnea spp. include, without limitation, U. barbata, U. dasypaga, U. filipendula,

U. florida, U. hirta, U. rubicunda, U. rubiginea, U. scabrida, U. sphacelata, U. strigosa,

U. subfloridana.

Examples of Cetraria spp. -in a broad sense- and Karnefeltia include, but are not limited to, C. aculeata, C. crespoae, C. Icelandica, C. ericetarum, and C. iberica (Karnefeltia merrillií.

Liguénic extract of the invention and its uses

In another aspect, the invention relates to a liquid extract, hereinafter "liquid extract of the invention", obtained by the method of obtaining the liquid extract described above.

The liquid extract of the invention is characterized in that it intactly comprises all the compounds and bioactive substances present in the liquid species in its fresh or natural state. This characteristic is a consequence of the method of obtaining used because, as explained, a physical process of sterilization (filtration sterilization) is used in the method of obtaining

which does not alter the physical, chemical or biological properties of the compounds and substances present in lichen.

As the person skilled in the art understands, the liquid extract of the invention may be part of a composition that in addition to comprising said liquid extract, comprises other compounds or substances that can enhance or facilitate the growth or isolation of microorganisms. Thus, in another aspect, the invention relates to a composition, hereinafter "composition of the invention", which comprises the liquid extract of the invention. Examples of other compounds or substances that may be present in the composition of the invention include, without limitation, a source of carbon, a source of nitrogen, a source of phosphorus, vitamins, antifungals or antibacterials according to interest, etc.

In the context of the present invention, the composition may be liquid or solid. In a particular embodiment, the composition is a culture medium.

The presence in the liquid extract of the invention of the biactive compounds and substances present in the liquid species in its fresh or natural state allows that, if this liquid extract is added to a culture medium, microorganisms associated with it can be isolated and cultured. liquid species more effectively than if the culture medium does not include the liquid extract.

Therefore, in another aspect, the invention relates to the use of the liquid extract of the invention and / or the composition of the invention, to isolate and / or cultivate the microorganisms associated with a liquid species of interest, wherein the Liquid extract is obtained from at least one liquid talus of the liquid species from which you want to isolate the microorganism (s). In a particular embodiment, the culture is carried out in a minimum medium supplemented with at least one liquid extract obtained from talos of the same population as the talo / s of the liquid species from which it is desired to isolate the microorganism / s. The term "population" has been previously defined.

In a particular embodiment, the microorganism associated with the liquid species is selected from the group consisting of bacteria, algae, filamentous fungi, yeasts and viruses of the previous liquid microorganisms.

Examples of non-lichen fungi associated with lichen include, but are not limited to, members of the Ascomycola (Bates el al., 2012. The Lichenologisl 44 (1): 137-146, doi: 10.1017 / S002428291 1000648. The lichenizing fungi have previously mentioned.

Examples of algae include, but are not limited to, green microalgae. The green microalgae that are usually associated with Ascolíquenes and Basidiolíquenes are, within the Chlorophyta, of the classes Trebouxiophyceae and Ulvophyceae, (for example: Trebouxia, Asterochloris, Coccomyxa, Dictyochloropsis sl eh / arel / a, Elliptochloris, Phycopeltis, Trentepois, Trentepois, etc .) or cyanobaclerias of the orders Chroococcales, Nostocales, and Stigonematales (for example: Nostoc, Rhizonema, Scytonema, Stigonema, etc.) [Friedl T. & Büdel B. 2008. Photobionts. In: Nash T. H. (Ed.), Lichen biology, Second edition. Cambridge University Press, Cambridge, pp. 9-26].

Examples of liquid microorganism viruses include, but are not limited to, Ivy (ivy) latent Cytorhabdovirus and Apple (apple tree) mosaic virus (ApMV). Both are types of double-stranded RNA viruses found in liquid algae of the genus Trebouxia (Petrzik, K. el al., 2014. Eur. J. Planl Palhol., 138 (3): 549-559).

Examples of bacteria include, but are not limited to, those pertaining to Actinomycetales, Badila / es, Burkholderiales, Caulobacterales, Rhizobia / es, RhodospiriIJa / es, Pseudomonadales, Sphingomonadales and Xanlhomonada / es (Cardinale el al., 2006; Cardinale al., 2008 (FEMS Microbio !. Eco !. 66: 63-71) Liba el al., 2006; Grube & Berg 2009 (Fungal Biology Reviews 23: 72-85.); Grube el al., 2009, 2014 ; Hodkinson & Lulzoni 2009 (Symbiosis, 49: 163-180.); Selbman on al., 2010; Bates on al., 2011 (App !. Environ. Microbe !. 77 (4): 1309-1314; Lasken & McLean , 2014).

In another particular embodiment, the liquid species of interest is selected from the group consisting of Pamolrema spp., Pseudevernia spp. and Ramalina spp. Examples of other genera and liquid species that can be used in the present invention have been previously described in the method of obtaining the liquid extract of the invention.

Method for isolating microorganisms associated with a lumen species of interest In another aspect, the invention relates to a method for isolating microorganisms associated with a liquid species of interest, hereinafter "method of isolation of the invention", which comprises

(to)

collect at least one liquid talus from the liquid species from which the microorganisms are to be isolated, under aseptic conditions,

(b)

Wash the liquid talus collected in step (a) extensively, with isotonic solution.

(and)

Carry out the disruption of the washed liquid thallus in step (b), and

(d)

cultivate, in a minimum culture medium supplemented with at least the liquid extract of the invention:

(i)

the product obtained from the disruption of the liquid talus of step (e), and / or

(ii)

the wash solution recovered after extensive washing of step (b)

where, in the case that they are cultivated (i) and (ii), they are grown independently, and the liquid extract is obtained from the liquid species of interest from which the microorganisms are to be isolated.

In a first stage, the method of isolation of the invention comprises collecting at least one liquid thallus from the liquid species from which the microorganisms are to be isolated, under aseptic conditions.

The collection of the thallus of the liquid species of interest has to be done under aseptic conditions, that is, under the appropriate collection conditions so that the lichens are not contaminated with other microorganisms outside the lichen itself. As the person skilled in the art understands, one or more talos of the same liquid species can be collected. Lichens can grow on a wide variety of substrates, such as tree bark, rock subsurface, soil, artificial materials (glass, uralite, tiles, cement, etc.), etc., so that for To obtain the liquid talos you must first collect them from your surroundings. For this, scalpels, knives, knives, scissors, gloves, tweezers, etc. can be used. and once the talos are collected, they can be stored in bags, petri dishes or plastic tubes for transfer to the laboratory. In order to achieve said aseptic collection conditions, all the aforementioned material can be sterilized or decontaminated before being used. Once you have collected the liquoric talus (s) from your environment, these are taken to the laboratory to be processed.

An essential feature of the method of isolation of the invention is that the liquid extract used in the culture medium must have been obtained from the same liquid species from which the microorganism (s) is to be isolated. For example, if the objective is to isolate the microorganisms associated with the liquid species Pseudevernia furfuracea (L.) Zopf, the liquid extract with which the minimum medium will be supplemented will be obtained from the talus / s of a species of P. furfuracea. Another example may be Ramalia farinacea (L.) Ach. o Parmotrema spp. , in such cases the culture media would be supplemented with liquid extracts of the respective lichens.

In a particular embodiment of step (a), the collected liquid tali belong to the same population of the liquid species. The term population has been previously described. Thus, in the isolation of microorganisms associated with lichen, a liquid extract obtained from talus of lichen from the same population as the lichen from which you want to isolate and cultivate the microorganisms can be added to the minimum medium.

In a second stage, the method of isolation of the invention comprises extensively washing the liquid thallus collected in step (a) with an isotonic solution. In the present invention, "extensive washing" or "extensive washing" is understood as washing, which lasts from 60 to 90 minutes. In a particular embodiment, extensive washing of the liquid thallus comprises washing for at least about 60 minutes, preferably about 90 minutes. Any sterile isotonic solution can be used in the washing of the liquid thallus collected in step a). However, in a particular embodiment, the washing is carried out with a sterile isotonic solution comprising a surfactant. In another particular embodiment, the sterile isotonic solution is Ringer solution. In another still more particular embodiment, the surfactant is Tween 20. Both definitions and examples of isotonic solutions and surfactants have been previously described in the present description and are applicable to the present inventive aspect.

Optionally, after extensive washing of the liquid talus with isotonic solution, and separating it from the washing solution obtained in step (b), it is possible to remove possible remains of the wash with sterile distilled water by means of, for example, a rinse.

Next, the disruption of the liquid thallus is carried out. The term "disruption" has been previously defined in the present description. The disruption can be carried out by crushing, dilating, crushing, etc. However, in a particular embodiment, the disruption of the liquid thallus is carried out by crushing in a sterile antioxidant maceration buffer (AMB of its initials in English Antioxidant Maceration Buffer) and under aseptic conditions. The technical effect associated with this crushing procedure is to obtain a greater amount of isolated microorganisms than through the use of other disruption techniques (crushed / dilated). Likewise, the use of antioxidant maceration buffer prevents possible oxidation reactions that could occur during the processing of the material and that could inhibit microbial growth.

Once steps (a) to (c) of the method of isolation of the invention have been carried out, step (d) is carried out which comprises cultivating, in a minimum culture medium supplemented with at least the liquid extract of the invention:

(i)

the product obtained from the disruption of the liquid talus of step (c), and / or

(ii)

the wash solution recovered after extensive washing of step (b)

where, in the case that they are cultivated (i) and (ii), they are grown independently, and the liquid extract is obtained from the liquid species of interest from which the microorganisms are to be isolated.

The implementation of the method of the invention requires that the minimum medium be supplemented with a liquid extract obtained from the talus of the liquid species of interest, that is, from the lichen talus from which the microorganisms are to be isolated. For example, as previously explained for previous inventive aspects, if the objective is to isolate the microorganisms associated with the P. furfuracea liquid species, the liquid extract with which the minimum medium is supplemented will be obtained from talols of said species . Another example may be

R. farinacea or Parmotrema spp., In such cases the culture media would be supplemented with liquid extracts of the respective liquid species. In a particular embodiment, the culture stage (d) is carried out in a minimum medium supplemented with at least one liquid extract obtained from talos of the same population as the talols of the liquid species collected according to stage ( to). The term population has been previously defined.

In the present invention, "minimum medium" means any mineral base medium comprising the basic compounds to allow the growth of microorganisms. Examples of minimum media include, but are not limited to, minimum AB media (Chillan al., 1974, Proc. Nall. Acad. ScL USA, 71: 3672-3676): K, HPO "3 g; NaH, PO, 1 g ; NH, CL 1 g; MgSO, -7H, O 0.3 g; KCI 0.15 g; CaCl, 0.01 g; FeSO, 7H, O 2.5 mg, per 1 L distilled water; glucose 0 , 5%), half M9, half MBMA, half M63 and half MA More details can be found in Santander on al., 2014 (FEMS Microbiol. Ecol. 90 (3): 895-907, doi: 10.1111 / 1574-6941.12444 In order to carry out the method of isolation of the invention it is not necessary that the minimum medium carries a carbon source, since the presence of the liquid extract of the invention is sufficient, however, in a particular embodiment, the minimum medium comprises one or more carbon sources which, in another even more particular embodiment, said carbon source is 0.5% glucose and / or mannitol.

Also, depending on the type of microorganism that is to be isolated (bacteria, algae, filamentous or unicellular fungi (yeasts) and / or viruses), the minimum culture medium may be supplemented with other components. For example, if the microorganism to be isolated by the method of isolation of the invention is a liquid bacterium (that is, associated with a lichen), the culture medium will have to be supplemented with other compounds that prevent the growth of other microorganisms that may interfere with the growth of the bacteria. Thus, the minimum culture medium can be supplemented with at least one antifungal. In the present invention "antifungal" means any substance that has the ability to prevent the growth of some types of fungi or even cause death. Examples of antifungals include, but are not limited to, polyenes (e.g. amphotericin 8), pyrimidines (e.g. fluorocytosine), imidazoles (e.g. miconazole), triazoles (e.g. fluconazole), cardines and cycloheximide. In a particular embodiment, step cultivation

(d) it is carried out in a minimum culture medium supplemented with at least the liquid extract and with at least one antifungal, preferably, natamycin.

On the other hand, if the microorganism that you want to isolate from the liquid talus is a virus associated with a liquid microorganism (bacteria, algae, filamentous fungi or yeasts), then the culture medium will be supplemented with a lawn of the liquid microorganism so that the virus I can grow Thus, in another particular embodiment, the cultivation of step (d) is carried out in a minimum culture medium

supplemented at least with the liquid extract and with the liquid microorganism of interest.

In a particular embodiment, the microorganism associated with the liquid species of interest is selected from the group consisting of bacteria, algae, filamentous fungi, yeasts and viruses of the aforementioned liquid microorganisms. Examples of fungi and algae associated with liquid tali have already been described in previous paragraphs.

Examples of liquid species that can be used to isolate microorganisms associated with them have been mentioned in previous inventive aspects. In a particular embodiment, the lichen tali of the lichen species is selected from the group consisting of Pamotrema spp. , Pseudevernia spp. and Ramalina spp.

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 examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Species of lichen fruit, Pseudevernia furfuracea, in bark of Pinus sylvestris. Scale bar = 1 centimeter.

Figure 2. Improved recovery of bacteria associated with Pseudevernia furfuracea with a modified isolation protocol and minimal culture medium. The average of the counts of cultivable bacteria (CFU / g) ectoliquénicas (white bars) and endoliquénicas of P. furfuracea (gray bars) in the culture media KB, 8BM and MMS after 7 days of incubation at 25 ° C is shown . Samples of liquid tali were taken in spring 2012 from several specimens of Pinus sylvestris from the Sierra de Javalambre (Teruel, Spain). Each bar represents the average value of the counts in each culture medium of three different talus analyzed in independent experiments. The standard deviation of the data is indicated by vertical lines.

.

Figure 3. Comparison of the growth of bacterial strains of Pseudevernia furfuracea in minimal medium enriched with lichen versus without lichen with or without carbon sources. Representative images showing the growth of selected bacterial strains isolated from P. furfuracea in AS medium supplemented or not with glucose (G), mannitol (M), or both (GM) and / or lichen extract (L) after 96 incubation hours at 25 ° C. Some details of the differential growth of some bacterial strains are indicated with black asterisks. All strains tested grew in the KB control culture medium (data not shown).

Figure 4. Effect of culture media enriched with lichen extract and incubation time on the growth of bacterial strains of Pseudevernia furfuracea. The graph of stacked growth columns of 139 bacterial strains of P. furfuracea in the minimum AS medium supplemented or not with carbon sources (glucose, G; mannitol, M; or glucose and mannitol, GM) and / or lichen extract ( L) after 24 hours (a) and 96 hours (b) of incubation at 25 ° C. The growth of bacteria in each medium tested was compared with the control in the KS medium, which was arbitrarily set at 100% growth. The growth results were grouped into three categories (no growth, growth at the point of inoculation and increased growth). The data shown come from an experiment performed in duplicate. Significant differences (p <0.001) between the two culture media are indicated by asterisks located above the stacked columns. Significant differences (p <0.001) were also found between 24 hours (a) and 96 hours (b) of incubation.

Figure 5. Effect of lichen-enriched media on the recovery of bacteria associated with Pseudevernia furfuracea from a thallus sample. The average of the counts of cultivable bacteria (CFU / g) ectoliquénicas (a) and endoliquénicas of P. furfuracea (b) is shown in the culture media AS, ASG and A8GM enriched or not with lichen extract and in the medium K8 , after 3 days of incubation at 25 ° C. The talus sample was taken in spring 2012 from a copy of Pinus sylvestris from the Sierra de Javalambre (Teruel, Spain). Each bar represents the average value of the counts made in duplicate in each culture medium of the same talus sample. The standard deviation of the data is indicated by vertical lines. Significant differences are indicated by different letters above the error bars (* p <0.005; ** p <O, 01).

Figure 6. Effect of lichen-enriched media and the time of

incubation in the recovery of bacteria associated with Pseudevernia furfuracea from samples composed of different talos. The average of the counts of cultivable bacteria (CFU / g) ectoliquénicas (Figures 6a, Be) and endoliquenicas of P. furfuracea (b, d) of samples composed of five different talos, in the culture media enriched with lichen with high (ABGML) and low (ABL) nutrient content, in the lichen-free medium with high nutrient content (ABGM) and in the nutrient-rich KB environment, by increasing the incubation time up to 15 days at 25 ° C. Samples of liquid tali were taken in the fall of 2012 (Figures 6a, 6b) and in the summer of 2013 (Figures 6c, 6d) of several specimens of Pinus sylvestris from the Sierra de Javalambre (Teruel, Spain). Each point represents the average value of triplicate counts in each culture medium of the same sample composed of different talos. The standard deviation of the data is indicated by vertical lines.

Figure 7. Effect of two antifungals on the growth of filamentous fungi from samples of Pseudevernia furfuracea. Two representative examples of the effect of cycloheximide (C) and natamycin (N) in the nutrient-rich medium KB on the growth of two filamentous fungi, frequently isolated from samples of liquid talus, are shown after 7 days of incubation at 25 ° C

Figure 8. Effect of cycloheximide (e) and natamycin (Nat) on the recovery of bacteria from Pseudevernia furfuracea. The average of the ectoliquénica cultivable bacteria counts (CFU / ml) of P. furfuracea is shown in both nutrient-rich culture medium (KB) and minimum medium (ABGM), supplemented with cycloheximide or natamycin. H, filamentous fungi that invade the plates of culture medium in a period of 3-5 days. H ', filamentous fungi that invade the plates of culture medium in a period greater than 7 days. Each bar represents the average value of the counts in each culture medium of two different P. furfuracea talus analyzed in duplicate. The standard deviation of the data is represented by vertical lines. Asterisks indicate statistically significant differences (** p <0.01; *** p <0.0001).

Figure 9. Effect of washing time on the recovery of ectolygenic bacteria from Pseudevernia furfuracea. The average counts are shown

of cultivable bacteria (U Fe / mi) ectoliquénicas of P. furfuracea both in culture medium rich in nutrients (KB) and minimum medium (ABGM), both supplemented with natamycin, after 3 days of incubation at 25 ° C. Each symbol represents the average value of three counts in each culture medium of a talus composition of P. furfuracea. The standard deviation of the data is represented by vertical lines.

Figure 10. Effect of different disinfection treatments and the processing of the liquid talus in the recovery of endolygenic bacteria from Pseudevernia furfuracea. The average of the endoliquenic cultivable bacteria (CFU / ml) counts of P. furfuracea is shown in both nutrient-rich culture medium (KB) and minimum medium (ABGM), both supplemented with natamycin, after 7 days of incubation at 25 ° C. Details on the type and / or disinfection time tested are shown in the figure. The types of processing compared were dilated (Dil.) Versus crushed (Mach.). Each bar represents the average value of three counts in each culture medium of a talus composition of P. furfuracea. The standard deviation of the data is represented by vertical lines. Asterisks indicate statistically significant differences (* p <O, 05; ** p <O, 01; *** p <O, 0001).

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors that demonstrate the effectiveness of the product of the invention.

Example 1 Isolation of bacteria associated with liquid species by using a liquid extract

MATERIAL AND METODDS

Samples of lichen instead of sampling and sampling the talus of lichen Pseudevernia furfuracea (Figure 1) were obtained from an area of Pinus sylvestris forest in the mountains of the Sierra de Javalambre, located in the Gúdar-Javalambre region, in the province from Teruel (Aragon, Spain). Initial sampling was carried out in May 2012. Thereafter, new samples were taken at the same place. In each sampling, lichen samples were collected from at least five randomly selected trees within an area of about 50 meters. Physiologically active specimens were collected that were carefully removed from the tree bark using sterile gloves, then transferred to sterile plastic Petri dishes that were stored in individual plastic bags. The different sampled liquid talus were transported to the laboratory and kept at 4 ° C until processing within 24 hours after sampling. The temperature of each sampling was recorded in situ and the humidity data were obtained from the Spanish Meteorological Agency (AEMET).

Bacteriological analysis

For the isolation of bacteria associated with P. furfuracea, samples of liquid talus were initially analyzed as follows. Briefly, each individual thallus was washed in sterile distilled water for 1 minute and with the help of a sterile scalpel 0.2g subsamples were cut into sterile Petri dishes. These subsamples were dilated in sterile saline (0.9% NaCl in distilled water, pH 7.0) (SS) and then serial dilutions were made in sterile phosphate buffered saline (PBS) (10 mM, pH 7 , 0 (NaCI8 g; KCI 0.2 g; Na, HPO, • 12H, O 2.9 g; KH, PO, 0.2 g; distilled water up to 1 L). Both of the talus dilatation samples and of its dilutions, 100 IJI aliquots were seeded in the non-selective solid medium King B (KB) (King et al., 1954; J. Lab. Clin. Med. 44: 301-307) routinely used for the isolation of bacteria associated with plants, after supplementation with cycloheximide (50 mg I mL) (Biosca et al., 2003; Phytopathology 93: 485-492, modified according to Ishimaru & Klos, 1984, Phytopathology. 74: 1342-1345) to prevent the growth of Fungi Plates were incubated for a week at 25 ° C in dark conditions and the bacterial colonies that appeared in them were counted to determine the co-forming units Lonias per gram (CFU / g) of thallus. Morphologically different colonies were purified in the same medium and cryopreserved at -80 ° C in 25% (v / v) sterile glycerol.

Modification of the bacterial isolation protocol to improve the recovery of bacteria associated with Po furfuracea To try to improve the recovery of bacteria associated with lichen P. furfuracea, two modifications of the bacterial isolation protocol were tested by analyzing three different thallus samples in independent experiments. First, to increase the recovery, mostly, of ectoliquenic bacteria, after a brief 1-2 minute wash in sterile SS, the samples were subjected to extensive washing in 250 mL flasks with 50 mL of sterile Ringer's solution ( Schaad et al., 1990; Methods in Phytobacteriology. Z. Klement, K. Rudolph & DC Sands (eds.) Akadémia Kiadó, Budapest, pp. 153-190.) Supplemented with 0.05% of Tween 20 surfactant (RST ), with stirring for 90 minutes at 200 rpm at room temperature (Biosca et al., 2011; In: IV International Conference on Environmental, Industrial and Applied Microbiology, BioMicroWorld 2011). Second, to increase the recovery of endolygenic bacteria, these same samples of washed talus were subjected to a surface disinfection (with 70% ethanol (v / v) for 1 minute), washed twice for 5 minutes in distilled water sterile and dilated in 3 mL of antioxidant maceration buffer (AMB) (Gorris et al., 1996; Acta Hortic. 411: 47-51); to prevent oxidative stress that could occur during the processing of the liquid talus and that could affect the isolation of liquid bacteria. Each sample of talus dilacerated was shaken slightly for 5-10 minutes on ice before it was planted in the culture medium plates. Aliquots of 100 IJI were sown in duplicate in KB plates, both of the talus washing samples and the thallus dilatation samples and their respective dilutions. Since some liquid bacteria, such as Methylobacterium sp., Grew during the isolation of P. furfuracea microalgae in the BBM minimum medium (from the initials in English Bold's Basal Medium) used for the growth of microalgae (Bischoff & Bold, 1963. Some soil algae from enchanted rack and related algal species. Phycological Studies IV., Univ. No.

6318, Texas, p. 1-95) in previous studies, this culture medium together with the Methanol Mineral Sales (MMS) medium (Green, 2006. Methylobacterium. Prokaryotes 5, pp. 257-265), selective for Methylobacterium, were included in these trials after add cycloheximide, as described above. All inoculated plates were incubated for one week at 25 ° C in dark conditions, noting the number of bacterial colonies that developed in all culture media. The populations of cultivable endolygenic and endolygenic bacteria were estimated by calculating the average values of CFU / g of samples of washed and disinfected and dilated talos, respectively. Colonies with different colonial morphology were randomly selected from each culture medium and purified and cryopreserved as mentioned above.

Development of culture media enriched with lichen extract To further increase the recovery of cultivable bacteria associated with Pseudevernia furfuracea, attempts were made to simulate the nulricional conditions of this lichen by developing several minimum culture media enriched with extracts of P talus furfuracea For this purpose, the minimum medium AB (Chiltan el al., 1974, cited ad supra) was included, which was supplemented or not with glucose (G) (main source of photobiont carbon) (ABG) and / or mannitol (M ) main carbon source of mycobiont) (ABM and ABGM), and / or lichen extracts (L) (ABL, ABLG, ABLM and ABLGM). The different AS culture media were prepared by adding sterilized glucose and / or mannitol by filtration (each at 0.5% (w / v) according to Cardinale et al. (2006, cited ad supra) and / or extracts of fresh lichen (also 0.5% (v / v ».) For the preparation of lichen extracts, the P. furfuracea stains were washed briefly with sterile distilled water, then washed with sterile RST for 30 minutes at 200 rpm at room temperature, they were then crushed in RST (5%) (w / v) in a blender and sterilized by filtration (including the use of polycarbonate prefilters with a pore size of 0.8 microns, Millipore) The minimum BBM and MMS media supplemented or not with lichen extracts were also used for some tests All solid culture media were prepared with 1.5% bacterial agar (plv) (Pronadisa) and supplemented with cycloheximide as described above

Evaluation of lichen-enriched culture media on the growth of bacterial isolates of P. furfuracea To investigate the effect of adding lichen extract to the minimal media on the growth of a collection of bacterial isolates of P. furfuracea, initially 20 strains were tested in the AB medium (supplemented or not with carbon sources and / or lichen extract) and BBM and MMS (with and without lichen extract). Based on these results, the AB medium was selected as the minimum baseline medium (modified or not with glucose and / or mannitol and / or lichen extract) for the performance of new tests with a collection of 139 liquid bacterial strains obtained in this I study and in others of our laboratory. In addition, for comparison purposes, eight reference heterotrophic bacterial strains of the Spanish Type Culture Collection (CECT) (BaciJlus cereus CECT 495, Enferobacter cJoacae CECT 194, Enterococcus faecalis CECT 481, Escherichia coN CECT 101, Micrococcus luteus CECT 245, were included Pseudomonas fluorescens CECT 378, Salmonella enterica CECT 443 and Serratia marcescens CECT 159). All strains were inoculated in all media enriched or not with lichen, using the Ka medium, in which they were usually purified, as growth control, which was arbitrarily set as a 100% growth. Bacterial inoculums were prepared from cultures grown in Ka plates at 25 ° C for 48 hours and resuspended in sterile PBS (approximately 108 CFU / ml). These bacterial inoculums were inoculated with a multi-inoculator (Denley multipoint inoculator), at least in duplicate, in all the aforementioned media. On the other hand, to determine the effect of incubation time on bacterial growth, the plates were incubated for 96 hours at 25 ° C and growth and coloration were recorded after 24 hours and 96 hours of incubation. For subsequent statistical analysis, the growth results were grouped into two (no growth versus growth) or three categories (no growth, growth at the inoculated point and higher growth (abundant growth at the inoculated point).

Improvement of the cultivability of the bacteria associated with P. furfuracea from individual thallus samples In order to evaluate the protocol of isolation of bacteria and the culture media enriched with lichen in the improvement of the cultivability of both ectoliquénicas and endoliquénicas bacteria of P. furfuracea tali, new thallus subsamples (0.2 g) were analyzed according to the methodology described above. Subsequently, aliquots (100 IJI) of talus wash and dilated thallium were sown in duplicate in the enriched media or not with lichen extract (with or without glucose or glucose and mannitol) and the KB medium, and incubated at 25 ° C for 3 days. After incubation, the bacterial colonies were counted in all culture media, and the populations of ectoliquenic and endolygenic arable bacteria were estimated by calculating the mean values of CFU / g of thallus lavage and dilated of thallus washed and disinfected, respectively. A random selection of colonies that showed different morphologies on the isolation plates were purified and cryopreserved as already mentioned. The remaining wash and dilated thallus were also cryopreserved for further analysis.

Improvement of the cultivability of bacteria associated with P. furfuracea from samples composed of different talos

To validate the methodology developed in this study in different samples of talos, new samples of lichens were taken from the same locality sampled in May 2012, but from different specimens collected from P. sylvestris, in two

additional sampling in October 2012 and September 2013. In this case, for each sampling carried out, samples of 1 9 composed of different talos of P. furfuracea (subsamples of 0.2 9 of five talos of different pines) were analyzed, instead of analyzing individual talos. Ectoliquénic and endoliquén cultivable bacterial counts of P. furfuracea were determined in triplicate in culture media enriched with lichen, ABLGM, with high nutrient content (with two carbon sources, glucose and mannitol) and ABL with low nutrient content (only with

the lichen extract nutrients), and in the medium without lichen with high ABGM nutrient content and the KB nutrient-rich medium, the latter for comparative purposes. The plates were incubated as described above and the number of colonies that appeared in them was recorded at different time intervals up to 15 days, unless the number of colonies was too high or filamentous fungi appeared on the media plates of cultivation In addition, to study the effect of the isolation medium on the diversity of the recovered bacteria, the different colonial morphologies observed in the different culture media at the end of the experiment were also recorded. Colonies that showed different phenotypes were randomly selected, mostly purified on KB plates and cryopreserved as described above.

Optimization of the isolation protocol to improve the recovery of bacteria associated with P. furfuracea In order to advance in the optimization of the method of recovery of liquid bacteria developed, two additional modifications of the bacterial isolation protocol were tested by analyzing new samples of talos in independent experiments. First, to reduce the growth of contaminating filamentous fungi that hinder the recovery of slower-growing bacteria, another antifungal was selected other than usual and the effect of both antifungals on the growth of a selection of 7 filamentous fungi was compared. They frequently grow on bacteria isolation plates from samples of liquid talus. Cycloheximide (50 mg / L) is the most widely used antifungal to reduce or prevent fungal growth during the isolation of bacteria from environmental samples, and natamycin (21.6 mg / L, according to Pedersen, 1992, Appl. Environ. Microbiol. 58 (3): 1064-1066), described as an antifungal more effective than cycloheximide, without health risks (used in the food industry) and without effects on bacterial growth, unlike cycloheximide . These tests were initially carried out in plates of the general culture medium rich in KB nutrients, where the selected fungi grow rapidly. Next, the effect of the addition of these antifungals on the recovery of liquid bacteria in both KB and the minimum medium (ABGM) was tested.

Secondly, in order to increase the recovery of endolygenic bacteria that seemed to be affected by the disinfection step: i) the effect of the washing time on the recovery of ectoliquenic bacteria was evaluated both in the nutrient-rich culture medium ( KB) as in the minimum medium (ABGM), both supplemented with natamycin, and ii) the effect of disinfection of the liquid talus with 70% ethanol (v / v) at two different times (30 seconds and 1 minute) was tested. or with 8% hydrogen peroxide (v / v) for 5 minutes, including subsamples that were processed without undergoing any disinfection treatment. At the same time, the effect of the type of thallus disruption in the AMB antioxidant buffer was also investigated, comparing the processing thereof by dilating (previously explained) against crushing, inside a sterilized plastic bag, with a rubber mace (Ordax el al., 2009, J. Appl. Microbiol. 107 (1): 106-16). In both cases, after thallus disruption, they were incubated on crushed ice for 5-10 minutes. After said incubation, serial decimal dilutions of the extracts were made in AMB, aliquots of 100 IJI were sown in duplicate both directly from said extracts and from their respective dilutions in the KB and ABGM culture media with natamycin. All inoculated plates were incubated for one week at 25 ° C in dark conditions, noting the number of bacterial colonies that developed in the culture media. In this case, the populations of cultivable endolygenic bacteria were estimated by calculating the average values of CFU / g.

Statistical analysis Data from the counts of arable bacteria were expressed as the means of two or three determinations for each bacteriological analysis and culture medium, and were normalized by logarithmic transformation. The statistical significance of the differences observed between two standardized means was assessed by a Student's t-parametric test and in the case of three or more means with an ANOVA analysis followed by a post-hoc Bonferroni or Dunnet analysis. Categorical variables were compared using the Chi-square test. The multivariate analysis was performed by means of a logistic regression model to determine the effect of the addition of lichen extract to the culture media, the incubation time and the bacterial isolation protocol, in the growth of bacterial isolates of P furfuracea P values less than 0.05 were considered significant. Data were analyzed with the SPSS 19.0 program (SPSS Statistics 18M).

RESULTS

Bacteria isolation

i) Initial analysis The isolation of Methylobacterium sp. In the minimal medium BBM used for the isolation of microalgae from Pseudevernia furfuracea was the beginning of an investigation to study the cultivable bacteria associated with the lichen species

P. furfuracea. A preliminary bacteriological analysis of P. furfuracea thallus samples, by dilating individual talos in sterile SS, showed similar results, only few colonies grew in the non-selective KB culture medium after 7 days of incubation, yielding low bacterial counts (102 CFU / g of fresh talus). Due to the low number of bacterial colonies recovered, two approaches were followed to try to improve the recovery of bacteria associated with P. furfuracea. First, the method of isolating bacteria from the thallus samples was modified and, secondly, culture media enriched with lichen extract was developed (see below).

ii) Improved recovery of bacteria associated with P. furfuracea by modification of the bacterial isolation protocol. To improve the recovery of bacteria associated with P. furfuracea, two modifications to the isolation protocol were tested: extensive washing of the

2.

Thallus samples with a surfactant (Tween 20) to increase the recovery of ectoliquenic bacteria, and the use of an antioxidant buffer (AMB) to dilate the thallus samples and improve the isolation of endolygenic bacteria. First, when the isolation of endolygenic bacteria from disinfected and dilated talus in the AMB antioxidant buffer versus SS was compared, the results showed that the average bacterial counts in KB were higher in AMB (103 CFU / g of fresh thallus) ( Figure 2) than in SS (102 CFU / g of fresh talus) (data not shown), for the same incubation period (7 days). In addition, bacterial colonies isolated with AMB appeared before in KB medium plates after 3 to 5 days of incubation.

Secondly, when comparing the isolation of ectoliquenic bacteria, estimated by the washing of fresh talos, against the endoliquénicos of talus dilatation (Figure 2), the average counts of arable bacteria were lower for ectoliquénicas than for endoliquénicas in the three culture media tested. In the KB medium, the average counts of ectoliquenic bacteria were 3.3 x 102 CFU / g of fresh washed thallus, being lower than those obtained with endolygenic bacteria (1 x 103 CFU / g of fresh dilated thallus), after 7 days of incubation (Figure 2). Interestingly, the inclusion of the minimum BBM and MMS media for comparative purposes yielded significantly higher endolygenic bacteria counts (from 1.8 to 2.1 x 103 CFU / g of fresh dilated thallus, respectively) than those obtained in the medium nutrient-rich KB (1 x 103 CFU / g of dilated fresh thallus) after 7 days (Figure 2), but the colonies were smaller and appeared 2 to 4 days later than in the KB plates. Similarly, the mean counts of ectoliquenic bacteria from fresh washed talus were also higher in BBM (6.7 x 102 CFU / g) than in KB plates (3.3 x 10 'CFU / g), but no in the MMS medium (1.3 x 10 'CFU / g) (Figure 2). The growth in minimal medium without a carbon source (BBM) or with methanol (MMS) alone was probably at the expense of the nutrients present in the washes or of the thal dilates used as inoculum in the isolation plates. There was no statistical support for the differences found between the average bacterial counts obtained in KB, BBM and MMS (p> 0.05), nor for those observed between ectoliquénicos bacteria against the endoliquénicas in each culture medium, probably due to the variability of the counts obtained with different talos.

Ii) Culture media enriched with lichen improves the growth of bacterial strains of P. furfuracea.

Because the increase in the recovery of bacterial colonies of liquid tali in a minimal medium could be due to the lichen extract used as inoculum during bacterial isolation, the effect of adding lichen extract to the minimum medium (supplemented or not with carbon sources) in the growth of a collection of bacterial strains of P. furfuracea. The results of preliminary experiments with 20 bacterial strains cultured in ASG, ABM, ABGM, BBM and MMS media enriched or not with lichen extract, showed faster and greater growth in lichen-enriched culture media, with better results with AS media with respect to SSM and MMS media (data not shown). Figure 3 shows representative images of the growth of bacterial strains isolated from P. furfuracea in the minimum AS medium supplemented or not with carbon sources and / or lichen extract. For some of the strains tested, greater growth was observed in all culture media enriched with lichen and carbon sources compared to culture media without lichen, supplemented only with carbon sources. In addition, for some strains, growth was higher in the medium with high nutrient content, ASLGM, enriched with lichen extract and with glucose and mannitol. Interestingly, the source of carbon used in the AS culture medium not only affected the growth of some strains, but also the pigmentation of some of them; because in the AS medium with glucose (alone or with mannitol) some strains showed a stronger pigmentation (yellowish or pinkish), regardless of the presence of lichen extract.

To confirm these initial results, further experiments were performed with all AS culture media using 139 bacterial strains isolated from P. furfuracea and eight bacterial reference strains from non-liquid sources. The effect of incubation time on bacterial growth was also investigated after 24 hours and 96 hours of incubation. As shown in Figure 4, when each of the culture media enriched with lichen was compared with the corresponding media without lichen extract, and the growth results were grouped into three categories: i) no growth, ii) growth in the inoculated point and iii) growth increase, significant differences (p <O, 001) were observed both after 24 hours (Figure 4a) and after 96 hours (Figure 4b) of incubation. Thus, a higher percentage of strains grew in the culture media enriched with lichen against the culture media without lichen, and after 96 hours versus 24 hours of incubation. On the other hand, significant differences (p <O, 001) were also found between the different culture media enriched with lichen, with a higher percentage of strains showing higher growth in the ABLGM medium with high

5 nutrient content (20.4%), followed by ABLM (19.7%) and ABLG (16.4%) culture media, and a lower percentage in the medium with low ABL nutrient content (2.7 %), after 96 hours of incubation (Figure 4b).

When the results were analyzed by grouping them into two categories (growth

10 versus non-growth), growth in lichen-enriched media versus lichen-free culture media, the Chi-square test revealed that the percentage of strains that grew in a lichen-enriched culture medium (91.7% ) was significantly higher (p <0.001) than in the same medium without lichen extract (66%) after 96 hours of incubation. In addition, lichen strains grew

15 significantly earlier (p <O, 001) in the presence of lichen extract (75.3% after 24 hours) than in the culture media without lichen (38.3% after 24 hours of incubation). In this sense, a logistic regression analysis revealed that the use of a culture medium with lichen extract with respect to a culture medium without lichen multiplies by five the probability that the associated lichen bacteria grow

20 (Probability ratio (odds ratio) = 5.2, 95% confidence interval = 4.2 to 6.4; p <0.001) (Table 1), regardless of the incubation period and the processing protocol used for the bacteria isolation.

Table 1. Logistic regression model of the probability of bacterial growth

Factors

p (SE) Probability ratio (odds ratio) [95% le¡ P value

Medium with lichen

If not'

1.64 (0.1) 5.18 (4.21-6.37) <0.001

Incubation time

96 hours 24 hours

1.19 (0, 1) 3.28 (2.68-4.01) <0.001

Isolation protocol

Dilacerated Wash ·

0.14 (0.12) 1.15 (0.9 1-1.4) 0.25

Constant

-1, 69 (0.16)

* Reference category

As expected, the incubation time multiplies by 3 the probability that bacteria associated with lichens grow (odds ratio) = 3.3, 95% confidence interval = 2.7-4.0, p <O, 001) (Table 1), regardless of the presence of lichen extract in the medium and the protocol for the isolation of bacteria. However, the protocol used for the isolation of bacteria from liquid thallus (dilated versus washing) did not have a significant effect on bacterial growth (p> O, 05) (Table 1).

Furthermore, when the growth of non-liquid reference bacterial strains in lichen-enriched culture media was compared with that of lichen-free culture media, again a higher percentage of strains grew in lichen-enriched culture media ( 78.1%) compared to those that did not contain lichen extract (59%) after 96 hours of incubation. In addition, an auxotrophic reference strain was able to grow in all culture media enriched with lichen but not in any of the culture media without lichen tested. However, in contrast to the results obtained with the bacterial strains isolated from P. furfuracea, no significant differences were found between the culture media enriched with lichen versus those that did not contain lichen extract (p> 0.05).

iv) Culture media enriched with lichen improves the cultivability of bacteria associated with individual P. talfuracea tali. The next step was to test the means enriched with lichen and the modified bacterial isolation protocol with new subsamples of the P. furfuracea thallus. As shown in Figure 5, just after 3 days of incubation at 25 ° C, the ectoliquénicas and endoliquénicas bacteria of the P. furfuracea talus, recovered in higher numbers in all the culture media enriched with lichen (the counts media ranged from 9.5 x103 to 1.8 x104 CFU / g of fresh washed talus and 3.9 to 5.7 x104 CFU / g of fresh thallus dilated), followed by non-lichen culture media (mean counts varied from 5 x1 03 to 1.0 x104 CFU / g of fresh washed thallus (except for the AS medium where no colonies were recovered) and from 9.5 x103 to 3.7 x104 CFU / g of fresh dilated thallus), and therefore In general, in lower numbers on the KB plates (the average counts ranged from 7 x 103 CFU / g of fresh talus washed to 1.0 x104 CFU / g of fresh dilated talus) used for comparative purposes. On the other hand, bacterial counts in minimal culture media with high nutrient content (with and without lichen extract) were higher than those obtained in minimal culture media with low nutrient content, especially when compared with AB culture medium, both for ectoliquenic and endolygenic bacteria (Figure 5), which also grew earlier in the minimal culture media with high nutrient content without lichen than in the minimum AB medium. In this sense, the endoliquénic colonies in the culture medium AB were only observed after 3 days of incubation, obtaining the average counts of lower colonies. In the case of ABL media, with low nutrient content, and AB, bacterial colonies grew more slowly than in other culture media, as did colonies of contaminating filamentous fungi that grew less than in other culture media. These differences were more pronounced with respect to the nutrient-rich KB medium.

Although the bacterial counts in the lichen-enriched media were higher than those obtained with the lichen-free culture media, there were no significant differences (p> O, 05) between the average counts obtained in each culture medium tested, nor between ectoliquénicas and endoliquénicas bacteria for each culture medium (p> O, 05). However, when the average values of CFU / g obtained with both ectoliquenic bacteria (Figure 5a) and endoliquénicas (Figure 5b) were analyzed by grouping the isolation media into two categories (media enriched with lichen against culture media without lichen), the results revealed significantly higher bacterial counts in the minimal media with lichen extract than in these same media without lichen, and in the KB medium (p <O, 05) (Figures 5a and 5b). When the mean counts obtained with ectoliquenic bacteria were compared against endolygenic bacteria after 3 days of incubation at 25 ° C, again the population sizes of bacteria were significantly higher in the dilatation of the thallus than in the superstructure of the thallus (p < Or, 001).

v) Improvement of the cultivability of the bacteria associated with P. furfuracea from samples composed of different talos through the use of culture media enriched with lichen and prolonged incubation periods. Based on the two results mentioned above, we decided to evaluate two culture media enriched with lichen, ABLGM (high nutrient content) and ABL (low nutrient content), together with the improved bacterial isolation protocol, to increase the cultivability of a broader range of bacteria associated with P. furfuracea, using samples composed of different talus of additional samples made in the fall of 2012 and summer 2013, including the non-lichen ABGM and KB culture media for comparative purposes. In addition, to favor the recovery of slow-growing bacteria in the ABL medium with low nutrient content, the incubation period lasted up to 15 days. The influence of the incubation period and of the isolation medium on the recovery of bacteria associated with P. furfuracea were investigated by determining the increase in the number of colonies, both of ectoliquénicas and endoliquénicas bacteria, from samples composed of different talos, over time. As shown in Figure 6, in general, the average bacterial counts increased rapidly within the first 3 days of incubation, similar to the results shown in Figure 5 with an individual thallus. Bacterial counts continued to increase with longer incubation periods until the number of colonies stabilized after approximately 7 to 15 days depending on the isolation medium used, regardless of the bacterial isolation protocol and the sample (composed of different talos ) analyzed. In general, the ABL culture medium, with lichen and low nutrient content, reached the highest counts after 15 days of incubation, followed by the medium with lichen and high ABLGM nutrient content and the ABGM culture medium without lichen, in those that counted stabilized a little earlier in some cases. In the nutrient-rich culture medium KB the bacterial counts stabilized much earlier (in a week) (Figure 6). Again, in the ABL medium, with low nutrient content, the bacterial colonies were smaller and the filamentous fungi grew more slowly and in smaller numbers than in the culture media with more nutrients ABLGM, ABGM and KB. In general, the average bacterial counts in the media enriched with lichen ABLGM and ABL were higher than those obtained with the ABGM culture media, without lichen, and KB, regardless of the sample analyzed, the bacterial isolation protocol and the period of incubation (Figure 6).

When comparing the average values of CFU / g of the ectoliquenic bacteria, estimated in washed talus, compared to those of the endolygenic bacteria in the dilated talos, these were higher (P <0.05) for the ectoliquénic bacteria, regardless of the sample analyzed, of the incubation period and of the culture medium used for isolation (Figure 6), being different from those obtained with the individual talus sampled in the spring of 2012. Interestingly, when comparing the cultivability of the ectoliquénicas bacteria against that of Endogenous bacteria between the two culture media enriched with lichen, the bacterial counts were higher in the ABLGM medium, with high nutrient content, than in the ABL medium, with low nutrient content, for ectoliquenic bacteria (Figures 6a and 6e), regardless of the sample analyzed and the incubation period. In this sense, the average bacterial counts of ectoliquenic bacteria ranged from 1.7 to 6.5 x 104 CFU / g in the ABLGM medium and 1.3 to 5.6 x 104 CFU / g in the ABL medium, after 15 days of incubation (Figures 6a and 6 e). In contrast, the ABL medium with low nutrient content yielded higher bacterial counts than the ABLGM medium, with a high nutrient content, for the endolygenic bacteria (Figures 6b and 6d), regardless of the sample analyzed and the incubation time. Thus, at the end of the experimental period, the average numbers of endogenous bacteria varied from 4.8 to 7.9 x 103 CFU / g in the ABL medium and from 1 to 1.5 x 10 'CFU / g in the ABLGM medium. (Figures 6b and 6d). These differences were also observed between ectoliquénicos bacteria against the endoliquénicas depending on the isolation medium in the case of the medium without lichen ABGM and the medium rich in nutrients KB, in which the average values of CFU / g were again lower than in the means enriched with lichen. In the ABGM culture medium, the average bacterial counts were higher than in the KB medium (Figures 6a and 6c), in the case of the ectoliquenic bacteria while the opposite happened for the endoliquenic bacteria (Figures 6b and 6d). After 15 days of incubation, the counts of ectoligenic bacteria varied from 9.5 x 103 to 4.7 x 10 'CFU / g in the ABGM medium and from 7.7 x 10' to 3.9 x 10 'CFU / g for KB medium (Figures 6a and 6c). On the contrary, the average values of endolygenic bacteria varied from 4 x 102 to 1 x 103 CFU / g in ABGM plates and from 1 to 1.25 x 103 CFU / g in KB plates (Figures 6b and 6d).

There was no statistical support for the differences observed between the average bacterial counts in each culture medium tested (P> 0.05), except between the ABLGM and ABGM media for the ectoliquenic bacteria in one of the samples, that of autumn. This could be due, at least in part, to the variability in bacterial counts on the isolation plates, which are more pronounced with the samples of talus dilatation than with those of talus washing.

Regarding bacterial diversity, initially analyzed by comparison of the number of morphologically distinct colonies isolated in culture media

enriched with lichen against culture media without lichen after 15 days of incubation, greater diversity was found in culture media enriched with lichen compared to culture media without lichen (data not shown). In addition, the identification of a selection of bacterial strains isolated from different P. furfuracea stems and identified by partial sequencing of the rDNA 168 bacterial gene has revealed a great diversity of cultivable bacteria, similar to that described in other lichens analyzed mostly by techniques independent of Cultivation such as mass sequencing (Cardinale et al., 2006, 2008; Liba el al., 2006; Grube & Berg, 2009; Grube el al., 2009; Hodkinson & Lutzoni, 2009; Selbman et al., 2010; Bates el al., 2011; Lasken & McLean, 2014; Grube el al., 2014).

vi) Optimization of the recovery of bacteria associated with P. furfuracea, Since the growth of filamentous fungi in the isolation plates of liquid bacteria hindered their recovery, especially in the case of slower growing bacteria, and despite Since all the culture media used contained the antifungal cycloheximide (actidione), it was decided to evaluate another more effective antifungal such as natamycin (pimaricin) and apparently without health risks (Pedersen, 1992, Appl. Enviran. Microbiol. 58 (3 ): 1064). For this reason, the effect of both antifungals on the growth of a selection of the 7 most frequent filamentous fungi in the isolation plates of the samples of liquid talus in the KB culture medium (the culture medium, of the tested ones, was investigated, in which fungi develop more quickly). As shown in Figure 7, the addition of natamycin to the KB culture medium was able to suppress the growth of the filamentous fungi tested, while with cycloheximide only a reduction in their growth was observed. Based on these results and the greater toxicity of cycloheximide, the next step was to carry out a comparative study of the effect of each of the two antifungals separately in the recovery of liquid bacteria both in nutrient-rich culture medium (KB) and in the minimum medium (ABGM). As can be seen in Figure 8, in the control culture medium without antifungals, the filamentous fungi invaded the KB plates in a period of 3-5 days, said reduced growth being seen when the culture medium was supplemented with cycloheximide. In the latter case, the growth of filamentous fungi required a period of more than 7 days to invade the KB plates. However, the addition of natamycin revealed an antifungal activity significantly higher than that of cycloheximide, inhibiting the growth of

filamentous fungi in both the KB medium and the ABGM medium for a period longer than 15 days. This period is considered as the minimum time of

incubation to favor the recovery of slow-growing liquid bacteria, especially in media with low nutrient content. Consequently, the addition of natamycin to the culture media constituted an optimization for the recovery of liquid bacteria, regardless of the culture medium used.

On the other hand, since the results of Figure 6 seemed to indicate that the cultivability of the endoliquenic bacteria could be affected by the disinfection step prior to the disruption of the liquid tali. And given that said step is performed after extensive washing of the same, the effect of the washing time on the recovery of ectoliquenic bacteria in culture media with natamycin (Figure 9) was confirmed first, as well as the elimination of them as a previous step to the analysis of endolygenic bacteria (Figure 10). The results of these tests, not only confirmed that the average counts of recovered ectoliquenic bacteria increased very significantly (p <0.000 1) as the washing time of the liquid talus (which stabilized between 60 and 90 minutes of washing, increased), regardless of the culture medium tested) (Figure 9), but also suggested the possibility of dispensing with the disinfection step that seemed to adversely affect the recovery of endoliquenic bacteria. In this sense, the next step was to investigate the effect of disinfection on the recovery of endolygenic bacteria in KB and ABGM media with natamycin from the washed talus 90 minutes (Figure 10). In addition, to try to standardize and expedite the processing of the liquid tali, the efficiency in the isolation of bacterial strains of talus processed by dilatation was compared, a process that involves about 5 -10 minutes per sample, with respect to crushing, which allows process a larger number of samples in less time (approximately 1.5 minutes / sample). As shown in Figure 10, when the isolation of endolygenic bacteria from washed subsamples of talus was compared to that of subsamples thereof, but disinfected, the mean counts of endolygenic bacteria in uninfected tali were significantly higher (p <0, 01) (between 1 and 2 logarithmic units, as appropriate). These results evidenced the negative effect of disinfection on the recovery of endolygenic bacteria, regardless of the type and time of disinfection tested, as well as the culture medium used. These results together with the

obtained after extensive washing of the liquid tali (60-90 minutes, Figure 9), demonstrated not only that after such washing it is not necessary to carry out a disinfection step but that said step negatively affects the cultivability and / or viability of the endoliquénicas bacteria and, therefore, to his recovery. 5 Additionally, when what was compared was the effect of the type of processing of the liquid talus on the recovery of endolygenic bacteria, specifically the dilated versus the crushed, both in AMB antioxidant buffer, the results showed that, in most of the cases, the average bacterial counts were significantly higher in the liquid talus processed by crushing

10 (p <0.05) (Figure 10) than in those who dilated, after an incubation period of 7 days. These results were independent of both the culture medium used and the disinfection and were subsequently confirmed with samples of new liquid talus of several species and with culture media supplemented with the respective liquid extracts (data not shown).

3.

Claims (30)

1. Method for obtaining a liquid extract comprising:

(to)

collect at least one liquid talus from a liquid species of interest,

(b)

wash and proceed to the disruption of the liquid thallus with a sterile isotonic solution, and

(and)

sterilize by filtration the product obtained after carrying out step b).

2.

Method according to claim 1, wherein the collected liquid tali belongs to the same population of the liquid species.

3.

Method according to claim 1 or 2, wherein the sterile isotonic solution of step b) is supplemented with a surfactant.

Four.

Method according to claim 3, wherein the surfactant is Tween 20.

5.

Method according to any of claims 1 to 4, wherein the sterilization by filtration of step e) is carried out with a prefilter and a filter, wherein the pore size of the filter is between 0.22 and 0, 45! -1m.

6.

Method according to any one of claims 1 to 5, wherein the disruption of the liquid thallus is carried out by crushing.

7.

Method according to any one of claims 1 to 6, wherein the isotonic solution is Ringer solution.

8.

Method according to any one of claims 1 to 7, wherein the liquid species is selected from the group consisting of Pamotrema spp., Pseudevernia spp. and Ramalina spp.

9.

Method according to any one of claims 1 to 8, wherein the liquid thallus is preferably fresh or frozen.

10.

A liquid extract obtained according to the method of any one of claims 1 to 9.

eleven.

A composition comprising the liquid extract according to claim 10.

12.

Composition according to claim 11, wherein the composition is a culture medium for microorganisms.

13.

Method for isolating microorganisms associated with a liquid species of interest comprising

(to)

collect at least one liquid talus from the liquid species from which the microorganisms are to be isolated, under aseptic conditions,

(b)

Wash the liquid talus collected in stage (a) extensively,

(and)

Carry out the disruption of the washed liquid thallus in step (b), and

(d)

cultivate, in a minimum culture medium supplemented with at least one liquid extract according to claim 10:

(i)

the product obtained from the disruption of the liquid talus of step (c), and / or

(ii)

the wash solution recovered after extensive washing of step (b)

where, in the case that they are grown (i) and (ii), they are grown independently, and the liquid extract is obtained from the liquid species of interest from which the microorganisms are to be isolated.

14.

Method according to claim 13, wherein the culture step (d) is carried out in a minimum medium supplemented with at least one liquid extract obtained from talos of the same population as the thallus of the collected liquid species according to stage (a).

fifteen.

Method according to claim 13 or 14, wherein the washing is carried out with a sterile isotonic solution comprising a surfactant.

16.

Method according to any one of claims 13 to 15, wherein the extensive washing of the liquid thallus comprises washing for at least about 60 minutes, preferably about 90 minutes.

17.

Method according to claim 15 or 16, wherein the sterile isotonic solution is Ringer solution.

18.

Method according to any of claims 15 to 17, wherein the surfactant is Tween 20.

19.

Method according to any one of claims 13 to 18, wherein the thallus disruption comprises crushing the liquid talus in a sterile antioxidant maceration buffer and under aseptic conditions.

twenty.

Method according to claim 17, wherein the sterile antioxidant maceration buffer is sterile AMB buffer.

twenty-one.

Method according to any of claims 13 to 20, wherein the minimum culture medium comprises one or more carbon sources.

22

Method according to claim 21, wherein the carbon source is glucose and / or mannite!

2. 3.

Method according to any one of claims 13 to 22, wherein the microorganisms associated with the liquid species of interest are selected from the group consisting of bacteria, algae, filamentous fungi, yeasts and / or viruses of liquid microorganisms.

24.

Method according to claim 23, wherein to isolate liquid bacteria, the culture of step (d) is carried out in a minimum culture medium supplemented with at least the liquid extract and at least with an antifungal, preferably, natamycin.

25.

Method according to claim 23, wherein in order to isolate virus from a liquid microorganism, the culture of step (d) is carried out in a minimum culture medium supplemented at least with the liquid extract and with the liquid microorganism of interest.

26.

Method according to any one of claims 13 to 25, wherein the liquid thallus is selected from the group consisting of Pamotrema spp., Pseudevemia spp. and Ramalina spp.

27.

Use of a liquid extract according to claim 10 and / or the composition according to claim 11 or 12, to isolate and / or grow microorganisms associated with a liquid species of interest, wherein the liquid extract is obtained from at least , a liquid talus of the liquid species from which you want to isolate the

5 microorganisms 28. Use according to claim 27, wherein the culture is carried out in a minimum medium supplemented with at least one liquid extract obtained from talos of the same population as the liquid thallus of the liquid species of which be 10 want to isolate microorganisms. 29. Use according to claim 27 or 28, wherein the microorganism associated with the liquid species is selected from the group consisting of bacteria, algae, filamentous fungi, yeasts and / or virus of liquid microorganisms. 30. Use according to any of claims 27 to 29, wherein the liquid species of interest is selected from the group consisting of Pamotrema spp. , Pseudevernia spp. and Ramalina spp.