METHODS FOR THE CULTURE CONSERVATION OF EDIBLE AND MEDICINAL FUNGI

The maintenance and conservation of strains of microorganisms such as bacteria, yeast, and fungi are vital for the advancement and development of various life science areas, impacting studies in genetics, biodiversity, bioprospecting, biotechnology, medicine, veterinary, environment, food security, nutrition, among others. The preservation of this biological material has achieved the safeguarding of industrial potential and the possibility of researching new functions and the use for the benefit of humanity. Various culture preservation methods have been developed over the years, such as sterile distilled water, cryopreservation, freeze-drying, sub-culture, and sterile mineral oil; these allow storing strains of various microorganisms under appropriate conditions and for long periods. For the case of edible and medicinal fungi, the most used conservation methods are cryopreservation and sterile distilled water, guaranteeing the stability of the characteristics of these fungi, their viability, and their biological potential over time; however, the need to continue evaluating different methods and applications in this type of fungus persists.


INTRODUCTION
Microbial collections are considered centers of biological resources, maintaining strains of different microorganisms in optimal conditions for research, biotechnological, and industrial applications (Hu et al., 2014;Sharma et al., 2017). In order to store under controlled conditions and for a specific time such organisms, it is necessary to apply preservation methods that ensure the viability, availability, and safeguarding of all phenotypic, genotypic, and potential characteristics industrial of the preserved strains ( For several years different culture conservation methods have been developed for the maintenance and conservation of microorganisms such as bacteria and fungi, like freeze-drying (lyophilization), cryopreservation, sub-culture (periodic transfer), sterile distilled water, mineral oil layer, drying on filter paper, drying in soil, sand, silica gel, among others (García et al., 2000;Ryan et al., 2004;Weng et al., 2005;Gato, 2010). These methods allow that biological material to be available and stable for varying periods; however, the choice and application of one method or other depends on the type of microorganism to be preserved, the time they wish to keep preserved, the resources, laboratory procedures, and staff in charge (Rico et al., 2004;Smith et al., 2012). In Table 1, the main conservation methods of fungi can be seen along with their advantages and disadvantages. In general, studies for the maintenance of microbial collections have shown the effects of methods over a given time on the phenotypic, genotypic, feasibility, stability, purity, and reproductive capacity of the strains mainly in filamentous fungi, yeasts, and bacteria of importance for the industry, medicine, agriculture, and the environment. However, the development and research of preservation methods for basidiomycetes have not been studied as extensively as in other types of microorganisms, despite the importance that these fungi have in nature, because they are responsible for cycles nutrient replacement, contributing to increased soil fertility; as well as the different secondary metabolites with properties of biotechnology interest and applications in different industry sectors (Cortés et al., 2013;Ladislav Homolka, 2014;Eichlerová et al., 2015). Therefore, this document's objective is to make available to the scientific community a review of the main works carried out on the conservation of basidiomycetes fungi, with an emphasis on edible and medicinal fungi. Decreases the tendency to pleomorphism by some species.
It is simple, safe, and inexpensive, does not require personnel specialized.
It maintains viability, purity, and stability for extended periods.
It also allows to preserve fungi that cannot be preserved by liquid nitrogen or freeze-dried.
Has been successfully used to preserve basidiomycetes.
The strains can degenerate during storage time.
Conservation times are variable and will depend on the fungus to be preserved. Burdsall et al. (1994) Bueno et al.  (2017) Cryopreservation Is a reliable method for long-term storage of microorganisms.
It is considered an expensive technique due to the requirement of space and specialized There is a low risk of changes in the viability and genotype of the microorganisms.
The method is used for the long-term preservation of fungi.
This method applies to a broad spectrum of fungi. equipment.
It requires a bioprotectant to avoid excessive dehydration of the cells.
Need a constant supply of liquid nitrogen. Hu et al. (2014) S.K. Singh (2017) Ladislav Homolka (2014) Freeze-drying The maintenance of the culture is low cost.
The method is applied to a wide range of microorganisms.
The method can be complicated and requires specialized equipment.
It requires a bioprotectant to avoid extracellular and intracellular crystallization.
Limited application in basidiomycetes.
Fungi can lose its viability.
Ladislav Homolka (2014) Hu et al. (2014) Sub-culture Useful for small collections of fungus that do not require long-term storage.
The storage time is less than a year.
It requires time and labor because it needs subculturing every few months so that it can be expensive.
There is a risk of contamination with other microorganisms, so it should be continuously checked.
Increase the danger of changes in morphology and physiology of the cultured fungus. Iqbal et al. (2017) S.K. Singh (2017) Cui et al. (2018) Sterile mineral oil It is a friendly method for laboratories with limited resources.
It has been used in different fungi, remaining viable for 20 years or more.
In this technique, the fungi continue to grow, and therefore there is a risk of mutation in adverse conditions.

Sterile distilled water (Castellani method)
It is a widely used technique for conserving microorganisms such as filamentous fungi, yeasts, and some bacteria due to the high percentages of viability obtained over time (García et al., 2000). As seen in figure 1, the method consists of suspending agar discs or blocks with the colonies of the microorganism in tubes or vials with sterile distilled water, and these are sealed by screw caps, rubber or cotton caps and stored at room temperature (between 20 and 25 °C) or in cold storage between 4 and 5 °C (Castellani, 1963;Nakasone et al., 2004;S.K. Singh, 2017). It is the most preferred method for the maintenance of fungal culture because decreases the tendency to pleomorphism by some species, prevents the attack of mites, is simple, safe, and inexpensive, does not require personnel specialized and maintains viability, purity, and stability for extended periods; also can be applied in fungi that cannot be preserved by liquid nitrogen or freeze-dried (  In 1963 Aldo Castellani, in his publication "Further researches on the long viability and growth of many pathogenic fungi and some bacteria in sterile distilled water", showed to the scientific community the progress made by the application of its method, which consisted of preserving strains of filamentous fungi, yeasts, and some bacteria in test tubes with sterile distilled water (8 to 10 mL) properly capped, kept at room temperature for different periods (one year or more), and subsequently recovered in test tubes with dextrose agar to assess the efficiency of the method. The results obtained in their studies showed that these strains, after several years, maintained high percentages of viability, average growth, and no changes in their morphology (Castellani, 1963 evaluated sterile distilled water in the medicinal fungus Humphreya coffeata, using filter paper discs inoculated with the fungus, guaranteeing high viability of the culture for 18 months, without visible morphological changes, contamination by bacteria or other fungi. As observed, sterile distilled water has allowed the conservation of different culture of edible and medicinal fungi between 2.5 months and 48 years, depending on the type of fungus and the conditions of applying the technique.

Cryopreservation
Cryopreservation is a technique that allows a cell suspension to be stored at a temperature equal to or below the freezing point. Depending on the temperature, this method of preservation can be classified into ordinary freezing (-5 to -20 °C), ultra-cold freezing (-50 to -80 °C), and freezing with liquid nitrogen (-150 to -196 °C) (Hernández et al., 2014). It is a long-term preservation method that allows storage microorganisms between one year and twenty years; it also guarantees the viability, purity, and genetic stability of the stored strains. It is one of the most recommended techniques for conserving fungi that cannot be freeze-dried Cryopreservation has some disadvantages such as high cost of the equipment required, energy cost, the need to maintain a constant supply of nitrogen (when applied), the danger that some mechanical and electrical failure will destabilize the temperature as well same specific conditions for the transport of the strains and mycetes by rapid freezing at -85 °C with glycerol and ethylene glycol as cryoprotectors at different concentrations; as well as these fungi were preserved using wood sawdust with 65 % moisture. The results obtained in this study showed that with 10 % of glycerol cryoprotectant, strains remain viable and stable for up to 10 years even after defrosting the strains at room temperature and subjecting them to processes of alternating freezing and thawing. Regarding studies concerning the conservation of edible fungi, the researchers Ohmasa et al. (1996); evaluated the effect of three cryopreservation protocols on the yields of fruiting bodies of the Flammulina velutipes after having preserved two strains (FMC224 and FMC225) at three temperatures (-20, -85 and -196 °C), and with three cryoprotectants (glycerol, dimethylsulfoxide, and polyethylene glycol) for seven years. The results showed that 9 of the 11 strains conserved did not show significant changes in viability, biological efficiency, the weight of fruiting bodies, and genetic stability. Ladislav Homolka et al. (2006); developed a new method for the conservation of 442 strains of basidiomycete fungi of different species; by the cryopreservation of the fungal mycelium using agricultural grade perlites as support. The cultures were stored in cryotubes (1.8 mL) for 48 h up to 3 years in liquid nitrogen; after this time, the ability to maintain laccase production, growth, and the macro and microscopic characteristics of the conserved strains were evaluated. They obtained that 100 % of the strains maintained the viability, purity, morphological characteristics, and laccase production during the evaluated period. In 2007, these same authors evaluated the capacity of 50 strains of basidiomycete fungi preserved by the perlite protocol to survive three successive cycles of freezing and thawing, obtaining that in the first cycle, 100 % of the strains evaluated kept viability, purity, macro, and microscopic characteristics, shape, and speed of growth, as well as the production capacity of laccase, for cycles 2 and 3, 96 % of the strains survived while maintaining the same characteristics mentioned above. Therefore, they assured that with this cryopreservation method, the difficulties caused by the interrupted supply of liquid nitrogen or electrical energy during storage could be overcome without affecting the preserved culture's survival and quality (Ladislav Homolka et al.,  2007).
In a study on the vitality and genetic stability of mycelium in 15 species of whiterot fungi, different cryopreservation protocols were evaluated at -80 °C and lyophilization. These included variables such as culture medium, cryoprotectants, time, number of infusions, and origin of the samples. The results showed that it is possible to perform adequate conservation of the basidiomycete strains by these techniques; however, in lyophilization, morphological changes occurred in two isolates of Ganoderma adspersum, something that did not occur with freezing pearls and plastic straws for the cryopreservation of Pleurotus ostreatus and Trametes versicolor; these strains were frozen at -70 °C at a speed of 1 °C min -1 using 5 % glycerol as a cryoprotectant and then stored in liquid nitrogen for 12 years, in order to assess the viability, growth rate, morphological characteristics, laccase enzyme activity, and decolorization test. As a result of this investigation, they obtained that 100 % of the strains conserved by these methods managed to remain viable, retained their macro and microscopic characteristics, and maintained their enzymatic activity.

Freeze-drying or lyophilization
It is a long-term conservation method that guarantees the genetic stability and viability of the organisms preserved for periods of 10 or more years, also prevents the occurrence of successive generations (Arencibia et al., 2008). The method stops the microorganism's metabolism and extracts water from the frozen cells by sublimation of the ice under high vacuum conditions (Morales et al.,  2010). The culture of the microorganism is carried out in Petri dishes with potato dextrose agar or malt extract agar, then spore or hyphae suspensions are transferred in glass ampoules, these are frozen at -70 °C for 4 to 6 h, then lyophilized and later stored in cold storage or room temperature (S.K. Singh, 2017). The dehydration of the cells, necessary to avoid intracellular crystallization during freezing, is regulated through the cooling rate and depends on the cell's size and the thickness of the cell wall (Ladislav Homolka, 2014). The addition of a cryoprotective agent is required to prevent the cells from suffering some damage, such as monosodium glutamate, glucose, sucrose, trehalose, skim milk, inositol; other cryoprotectants such as glycerol, dimethylsulfoxide, 1,2 propane-diol, ethylene glycol, ethanol, methanol, polyethylene glycol are used; however most of these are toxic ( In some cases, the lyophilization of basidiomycetes has been successful, which has increased interest in this method. Tan et al. (1991) evaluated the method in 4 ascomycete strains (Alternaria bataticola, A. dianthicola, Cercospora autensis, and Chaetomium balloonsum) and two strains of basidiomycetes (Coprinopsis sp and Schizophyllum commune); finding that all strains were recovered after the lyophilization process with differences in the survival rate which was lower in basidiomycetes compared to the ascomycetes evaluated; the rate increased in both phylum when incubated in media containing trehalose. Croan et al. (1999); applied this method of preservation in Pleurotus ostreatus, P. populinus, and P. pulmonarius, which showed an intense colonization mycelial growth and more efficient substrate after preservation compared to non-lyophilized, at cold storage (4 °C), 57 % of the isolates survived for 2.5 months at 15 °C, and 92 % of the isolates survived ten months. the results showed that the growth rate of the fungus was not affected by age and sub-culture, maintaining vitality for 12 months.

Sub-culture
It is considered a short-term conservation technique since microbial cultures must be reactivated and renewed periodically to maintain the characteristics that make them important; therefore, these time intervals depend on the type of microorganism to be preserved. Some microorganisms need to be transferred to new culture media after days, weeks, or months (usually in less than a year), while other culture preservation methods managed to conserve for months or years; however, it is one of the most commonly used techniques for basidiomycete fungi since they cannot be easily preserved by other methods ( It is a method that has several drawbacks, including the selection of phenotypic variants (pleomorphic growth) and mutant gene, the loss of pathogenicity, virulence, or sporulation. It is a universally applicable technique currently used in most low-budget collections or as a second maintenance method. Generally, the storage temperature in this method is ambient; and one way to avoid performing several cultures is cold storage or using wet perlite; which can be an alternative method that has shown excellent results in maintaining strains for up to four years (

Sterile mineral oil
It is an alternative method commonly used to conserve mycelium or fungi that are not sporulated or have poor sporulation, which is susceptible to lyophilization and freezing; also, when deep freezing is not an option, due to its high cost. With this technique, the culture can maintain its viability for several years or even up to 32 years at room temperature or temperatures between 15 °C and 20 °C (Nakasone et al., 2004;Ladislav Homolka, 2014).
It is a method of suppressing evaporation. It consists of completely covering the culture in a tube (after its development in a solid medium), with a layer of sterile mineral oil or sterile petroleum jelly (1 cm), tubes are stored in a vertical position at cold storage or room temperature, preventing the evaporation of the water contained in the culture medium and avoiding the increase of osmotic pressure (S.K. Singh, 2017). It is a simple, economical, and fast method, which does not require expensive equipment, maintains the viability of most species, and reduces mite contamination; likewise, strains can be kept at room temperature or refrigerated between 1 to 47 years (

Recent methods and future trends
Edible and medicinal mushrooms represent an alternative to obtain nutrients or active compounds in the diet, being a possible strategy for reducing food insecurity (Cuesta et al., 2017), evaluating their impact on health and nutrition, promoting industrial production, and the application and research of culture conservation methods. In addition to the previously mentioned methods, it is essential to evaluate other preservation techniques that have been applied mainly to entomopathogenic and phytopathogenic fungi, showing promising results in viability and storage time, which could be evaluated in edible and medicinal mushrooms. Among these techniques are the gelatin method (Igor Forigo et  It is also essential to continue studying traditional methods and their application in edible and medicinal fungi, continuing with the standardization of parameters, adaptation, and fungal species evaluation.

CONCLUSION
Edible and medicinal mushrooms have various industrial applications, this requires adequate knowledge of different conservation methodologies that allow preserving all their genetic potential and avoid the deterioration of essential strains, as well as knowing what method is applied for each fungus depending on their particular characteristics. The methods that have shown the best results for the conservation of edible and medicinal fungi are sterile distilled water (Castellani method) and cryopreservation; however, the choice will depend on the type of microorganism and the economic capacity and infrastructure of the laboratory. Freeze-drying is a simple, fast method that allows long-term preservation; however, equipment, infrastructure, and maintenance costs must be considered, and some mycelial forms do not survive, which requires prior studies to be implemented. Subculture is a short-term method suitable for low-budget laboratories or as the first conservation method in a new laboratory, but the changes that the microorganism may have during storage and the time of dedication for the maintenance of the strains should be considered. Finally, the conservation of edible and medicinal mushrooms in sterile mineral oil requires further study to be recommended for the preservation of this type of microorganisms.