Journal of Microbiological Methods 99 (2014) 81–83
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
A new method for the preservation of axenic fungal cultures Xiaojia Hu a, Gordon Webster b, Lihua Xie a, Changbing Yu a, Yinshui Li a, Xing Liao a,⁎ a b
Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062, China Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3AT, UK
a r t i c l e
i n f o
Article history: Received 6 February 2014 Accepted 14 February 2014 Available online 21 February 2014 Keywords: Cryopreservation Fungi Aspergillus Trichoderma Culture collection
a b s t r a c t Microbiological work requires a reliable source of cultures that are not only well defined and taxonomically determined, but are also adequately preserved without changes in their morphological, physiological and genetic traits. Here we describe an easy, cost effective and rapid method for reliably preserving filamentous fungi on cellophane pieces at −80 °C for use in laboratory culture collections. © 2014 Elsevier B.V. All rights reserved.
The aim of a culture collection is to preserve and maintain a microbial culture in a viable state without changes to any of its genetic, physiological or morphological characteristics, preserving the strain as a principal source of material for subsequent research, teaching and biotechnological processes. Filamentous fungi, are frequently kept by routine subculturing from staled to fresh media (Fennell, 1960), a method that is time consuming, prone to contamination, and does not prevent genetic and physiological variations during long-term maintenance (Homolka, 2013). To overcome these limitations other methods of storage have also been developed to preserve fungal strains: (1) Ambient temperature preservation, including storage in sterile water (Boesewinklel, 1976), mineral oil (Perrin, 1979), silica gel (Perkins, 1962) and sand/soil (Smith and Onions, 1994). These methods are simple, and operate easily, but the transfer times are often short, and strains degenerate over time. (2) Freeze-drying (lyophilisation) preservation, some strains can be long preserved by this method, but the method is complicated, timeconsuming, requires expensive equipment (Smith, 1983) and often strain specific (Homolka, 2013). (3) Cryopreservation (ultra-low temperature freezing), preservation temperature is generally at −80°C in an ultra-low temperature freezer or at −196 °C using liquid nitrogen. Strains can be long preserved by this method, but the fungal culture must be prepared with a cryoprotective agent, and the choice of cryoprotectant is a matter of experience as it varies according to the organism being curated. In addition, when storing cultures in liquid nitrogen, the need to ensure an adequate supply of liquid nitrogen can also be a disadvantage (Smith and Thomas, 1998).
⁎ Corresponding author. Tel.: +86 27 86712256; fax: +86 27 86816451. E-mail address: [email protected] (X. Liao).
http://dx.doi.org/10.1016/j.mimet.2014.02.009 0167-7012/© 2014 Elsevier B.V. All rights reserved.
Here we describe a novel, highly efficient method based simply on sterile cellophane squares (CPS), for the long-term preservation and fast revival of filamentous fungi. Sterile cellophane with permeability characteristics that allow growth of fungi when placed on the surface of solidified media is commonly used in morphological and biochemical studies of fungal hyphae (Dusseau, 1938; De Araújo and Roussos, 2002) and preparation of fungal material for rapid DNA extraction (Cassago et al., 2002). Cellophane (1.0 m2; Shanghai Bio-Tech Co., Ltd.) was cut into squares (40–50 mm2) and autoclaved in glass Petri dishes (90 mm diameter) for 20 min at 121 °C. After autoclaving, the CPS were soaked in sterile water to prevent sticking and then placed flat on the surface of agar-solidified medium (Potato Dextrose Agar; PDA) in plastic Petri dishes (90 mm diameter) with the aid of sterile forceps. Fungal spores (103 spores ml− 1) from a stock culture were used as inoculum (20– 30 μl) and placed directly on to CPS. However, it should also be noted that other methods of inoculation of CPS have also been undertaken with similar rates of success, and these included inoculating CPS with small agar-fungal pieces (5–10 mm2) and streaking with an inoculation loop containing hyphae and fungal spores. For all strains used in this study, an incubation period of 4–5 days at 28 °C was sufficient to obtain colonisation of the CPS, but growth time can be extended depending on the strain selected. The overgrown CPS was carefully removed from the PDA with the aid of sterile forceps and placed immediately into an empty sterile Petri dish and cut into smaller squares (10 mm2) using sterile scissors. Fungal CPS were then allowed to completely dry for 3 days in incubator at 28 °C. Dried fungal CPS were then placed into sterile 2.0 ml-volume cryovials and frozen at − 80 °C in an ultra-low temperature freezer for long-term storage. It should also be noted, that if desired, dried fungal CPS can also be stored at − 196 °C, as
82
X. Hu et al. / Journal of Microbiological Methods 99 (2014) 81–83
Fig. 1. Examples of fungal cultures revived after more than 10 years storage at − 80 °C using the CPS method. (A) Trichoderma viride strain Tv-36, (B) Trichoderma sp. TRI-1 and (C) Aspergillus sp. ASP-4. All strains tested were revived on PDA plates and grown at 28 °C.
experiments on several fungal strains (e.g. Trichoderma and Aspergillus species) show 100% viability after 50 days storage in liquid nitrogen (data not shown). The versatility of the CPS method described above was mainly tested for the long-term preservation of a collection of mycoparasitic strains, recently identified as biocontrol agents for Sclerotinia stem rot of oilseed rape (Hu et al., 2013): Aspergillus sp. ASP-4; Trichoderma viride strain Tv-36; Trichoderma sp. TRI-1, although several other phylogenetically diverse fungi were also tested: Aspergillus aculeatus, Aspergillus niger, Penicillium oxalicum, Rhizopus stolonifer, Mucor circinelloides and Neurospora crassa. The recovery rates of fungal strains preserved using the CPS method were then assessed by thawing 3 replicate fungal CPS per strain and placing them on to fresh PDA medium at yearly intervals, all fungal CPS inoculated PDA plates were incubated at 28 °C and observed for growth daily. Recovery of stored fungal cultures on PDA after freezing was observed for all fungal strains tested after only 2 days incubation, with all strains showing 100% growth and survival after one year storage at −80 °C (Fig. 1). In addition, all mycoparasitic strains of the plant pathogen Sclerotinia sclerotiorum (e.g. Aspergillus sp. ASP-4, Trichoderma viride Tv-36 and Trichoderma sp. TRI-1) which had been preserved at − 80 °C for more than 10 years also showed 100% survival (Figs. 1 & 2). These results suggest that the CPS method of cryopreservation is highly efficient and can be used to preserve a diverse range of fungal isolates, maintaining viability in fungi from several members of the
Ascomycota and Zygomycota. We believe that this method could be readily adapted to preserve and revive many other species of fungi as CPS can be applied to any agar-solidified medium that is optimal for the growth of a specific strain, although growth times on CPS may require adjustment. In addition, since this method is very simple and did not employ any complicated protocols (Homolka, 2013), use specific spore preparations (Ryan and Smith, 2007), utilize the addition of cryoprotectants (Prakash et al., 2013), require expensive equipment or use liquid nitrogen it can easily be utilized to provide inexpensive storage for small scale, low-budget culture collections. Inexpensive storage that can provide extended shelf-life to fungi used in biotechnology or biocontrol (e.g. Hu et al., 2013) is also of major importance commercially (Prakash et al., 2013).
Acknowledgement This work was funded by the National Natural Science Foundation of China for (Project 30870451), Special Fund for Agro-scientific Research in the Public Interest (Contract 201203096), and by Special Funds from the National Scientific Support Program of China (Contract 2010BAD01B05-03). Trichoderma viride strain Tv-36, Trichoderma sp. TRI-1 and Aspergillus sp. ASP-4 are held in the Agricultural Culture Collection of China (Beijing, China) as ACCC 32505, ACCC 32501 and ACCC 32502.
Survival (%)
100
75
50
25
0
*
*
*
*
*
*
*
*
Strain
1 year
2 to 5 years
6 to 10 years
10+ years
Fig. 2. Recovery rates (% survival) of fungal strains after 1, 2 to 5, 6 to 10, and more than 10 years storage at −80 °C on CPS. All strains tested were revived on PDA plates and grown at 28 °C; * indicates no data recorded.
X. Hu et al. / Journal of Microbiological Methods 99 (2014) 81–83
References Boesewinklel, H.J., 1976. Storage of fungal cultures in water. Trans. Br. Mycol. Soc. 66, 183–185. Cassago, A., Panepucci, R.A., Baião, A.M.T., Henrique-Silva, F., 2002. Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei. BMC Microbiol. 2, 14. De Araújo, A.A., Roussos, S., 2002. A technique for mycelial development of ectomycorrhizal fungi on agar media. Appl. Biochem. Biotechnol. 98–100, 311–318. Dusseau, A., 1938. Growing fungi on cellophane. Science 88, 412. Fennell, D.I., 1960. Conservation of fungus cultures. Bot. Rev. 26, 79–141. Homolka, L., 2013. Methods of cryopreservation in fungi. In: Gupta, V.K., Tuohy, M.G., Ayyachamy, M., Turner, K.M., O'Donovan, A. (Eds.), Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology. Springer, Germany, pp. 9–16. Hu, X., Webster, G., Xie, L., Yu, C., Li, Y., Liao, X., 2013. A new mycoparasite, Aspergillus sp. ASP-4, parasitizes the sclerotia of Sclerotinia sclerotiorum. Crop. Prot. 54, 15–22.
83
Perkins, D.D., 1962. Preservation of Neurospora stock cultures in anhydrous silica gel. Can. J. Microbiol. 8, 591–594. Perrin, P.W., 1979. Long-term storage of cultures of wood-inhabiting fungi under mineral oil. Mycologia 71, 867–869. Prakash, O., Nimonkar, Y., Shouche, Y.S., 2013. Practice and prospects of microbial preservation. FEMS Microbiol. Lett. 339, 1–9. Ryan, M.J., Smith, D., 2007. Cryopreservation and freeze-drying of fungi employing centrifugal and shelf freeze-drying. Methods Mol. Biol. 368, 127–140. Smith, D., 1983. Cryoprotecant and cryopreservation of fungi. Trans. Br. Mycol. Soc. 80, 360–363. Smith, D., Onions, A.H.S., 1994. The Preservation and Maintenance of Living Fungi, Second edition. CAB International, Wallingford, UK. Smith, D., Thomas, V.E., 1998. Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi. World J. Microbiol. Biotechnol. 14, 49–57.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
A new method for the preservation of axenic fungal cultures Xiaojia Hu a, Gordon Webster b, Lihua Xie a, Changbing Yu a, Yinshui Li a, Xing Liao a,⁎ a b
Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062, China Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3AT, UK
a r t i c l e
i n f o
Article history: Received 6 February 2014 Accepted 14 February 2014 Available online 21 February 2014 Keywords: Cryopreservation Fungi Aspergillus Trichoderma Culture collection
a b s t r a c t Microbiological work requires a reliable source of cultures that are not only well defined and taxonomically determined, but are also adequately preserved without changes in their morphological, physiological and genetic traits. Here we describe an easy, cost effective and rapid method for reliably preserving filamentous fungi on cellophane pieces at −80 °C for use in laboratory culture collections. © 2014 Elsevier B.V. All rights reserved.
The aim of a culture collection is to preserve and maintain a microbial culture in a viable state without changes to any of its genetic, physiological or morphological characteristics, preserving the strain as a principal source of material for subsequent research, teaching and biotechnological processes. Filamentous fungi, are frequently kept by routine subculturing from staled to fresh media (Fennell, 1960), a method that is time consuming, prone to contamination, and does not prevent genetic and physiological variations during long-term maintenance (Homolka, 2013). To overcome these limitations other methods of storage have also been developed to preserve fungal strains: (1) Ambient temperature preservation, including storage in sterile water (Boesewinklel, 1976), mineral oil (Perrin, 1979), silica gel (Perkins, 1962) and sand/soil (Smith and Onions, 1994). These methods are simple, and operate easily, but the transfer times are often short, and strains degenerate over time. (2) Freeze-drying (lyophilisation) preservation, some strains can be long preserved by this method, but the method is complicated, timeconsuming, requires expensive equipment (Smith, 1983) and often strain specific (Homolka, 2013). (3) Cryopreservation (ultra-low temperature freezing), preservation temperature is generally at −80°C in an ultra-low temperature freezer or at −196 °C using liquid nitrogen. Strains can be long preserved by this method, but the fungal culture must be prepared with a cryoprotective agent, and the choice of cryoprotectant is a matter of experience as it varies according to the organism being curated. In addition, when storing cultures in liquid nitrogen, the need to ensure an adequate supply of liquid nitrogen can also be a disadvantage (Smith and Thomas, 1998).
⁎ Corresponding author. Tel.: +86 27 86712256; fax: +86 27 86816451. E-mail address: [email protected] (X. Liao).
http://dx.doi.org/10.1016/j.mimet.2014.02.009 0167-7012/© 2014 Elsevier B.V. All rights reserved.
Here we describe a novel, highly efficient method based simply on sterile cellophane squares (CPS), for the long-term preservation and fast revival of filamentous fungi. Sterile cellophane with permeability characteristics that allow growth of fungi when placed on the surface of solidified media is commonly used in morphological and biochemical studies of fungal hyphae (Dusseau, 1938; De Araújo and Roussos, 2002) and preparation of fungal material for rapid DNA extraction (Cassago et al., 2002). Cellophane (1.0 m2; Shanghai Bio-Tech Co., Ltd.) was cut into squares (40–50 mm2) and autoclaved in glass Petri dishes (90 mm diameter) for 20 min at 121 °C. After autoclaving, the CPS were soaked in sterile water to prevent sticking and then placed flat on the surface of agar-solidified medium (Potato Dextrose Agar; PDA) in plastic Petri dishes (90 mm diameter) with the aid of sterile forceps. Fungal spores (103 spores ml− 1) from a stock culture were used as inoculum (20– 30 μl) and placed directly on to CPS. However, it should also be noted that other methods of inoculation of CPS have also been undertaken with similar rates of success, and these included inoculating CPS with small agar-fungal pieces (5–10 mm2) and streaking with an inoculation loop containing hyphae and fungal spores. For all strains used in this study, an incubation period of 4–5 days at 28 °C was sufficient to obtain colonisation of the CPS, but growth time can be extended depending on the strain selected. The overgrown CPS was carefully removed from the PDA with the aid of sterile forceps and placed immediately into an empty sterile Petri dish and cut into smaller squares (10 mm2) using sterile scissors. Fungal CPS were then allowed to completely dry for 3 days in incubator at 28 °C. Dried fungal CPS were then placed into sterile 2.0 ml-volume cryovials and frozen at − 80 °C in an ultra-low temperature freezer for long-term storage. It should also be noted, that if desired, dried fungal CPS can also be stored at − 196 °C, as
82
X. Hu et al. / Journal of Microbiological Methods 99 (2014) 81–83
Fig. 1. Examples of fungal cultures revived after more than 10 years storage at − 80 °C using the CPS method. (A) Trichoderma viride strain Tv-36, (B) Trichoderma sp. TRI-1 and (C) Aspergillus sp. ASP-4. All strains tested were revived on PDA plates and grown at 28 °C.
experiments on several fungal strains (e.g. Trichoderma and Aspergillus species) show 100% viability after 50 days storage in liquid nitrogen (data not shown). The versatility of the CPS method described above was mainly tested for the long-term preservation of a collection of mycoparasitic strains, recently identified as biocontrol agents for Sclerotinia stem rot of oilseed rape (Hu et al., 2013): Aspergillus sp. ASP-4; Trichoderma viride strain Tv-36; Trichoderma sp. TRI-1, although several other phylogenetically diverse fungi were also tested: Aspergillus aculeatus, Aspergillus niger, Penicillium oxalicum, Rhizopus stolonifer, Mucor circinelloides and Neurospora crassa. The recovery rates of fungal strains preserved using the CPS method were then assessed by thawing 3 replicate fungal CPS per strain and placing them on to fresh PDA medium at yearly intervals, all fungal CPS inoculated PDA plates were incubated at 28 °C and observed for growth daily. Recovery of stored fungal cultures on PDA after freezing was observed for all fungal strains tested after only 2 days incubation, with all strains showing 100% growth and survival after one year storage at −80 °C (Fig. 1). In addition, all mycoparasitic strains of the plant pathogen Sclerotinia sclerotiorum (e.g. Aspergillus sp. ASP-4, Trichoderma viride Tv-36 and Trichoderma sp. TRI-1) which had been preserved at − 80 °C for more than 10 years also showed 100% survival (Figs. 1 & 2). These results suggest that the CPS method of cryopreservation is highly efficient and can be used to preserve a diverse range of fungal isolates, maintaining viability in fungi from several members of the
Ascomycota and Zygomycota. We believe that this method could be readily adapted to preserve and revive many other species of fungi as CPS can be applied to any agar-solidified medium that is optimal for the growth of a specific strain, although growth times on CPS may require adjustment. In addition, since this method is very simple and did not employ any complicated protocols (Homolka, 2013), use specific spore preparations (Ryan and Smith, 2007), utilize the addition of cryoprotectants (Prakash et al., 2013), require expensive equipment or use liquid nitrogen it can easily be utilized to provide inexpensive storage for small scale, low-budget culture collections. Inexpensive storage that can provide extended shelf-life to fungi used in biotechnology or biocontrol (e.g. Hu et al., 2013) is also of major importance commercially (Prakash et al., 2013).
Acknowledgement This work was funded by the National Natural Science Foundation of China for (Project 30870451), Special Fund for Agro-scientific Research in the Public Interest (Contract 201203096), and by Special Funds from the National Scientific Support Program of China (Contract 2010BAD01B05-03). Trichoderma viride strain Tv-36, Trichoderma sp. TRI-1 and Aspergillus sp. ASP-4 are held in the Agricultural Culture Collection of China (Beijing, China) as ACCC 32505, ACCC 32501 and ACCC 32502.
Survival (%)
100
75
50
25
0
*
*
*
*
*
*
*
*
Strain
1 year
2 to 5 years
6 to 10 years
10+ years
Fig. 2. Recovery rates (% survival) of fungal strains after 1, 2 to 5, 6 to 10, and more than 10 years storage at −80 °C on CPS. All strains tested were revived on PDA plates and grown at 28 °C; * indicates no data recorded.
X. Hu et al. / Journal of Microbiological Methods 99 (2014) 81–83
References Boesewinklel, H.J., 1976. Storage of fungal cultures in water. Trans. Br. Mycol. Soc. 66, 183–185. Cassago, A., Panepucci, R.A., Baião, A.M.T., Henrique-Silva, F., 2002. Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei. BMC Microbiol. 2, 14. De Araújo, A.A., Roussos, S., 2002. A technique for mycelial development of ectomycorrhizal fungi on agar media. Appl. Biochem. Biotechnol. 98–100, 311–318. Dusseau, A., 1938. Growing fungi on cellophane. Science 88, 412. Fennell, D.I., 1960. Conservation of fungus cultures. Bot. Rev. 26, 79–141. Homolka, L., 2013. Methods of cryopreservation in fungi. In: Gupta, V.K., Tuohy, M.G., Ayyachamy, M., Turner, K.M., O'Donovan, A. (Eds.), Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology. Springer, Germany, pp. 9–16. Hu, X., Webster, G., Xie, L., Yu, C., Li, Y., Liao, X., 2013. A new mycoparasite, Aspergillus sp. ASP-4, parasitizes the sclerotia of Sclerotinia sclerotiorum. Crop. Prot. 54, 15–22.
83
Perkins, D.D., 1962. Preservation of Neurospora stock cultures in anhydrous silica gel. Can. J. Microbiol. 8, 591–594. Perrin, P.W., 1979. Long-term storage of cultures of wood-inhabiting fungi under mineral oil. Mycologia 71, 867–869. Prakash, O., Nimonkar, Y., Shouche, Y.S., 2013. Practice and prospects of microbial preservation. FEMS Microbiol. Lett. 339, 1–9. Ryan, M.J., Smith, D., 2007. Cryopreservation and freeze-drying of fungi employing centrifugal and shelf freeze-drying. Methods Mol. Biol. 368, 127–140. Smith, D., 1983. Cryoprotecant and cryopreservation of fungi. Trans. Br. Mycol. Soc. 80, 360–363. Smith, D., Onions, A.H.S., 1994. The Preservation and Maintenance of Living Fungi, Second edition. CAB International, Wallingford, UK. Smith, D., Thomas, V.E., 1998. Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi. World J. Microbiol. Biotechnol. 14, 49–57.
