The strain Pleurotus ostreatus Florida f6, its 45 basidiospore-derived isolates (both monokaryons and dikaryons prepared in our laboratory), Trametes versicolor strain CCBAS 614 and 22 other T. versicolor isolates obtained from the sporocarps collected in distant localities were successfully preserved for 12 y using perlite and straw cryopreservation protocols. All tested isolates survived a 12-year storage in liquid nitrogen (LN) and their laccase production and Poly B411 decolorization capacity was preserved. Also mycelium extension rate and the types of colony appearance of individual isolates remained unchanged. Different cryopreservation techniques were also tested for the short time (24 h) and the long time (6 m) storage of the culture liquid with extracellular laccase produced by T. versicolor strain CCBAS 614. The results showed that 10 % glycerol was the most suitable cryopreservant. The absence of the cryopreservant did not cause high loss of laccase activity in the samples; the presence of DMSO (5 or 10 %) in LN-stored samples caused mostly a decrease of laccase activity. For the preservation of laccase activity in the liquid culture the storage in the freezer at -80 °C is more convenient than the storage in liquid nitrogen.
- MeSH
- fungální proteiny analýza metabolismus MeSH
- kryoprezervace metody MeSH
- lakasa analýza metabolismus MeSH
- mikrobiální viabilita MeSH
- Pleurotus chemie enzymologie růst a vývoj izolace a purifikace MeSH
- rostliny mikrobiologie MeSH
- Trametes chemie enzymologie růst a vývoj izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Basidiomycetes are used in industrial processes, in basic or applied research, teaching, systematic and biodiversity studies. Efficient work with basidiomycete cultures requires their reliable source, which is ensured by their safe long-term storage. Repeated subculturing, frequently used for the preservation, is time-consuming, prone to contamination, and does not prevent genetic and physiological changes during long-term maintenance. Various storage methods have been developed in order to eliminate these disadvantages. Besides lyophilization (unsuitable for the majority of basidiomycetes), cryopreservation at low temperatures seems to be a very efficient way to attain this goal. Besides survival, another requirement for successful maintenance of fungal strains is the ability to preserve their features unchanged. An ideal method has not been created so far. Therefore it is highly desirable to develop new or improve the current preservation methods, combining advantages and eliminate disadvantages of individual techniques. Many reviews on preservation of microorganisms including basidiomycetes have been published, but the progress in the field requires an update. Although herbaria specimens of fungi (and of basidiomycetes in particular) are very important for taxonomic and especially typological studies, this review is limited to live fungal cultures.
- MeSH
- Basidiomycota fyziologie MeSH
- mykologie metody MeSH
- ochrana biologická metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1 mM TBBPA within 4 days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2 mM TBBPA within 5 h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5 mM within 3 h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2',6,6'-tetrabromo-4,4'-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (Zalko et al., 2006), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity.
The macro- and micro-morphological features, mycelial extension rate, enzymatic activities and possible genetic changes were studied in 30 selected strains of basidiomycetes after 10-year cryopreservation on perlite in liquid nitrogen (LN). Comparisons with the same strains preserved by serial transfers on nutrient media at 4°C were also conducted. Production of ligninolytic enzymes and hydrogen peroxide was studied by quantitative spectrophotometric methods, whereas semiquantitative API ZYM testing was used to compare the levels of a wide range of hydrolytic enzymes. Our results show that cryopreservation in LN did not cause morphological changes in any isolate. The vitality of all fungi was successfully preserved and none of the physiological features were lost, even though the extension rate and enzyme activity were slightly affected. Moreover, sequence analysis of eight strains did not detect any changes in their genetic features after cryopreservation. These findings suggest that the perlite-based freezing protocol is suitable for long-term preservation of large numbers of basidiomycetes.
Cultures of 33 basidiomycete strains out of 35 tested were viable with unchanged characteristics after four years of maintenance on perlite in cryovials. These cultures can be a good substitute for agar cultures in long-term maintenance of fungi. For comparison, the storage under oil was evaluated but it turned out to be unsuitable for the majority of our cultures.
- MeSH
- Basidiomycota enzymologie růst a vývoj MeSH
- časové faktory MeSH
- fungální proteiny metabolismus MeSH
- kultivační média MeSH
- lakasa metabolismus MeSH
- mykologie metody přístrojové vybavení MeSH
- nízká teplota MeSH
- ochrana biologická metody přístrojové vybavení MeSH
- oleje MeSH
- oxid hlinitý MeSH
- oxid křemičitý MeSH
The viability, growth and morphology of 48 strains of Ascomycota (including 17 yeasts) and 20 strains of Zygomycota were determined after a 2-d and then after 1-year storage in liquid nitrogen using a new cryopreservation method with perlite as a particulate solid carrier. In case of Ascomycota, 45 strains (94 %) out of 48 survived both 2-d and 1-year storage in liquid nitrogen, respectively. In case of Zygomycota, all 20 strains survived both storage. In addition, 3 strains of Basidiomycota counted among yeasts were tested and all survived the 1 year storage. In all surviving cultures no negative effects of cryopreservation by this method have been observed after 1-year of storage in liquid nitrogen. The results indicate that the perlite protocol can be successfully used for cryopreservation of taxonomically different groups of fungi and also for fungi which failed to survive other routinely used preservation procedures.
Mycelial basidiomycete cultures on perlite in cryovials survived successfully three successive cycles of freezing, storage in liquid nitrogen (LN) and thawing without noticeable changes. This indicates that using perlite as a carrier for cryopreservation could in most cases overcome difficulties caused by interrupted supply of LN or electric power during the storage. Cultures on perlite can also be reused for successive inoculations.
