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Experience in using modern methods of long-term preservation of VKM fungi
Abstract
Results of preservation of VKM filamentous fungi using modern methods, such as freeze-drying and low temperature preservation including silica gel storage are presented. The analysis of the data accumulated over a long period of time has allowed to determinate optimum protocols of preservation of fungi from different taxonomical groups. For the first time the possibility of long-term (more than 20 years) guaranteed preservation of fungal cultures from 349 species and 162 genera is shown.
... 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. ...
... 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 timeless than 10 years [9]. ...
The results of successful long-term preservation by various methods (cryopreservation, lyophilization, preservation on anhydrous silica gel and in sterile soil) are presents for different taxonomic groups of fungi preserved in All-Russian Collection of Microorganisms (VKM), G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (IBPM RAS). For each species, data are given on the number of strains studied and the maximum time of maintaining vitality. The protocol of method “drying on anhydrous silica gel” with the following storage at different temperatures is given.
Fungi are eukaryotic, usually filamentous, spore-producing organisms and can be
obligate parasites, nonobligate parasites, or biotrophs, developing several interactions
with plants, animals, or the environment and can be used to produce food and
enzymes for industrial processes [27]. Most fungi species have microscopic structures
and studies on this organism group depend on various microscopy techniques
types. Stokes related the fluorescence phenomenon in 1852, describing a photon molecular absorption generating the emission of another photon with greater wavelength,
the principle from which it was possible to develop techniques of fluorescence
microscopy [20]. Therefore, natural or induced fluorescence characteristics have
been explored for organisms and macromolecules localization explaining several
types of biological phenomena, mainly by techniques of epi-fluorescence or laser
confocal microscopy. These techniques enable many morphological and physiological analyzes in cells and tissues, locating cellular components, interaction with plants, nuclear dynamics, reactive oxygen species accumulation, and cellular death. Fluorescence microscopy studies may include analyzes of fluorescence or autofluorescent samples. Some fungi like Basidiomycota are autofluorescent, and others such as Cercospora spp. produce fluorescent phytotoxins. Other studies types
are conducted by inducing fluorescence in the samples. This may occur through the
use of fluorochromes, immunofluorescence techniques, nucleic acids hybridization,
and molecular markers. Fluorochromes are molecules capable of specifically binding
to cellular components by inducing their fluorescence under the excitation of certain
wavelengths. These components can be the fungal cell wall, nuclei, chromosomes,
mitochondria, and others. Other fluorochromes may indicate physiological aspects,
such as cell death, accumulation of reactive oxygen species, or evidence of defense
reactions in plant tissues colonized by fungi. Immunofluorescence, also called
immunostaining, is a technique where fluorescent molecules are attached to antibodies
corresponding to antigens to which they will be located. Nucleic acid
hybridization, called FISH (fluorescent in situ hybridization), allows the use of
nucleic acid probes attached to fluorescent molecules. These probes are complementary
to target sequences, which can identify specific regions on chromosomes, the
expression of certain genes by mRNAs, or specific groups of organisms using
regions of phylogenetically important DNA. One of the most commonly used
molecular markers is fluorescent proteins such as GFP (green fluorescent protein).
By genetic transformation, the fluorescent markers genes are associated with the
genes whose products will be localized. Thus, the expressed proteins are localized
through the fluorescent protein anchored. In this chapter, protocols for fluorescence microscopy will be discussed in studies on fungi using only fluorochromes or autofluorescence of structures for localization techniques. All following procedures were done at the Electron Microscopy and Ultrastructural Analysis Lab at Federal University of Lavras, using an inverted Epi-Fluorescence Zeiss Axio Z.1 and an inverted Laser Confocal Zeiss LSM780 Observer Z.1 and Zen 2012 software.
This chapter presents methods for fungi and plants infected with fungi analysis using epi-fluorescence (EFM) and laser scanning confocal microscopy (LSM). Here are included fluorochrome-based methods for evidencing fungi structures as well as cell wall and nuclei, besides reactive oxygen species and cellular death staining. Plant dyes examples important for fungi-plant interaction studies and autofluorescent structures for certain fungi are shown.KeywordsLaser scanning microscopyEpi-fluorescence microscopyFungi-plants interactionLSM for 3D-fungi imaging
The field of fungal research is experiencing a paradigm shift with respect to technological advancement; however, the mainstay for any advanced research lies in accurate species delimitation. Identification of fungi mainly involves morphological features, which are not always produced in culture and are sometimes variable, which renders identification difficult. Therefore, morphology-based taxonomic approach, though considered indispensable, cannot completely be relied upon because of ambiguity in fungal identification. The DNA-based molecular approaches have proven extremely valuable to mycological venture like species identification, taxonomic classification, and phylogenetic studies. The use of nuclear ribosomal gene, i.e. ITS along with other gene markers (SSU, LSU, RPB1, RPB2, tef1, cal, and tub2, etc.), has been recommended for fungal species level identification and phylogenetic studies. However, the choice of gene target is variable among different groups of fungi. One has to go through multiple research articles, so as to find out the recommended gene targets and their PCR cycling conditions for their fungal group of interest. Therefore, we assembled all the required protocols at one place, starting from fungal DNA isolation, list of recommended gene targets for identification and phylogeny (for selected and most common fungal pathogens), their primer names, sequences and PCR cycling conditions, DNA PCR product purification, and DNA sequencing. We have also briefed about sequence analysis, fungal databases, methodologies, and softwares used for construction of phylogenetic trees. This chapter would be a one stop solution for every researcher especially for the beginners, who is working in the field of taxonomy, phylogeny and evolution of fungi.
Results of successful preservation experience is given for the taxonomic groups of fungi preserved in All-Russian collection of Microogranisms (VKM): the species names, conservation methods, storage time estimates.
We have evaluated the survival and potential morphological alterations of 45 species of pathogenic filamentous fungi that had been stored in sterile water following Castellani's method in the National Collection of Pathogenic Fungi (NCPF). Storage duration varied from 2 months to over 21 years. Ninety percent of stored organisms were shown to be viable. Viability was largely independent of the duration of storage, but did apparently vary to some degree in an organism-specific manner. In addition, certain fungi were shown to have undergone morphological alterations during storage, and exhibited significant degrees of pleomorphism upon revival. This was especially marked for several isolates of dermatophytes, where storage resulted in loss of recognisable colonial features, and overproduction of sterile mycelium with aberrant or no conidia. These findings suggest that while Castellani's method remains an easy and inexpensive method for long-term preservation of most fungi, water storage should be supplemented by a second storage method to increase the chances of retaining both viability and morphological stability over long periods.
1.
Of 18 species of fungi stored on loam, 17 remain viable for 1 year, and 2 for as long as 4 years.
2.
All the fungi that were preserved in soil remained in the original wild or primary state, whereas on agar the dermatophytes, Phoma sp., Phyllosticta sp. and Fusarium sp. became pleomorphic.
3.
In loam, the cells of Phoma, Fusarium, Phyllosticta and Hormodendrum b increased throughout the medium.
4.
This investigation suggests that soil storage of fungi in the laboratory would obviate the laborious periodic transfer of agar stock-cultures and would provide a rich source that may be drawn from repeatedly for years. Most important, it would prevent pleomorphic changes in the fungi.
Prior to 1985, cultures at the Center for Forest Mycology Research were maintained on 1.5% malt extract agar test-tube slants. This system not only made it necessary to transfer the entire collection ev-ery year but also permitted genetic change because continual growth occurred. In 1985, the method of storing fungal cultures in sterile distilled water in cryovials was introduced. This study reports on the use of this method for long-term fungal storage. For varying periods up to 7 years, 151 miscellaneous spe-cies of wood-decaying Basidiomycotina were stored in sterile distilled water. Water storage has numerous advantages: culture viability or growth rate is not sig-nificantly influenced; isolates can be stored longer; genetic stability is greater; the method is quick, easy, and inexpensive, and requires less space. The Center for Forest Mycology Research (CFMR) is home to the largest culture collection of wood-rotting Basidiomycotina in the world. The collection, started in 1932, contains more than 11000 secondary my-celium isolates of more than 1600 species and more than 3200 monobasidiosporous isolates taken from many of the same specimens as the dikaryons. Prior to 1985, the cultures were maintained on 1.5% malt extract agar test-tube slants, making it necessary to transfer the entire collection at least once every year.
Laccaria fraterna, a basidiomycetous, filamentous, non-sporulating (in vitro) fungus, was subjected to freeze-drying and tested for viability after rehydration. The optimization of the freeze-drying protocol included selection of the candidate fungus; optimizing physiological growth conditions and age; standardizing the protectant type and concentration; optimizing the pre-freezing method, freeze-drying run and extent of drying; choice of the rehydrant and the extent of rehydration. The culture retained its viability after lyophilization and when subjected to quality assurance tests gave consistent results similar to the non-lyophilized culture, indicating the stability of the product and the applicability of the developed process to freeze-dry vegetative mycelium of filamentous non-sporulating fungi.
The effects of preservation regime on secondary metabolite production in two genera of economically important fungi, Metarhizium anisopliae and Fusarium oxysporum, was assessed using thin layer chromatography and high performance liquid chromatography over a 2-year testing period. Five preservation regimes, commonly used in microbial culture collections throughout the world were investigated: continual sub-culture, lyophilization, storage of mycelial plugs in water, storage at –20 C and cryopreservation with liquid nitrogen. Preservation regime influenced secondary metabolite production in the test fungi. Changes in secondary metabolite profiles occurred after relatively short storage periods in most strains, irrespective of the preservation treatment used. Although no preservation treatment can be guaranteed to provide total stability of secondary metabolite production, cryopreservation was the best of the methods tested. Response to preservation and storage also differed between strains of the same species. Therefore, there is a need to develop new and existing preservation criteria with an emphasis on strain-specific criteria in order to reduce the prospects of instability in secondary metabolite production.
One hundred and ninety five strains of fungi were observed during freezing and thawing using a cryogenic light microscope. There was no obvious link between taxonomic position and their morphological response to freezing and thawing. The viability of seven of these strains was examined following freezing and thawing in the presence or absence of the cryoprotectants glycerol and dimethyl sulphoxide. Intracellular ice and hyphal shrinkage were not necessarily lethal events, but in many cases they affected the rate and quality of growth. Both cryoprotectants reduced shrinkage, shifted the cooling rate where intracellular ice formed in many cases, and improved the recovery of strains. The results presented aid the development of successful cryopreservation protocols.
Qualitative enzyme assays were used as a tool to investigate the stability of freeze-dried mycorrhizal fungi. Both lyophilized (L) and non-lyophilized (NL) mycelia of individual isolates showed identical response for all the enzymes tested (nitrate reductase, protease, pectinase, and nuclease). All the isolates showed positive nitrate reductase activity, except two isolates of Thelephora terrestris (both L and NL). Both L and NL cultures of individual isolates showed substrate specificity (between gelatin and casein) for protease activity. Though both L and NL mycelia of all the culture isolates grew upon pectin substrate, there was no pectinase activity expressed. RNAase activity was variously exhibited (little activity, little growth–no activity, and no growth–no activity) by individual test cultures. The consistencies in growth and enzyme activity of the cultures before and after lyophilization imply the stability of the freeze-dried vegetative mycelium.
The long-term preservation of valuable fungal cultures can be achieved in several ways and the choice of methodology can be problematical. Firstly, there is the decision whether to use a public service culture collection or `in-house' facilities. Secondly, the wide variety of preservation methods available often leads to confusion about which protocol(s) are best suited for specific fungi. No method can be universally applied to all fungi. Some species are notoriously difficult to preserve, whilst other fungi can be preserved by almost any method. A decision-based key has been devised, which uses questions related to fungal characters and user facilities and economics to determine the most appropriate method for long-term preservation of cultures. This key should facilitate the decisions of microbiologists when considering preservation of important fungal cultures.
This paper discusses the recovery of a sample of strains originally preserved on silica gel over the period 1970–1973. Fifty-three strains were tested of which 18 recovered, demonstrating survival for more than 20 years. The recovery of 26 of the strains is directly compared with that of replicates from oil storage and freeze-drying. A summary of storage of 421 strains by the silica gel technique is given, reporting survival of 64% for a quarter of a century or more. The technique is ideal for preserving sporulating fungi of the Ascomycota and many species of mitotic fungi for laboratories of limited facilities.
The aim of preserving a fungus is to maintain it in a viable state without change to its genetic, physiological, or anatomical characters. There are numerous methodologies available to preserve a fungus, but the two methods widely used by culture collections (biological or genetic resource centers) to achieve successful preservation are cryopreservation with liquid nitrogen using controlled-rate freezing and centrifugal freeze-drying. Generic methods are often used, but specific variations of a method may be required in order to achieve optimal stability. No single method can be applied to all fungi. More recently, techniques such as vitrification and encapsulation cryopreservation have been used to preserve recalcitrant fungi. The protocols described within this chapter have been developed over many years at one of the world's largest culture collections of filamentous fungi.