Keywords

1.1 Introduction

Microorganisms are fundamental materials for scientific and practical studies. Culture collections (biological resource centers) play a primary role in the stable preservation and long-term storage of microbial resources and ensure regular access to well-documented strains after a long time from their isolation for scientific or biotechnological use [32, 33].

Various methods of preservation of fungal cultures have been reported [13, 25, 29]. Freeze-drying (lyophilization) and cryopreservation methods are utilized for thousands of fungal strains in microbial collections all over the world [7, 12, 27]. Nevertheless, it is clear that the fungal strains of different species vary in the ability to survive after long-time storage preservation under laboratory conditions. Some of them are very difficult to maintain ex situ, whereas others could be easily and successfully preserved alive by using almost any conservation technique.

Storage methods for filamentous fungi result from the type and degree of sporulation. Spore-forming strains (as opposed to nonsporulating strains) can be effectively freeze-dried. Both types can be frozen and stored for long periods in liquid nitrogen or in a low-temperature refrigerator. The experience of long-term preservation of fungal strains shows that the duration of storage directly depends not only on the choice of the method but also on the laboratory protocol and temperature of subsequent cultures storage.

This chapter presents the methods of cryopreservation, freeze-drying, drying on silica gel, and preservation in sterile soil that are utilized in VKM fungal collection, accompanied by data on maximal storage time registered. The methods take into consideration the special features of cultures preserved as well as the equipment used.

VKM fungal collection (All-Russian Collection of Microorganisms, Russia) was established in 1955 and has a long-term experience in the preservation and storage of fungal cultures. Collection of filamentous fungi is currently composed of approximately 7000 strains (590 genera, 1600 species) belonging to species of the kingdoms Chromista (Oomycota) and Fungi (zygomycetous, ascomycetous and basidiomycetous fungi).

All the information on preservation methods for each VKM fungal strain is presented in the MS Access database. It keeps curated data on the strain numbers, preservation dates as well as inspection dates in various methods, and other technical information. Fields in the database table are presented in Annex 1. For operational analysis of these data, we use MS Access requests – «FunPreservEnd», «FunPreserv_Times», «FunPreserv_MaxTimes». The maximal preservation time is calculated automatically; the latest results (25.11.2019) are presented in Annex 2.

Preserved for many years fungi of various taxa retain their ability to produce different substances suitable as a material for industry and medicine. For instance, the zygomycetous fungus Cunninghamella japonica VKM F-1204D was found to be a promising lipid producer for biodiesel production [22]. Fungi of the genus Penicillium , which are supported in the collection for more than 40 years (VKM F-325, VKM F-691, VKM F-1823), are able to synthesize active compounds with diverse structures [10]. Aspergillus brasiliensis VKM F-1119, which was accepted by VKM 52 years ago, engaged in the vital process of biotransformation of artemisinin, uncial medicine for the treatment of tropical malaria [34]. Recently published data on the assessment of the effect of freeze-drying and long-term storage on the biotechnological potential of Aspergillus section Nigri strains show maintaining of biotechnological properties after preservation [19].

1.2 Cryopreservation of Filamentous Fungi

According to published data, the fast cooling rates followed by storage in liquid nitrogen at -196 °C allow secure and long-term preservation of some fungal cultures [21]. However, the ability to resist damage by freezing and warming differs considerably among genera/species and depends on their particular features (presence and type of sporulation, chemical composition of cytoplasmic membrane and cell wall, physiological state, etc.). Selection of optimal cryoprotectants, rates of cooling, and warming has enabled increasing the number and diversity of taxa preserved by this method [24, 28].

More than 75% filamentous fungi of VKM are stored using various cryopreservation protocols. Cultures with abundant sexual and nonsexual sporulation usually were preserved by using fast cooling rates followed by storage either in liquid nitrogen or in ultralow temperature freezers at −70 °C.

It was noticed that some cultures of zygomycetous fungi belonging to the genera Mortierella, Basidiobolus, Coemansia, and Entomophthora do not survive the ultrarapid freezing procedure even if they have abundant sporulation. Successful preservation of such strains was achieved by modification of the cryopreservation regime, for example, using slow programmed freezing. The same method was used either for nonsporulating fungi (basidiomycetous fungi) or zoosporeforming former fungi (Chromista, Oomycota).

According to our data, some parts of strains of Oomycota (20%), basidiomycetous fungi (4%), zygomycetous fungi (1%), and ascomycetous fungi (1%) did not survive cryopreservation at all freezing regimes and modification applied [9]. The strains most difficult to maintain belong to genera Dictyuchus and Phytophthora and to some species of Achlya and Saprolegnia. Similar situations have also been seen with some species of basidiomycetous fungi (Suillus, Amanita, Dictyophora, Mutinus, etc.). They are usually maintained by subculturing and preservation under mineral oil.

It has been suggested that those microbial cultures that are able to survive the freezing and a short storage will permanently stay in the vital state after any length of storage [20]. According to our data, this is not quite true: some strains of Achlya colorata, Achlya intricata, Clitocybe odora, Choanephora conjuncta, Conidiobolus thromboides, Kickxella alabastrina, Phanerochaete sanguinea, Rhodocollybia butyracea, and Saprolegnia terrestris have lost their ability to grow after 5–7 years of storage in liquid nitrogen, although they were in the viable state after 24 h of storage. The reason is not yet known. Nevertheless, the viability test showed that representatives of 311 species of fungi remain alive after 20–30 years of storage (Annex 2).

The cooling equipment being used in VKM is storage tanks “Bioproducts-0.5” with a capacity of 500 liters of liquid nitrogen and ultralow temperature freezers (−80 °C, Sanyo, Japan).

1.3 Freeze-Drying of Filamentous Fungi

Currently, freeze-drying is used to preserve approximately 85% of filamentous fungi maintained in VKM. Fungi from different taxonomical groups (zygomycetous fungi, ascomycetous fungi – both teleo- and anamorph) able to produce dormant structures (spores, sclerotia, etc.) usually survive freeze-drying [11]. According to our data, about 90% of strains of these fungal groups remain alive in this method. We noticed that the freeze-dried strains of 817 species stored at 5 °C for more than 20 years were in a viable state, and cultures of 289 species have been sustained for even 40–50 years of storage. Some species did not survive freeze-drying even when the sporulation is abundant, those are Conidiobolus coronatus, C. thromboides, Entomophthora thaxteriana, E. conica, E. dipterigena, Cunninghamella homothallica, and C. vesiculosa. Species of genus Botrytis (B. fabae and B. squamosa), forming only sclerotia as a dormant structure, remain in a vital state in freeze-drying only for rather a short time – less than 10 years [9].

Nonsporulating microorganisms from Oomycota and basidiomycetous fungi are not stored in VKM by freeze-drying, since sterile mycelia generally do not remain viable.

The equipment used in VKM for freeze-drying is the centrifugal freeze-dryer system Micromodulyo (Edwards, UK).

1.4 Drying in Sterile Soil of Filamentous Fungi

This simple and popular method for the preservation of fungi was applied at the beginning of the twentieth century [18]. Species of Aspergillus and Penicillium can be maintained by this way more effectively than other micromycetes. According to T.P. Suprun [31] who investigated the preservation of 78 Penicillium species (more than 1000 strains) in sterile soil for 7–10 years, the best preserved strains were representatives of Assymmetrica section. Less effectively preserved species were Biverticillata-Symmetrica, and the lowest effectiveness was observed with strains of the section Monoverticillata.

This method is also efficient for preservation of some human, animal, and plant pathogens with retaining their virulence [21]. For example, Alternaria japonica (syn. A. raphani), Fusarium oxysporum, and the species of Septoria (S. avenae, S. nodorum, S. passerinii, S. tritici) have retained their ability to infect a plant host after 2–5 years of storage [2, 8, 23]. Some degraded strains of micromycetes partly recuperated their lost qualities after preservation in soil [30].

According to our data, fungal strains of 167 species stored by this method are able to maintain viability for more than 30 years, and cultures of 87 species have been sustained for even 40–55 years of storage.

1.5 Drying of Filamentous Fungi on Silica Gel

Immobilized cells of microorganisms retain viability and biological activity at action of different stressors, as a rule, better than free ones [4]. Therefore, the preliminary drying of the cells on the adsorbent allows the microorganisms to remain viable for a longer time. As an adsorbent on which a suspension of microorganisms is applied for subsequent drying, silica gel (a dried gel of polysilicic acid with numerous pores) is most often used [5]. Silica gel promotes the dehydration of microorganisms and helps them to survive a thermal stress [24]. Since the silica gel can prevent all fungal growth and metabolism, the risk of any morphological, physiological, and genetic changes could be minimized [1].

Using of anhydrous silica gel particles for maintaining stock cultures of Neurospora crassa was suggested by D. Perkins in 1962 [17]. This new method has proved consistently useful and effective over several years.

At present, this method is widely used in relation to different taxa and ecological groups of fungi. So, the method was effective for the storage of entomopathogenic fungi of the order of Hypocreales for 2 years [3] and, in particular, Metarhizium anisopliae [6], as well as for fungi of many other taxa, including the spores of obligate biotrophic parasite Podosphaera fusca [16] and rust fungi, which cannot be grown on agar media [1].

As a disadvantage of the silica gel preservation method, researchers note that the time of storage is quite short (between 2 and 4 years) [14, 26]. But it is clear that the features of the methodological protocols can be crucial for the fungi preservation by this method, wherein the temperature at which frozen fungi are stored affects how long they could be preserved while remaining viable.

The method of storage on silica gel was introduced in VKM in the middle of the 1980s [24]. Our experience has shown that several groups of fungi can be preserved by this method without losing vitality for many years (Table 1.1).

Table 1.1 Drying of VKM fungal cultures on silica gel (storage time)
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The viability of more than 300 strains of zygomycetous fungi with various types of sporogenous structures (6 classes, 6 orders, 15 families, 35 genera, and 118 species) and near 300 strains of dark-colored anamorphic ascomycetous fungi with different types of conidiogenesis (7 classes, 18 orders, 34 families, 79 genera, and 164 species) (Table 1.2) was assessed from 1 to near 30 years of preservation (Figs. 1.1 and 1.2).

Table 1.2 Viability (%) of different taxa of VKM fungi after long-term preservation (30 years) at various temperatures on silica gel
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Fig. 1.1
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The long-term preservation of zygomycetous fungi on silica gel at different temperatures

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Fig. 1.2
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The long-term preservation of ascomycetous fungi on silica gel at different temperatures

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The analysis of the results testifies that this method has proved very successful for the storage of most of the investigated fungi within 3–7 years (Table 1.2).

Where it is desired to keep and constantly to renew cultures within 1–2 years, a temperature of 5 °C is perfectly applicable. More than 97% of the studied zygomycetous fungi and 94% of ascomycetous fungi were viable after storage. For long-term (more than 10 years) storage, however, this temperature is not reliable, since the viability of fungi in both groups is reduced to 57% and 55 % respectively.

A temperature of −12 °C is least favorable for long storage. Only 60% of zygomycetous fungi and 35% of ascomycetous fungi stored at such a temperature were viable after 10 years. After 17–20 years viability decreased to 10–13% and 4%, respectively (Fig. 1.1). Among zygomycetous fungi representatives of the classes Mortierellomycetes, Entomophthoromycetes, and Kickxellomycetes lost their vitality most rapidly at these temperatures. After 17 years of storage, their viability decreased to 0–4%. In contrast, the strains from the psychrotolerant species Helicostylum elegans and Thamnostylum piriforme and thermotolerant species Rhizomucor pusillus remained steady. Among dark-colored anamorphic ascomycetous fungi the best viability at temperature −12 °C after 20 years was found in strains of the genera Acrophialophora, Alternaria, Coniothyrium, Gonytrichum, Hormoconis, Paraconiothyrium, and Phialophora. After 30 years, only 1 strain (Hormoconis resinae) was viable.

The most acceptable temperature for the storage of mycelial fungi is the temperature −70 °C. In these conditions after 30 years of storage, 90% of strains were viable (Fig. 1.1).

The advantages of storing mycelial fungi at different temperatures on silica gel are obvious. On one hand, this method is so simple that the storage at 5 °C and −12 °C can be carried out for the most part in poorly equipped laboratories. On the other hand, the presence of a low-temperature refrigerator (−70 °C) means it is possible to support large numbers of cultures in a small area. The advantages of this method are also a minimum of preparatory work, the rapid reconstituted part of the stored material by transferring a few granules on appropriate culture medium, as well as the possibility of using the same vial without defrosting for a long time.

The cooling equipment being used in VKM is ultralow temperature freezers (−70–80 °C, Sanyo, Japan) and household refrigerators (5 and −12 °C).

1.6 Protocols

Protocols of cryopreservation, freeze-drying, and drying in sterile soil were described earlier [15].

1.7 Protocol of Drying on Silica Gel

1.7.1 Preparation of Sterile Silica Gel and Ampoules

  • Silica gel is pre-dried and sterilized by dry heat for 3 h at a temperature of 160 °C, conducting careful control of sterility.

  • Plastic ampoules (Nunc) (3 for each culture) are labeled and sterilized by autoclaving, at 121 °C for 20 min.

  • Sterile silica gel that has been washed with a concentration of cobalt chloride is placed in the ampoules to indicate the humidity. The cobalt chloride is deep blue when dry and turns pink when wet.

  • A sterile cotton ball is placed on top of the indicator.

1.7.2 Preparation of Cryoprotectant: 10% (v/v) Glycerol

  • Pour 5 mL of 10% glycerol into 12 mL glass tubes.

  • Sterilized by autoclaving at 121 °C for 20 min.

  • Stored at +5 °C for no longer than a month.

1.7.3 Preparation of Cultures

  • Grow sporulating fungal cultures on slant agar under optimal growth conditions and on suitable mediums (www.vkm.ru).

  • Wash off spores from agar surface with 5 mL of cool sterile 10% glycerol.

  • Titer of spores’ suspension should be not less than 106 spores/mL.

1.7.4 Silica Gel Inoculation

  • Add 75–100 silica gel granules (40 grade, 9–16 mesh) in a sterile Petri dish.

  • Add 1 mL spore suspension to sterile and dry silica gel.

  • Shake the Petri dish with the granules.

  • Put the Petri dish in desiccator and store in the refrigerator 12 h at 4–7 °C.

1.7.5 Filling of Vials

  • Add silica gel granules with fungal spores (20–25 pieces) to 3 plastic ampoules with a sterile spoon.

  • Place cryovials in the boxes and transfer them to the refrigerators (5 and −12 °C) and the ultralow temperature freezer (−70 °C).

1.7.6 Control of Viability

  • Place ampoule in a special metal container, thermostatic inside by expanded polystyrene, to prevent defrosting.

  • Transfer one granule of silica gel from ampoule on fresh suitable agar medium and incubate under optimal conditions.

  • The remaining granules were resealed and stored as described. Thus, each ampoule with fungal spores adsorbed on silica gel may be used repeatedly.

Result

The real storage time estimates obtained in VKM are given in Table 1 and Annex 2. They are not final data: the cultures are still being stored, and we expect to get longer storage times later on. Some cells of the table are empty; this is the case if the culture is not stored by this method.

There is at present clear that more than 98% of fungal cultures preserved by cryoconservation method remain viable after 20 years of storage. For lyophilization and storage in sterile soil methods, these figures after 30 years of storage are 95 and 85%, respectively. For long-term storage of fungal cultures on silica gel, the temperature −70 °C should be chosen. At this temperature, over 90% of spore-forming fungi retain their viability after 30 years of the experiment.

Conclusion

The conservation techniques used in VKM presents effective preservation of the stock of filamentous fungi from different taxonomic groups. The possibility and practical time estimates of secure long-term storage of fungal cultures belonging to 1600 species and 590 genera were shown. The represented information could be used as a reference for researchers intending to maintain pure cultures of microorganisms for a long time. The data produced are also accessible online on the VKM Web site.