versão impressa ISSN 0001-3714
Rev. Microbiol. v. 29 n. 3 São Paulo Set. 1998
Isabel Lara-Herrera, Gerardo Mata, Rigoberto Gaitán-Hernández *
Instituto de Ecología, Xalapa, Veracruz, México.
Submitted: July 11, 1997; Returned to authors for corrections: February 06, 1997;
Approved: July 23, 1998
Viability of 6 mushroom strains of the Pleurotus genus (2 from P. djamor var. djamor, 1 from P. ostreatus var. ostreatus, 2 from P. ostreatus var. columbinus and 1 from P. pulmonarius) after liquid nitrogen cryopreservation (-196º) was evaluated. The contact time for the mycelia of these strains with the cryoprotectant (glycerol) was studied 1, 2 and 3 hours before freezing. We also tested the effect of different times (5, 10 and 15 minutes) and temperatures (30, 45 and 60ºC) of the thawing system for mycelial recovery. The results showed a marked tendency toward faster mycelial recovery when samples were thawed at 30ºC, while at 60ºC no recovery was observed. A change in thawing and contact times with the cryoprotectant did not affect the results significantly, as the thawing temperature and strain employed affected.
Key words: Pleurotus, viability, liquid nitrogen, mushroom
INTRODUCTION Liquid nitrogen conservation is, at present, the most reliable and commonly employed method of storing the strains of certain basidiomycetous fungi. This system has the advantage that, because the metabolism of the stored strains is either slowed down or completely suppressed, the likelihood of changes in genetic material is lower; the storage period is, furthermore, theoretically limitless (2, 9). In the commercial sector, especially in the edible mushroom harvesting industry, this method provides a major advantage, as it preserves characteristics desirable in commercial strains (1, 15). Due, however, to the great variety of species that exist and to the fact that not all fungi respond similarly to freezing conditions (5, 16), it is important to conduct more extensive research to determine optimal conditions for nitrogen conservation. The Pleurotus genus has a cosmopolitan distribution and various species are commercially important because of their edibility. That is what prompted the present study, in which recovery after liquid nitrogen cryopreservation was examined in six strains of edible mushrooms of the Pleurotus genus.
MATERIALS AND METHODS Six strains of the Pleurotus genus were studied: two Mexican, P. djamor (Fr.) Boedijn var. djamor; one from Guatemala, P. ostreatus (Quél.) Quél. var. columbinus, and another from the United States; one from Japan, P. ostreatus (Jacq. : Fr.) Kumm. var. ostreatus and one German strain of P. pulmonarius (Quél.) Sing. All are registered and stored in the Fungus Strain Collection at the Instituto de Ecología, Veracruz, México as IE-111, IE-116, IE-9, IE-140, IE-129 and IE-4, respectively. The strains were maintained in Petri dishes on agar medium with malt extract (BIOXON).
For the cryopreservation experiments, the mycelium from each strain was grown in grains of sorghum (Sorghum vulgare Pers.) that had been previously soaked for 12 hours. The grains were placed in 90 mm diameter Petri dishes and sterilized at 121ºC for 45 minutes. Afterward, in aseptic conditions, they were inoculated with app. 1 cm2 fragments of colonized agar with the mycelium of the corresponding strain and incubated at 27 ± 1ºC in darkness until the mycelium had completely covered the grains of sorghum. The technique employed to prepare the material for freezing was a modification of that described by Hwang (4). We placed 1.2 ml of glycerol at 10% v/v in flasks of polypropylene and sterilized them at 121ºC for 15 minutes. Under aseptic conditions, 10 sorghum grains covered with mycelium from each of the strains were placed in a flask, sealed airtight and labelled. Using two flasks per treatment, we let the contact time being analyzed elapse according to the condition being evaluated. The flasks were subsequently placed in boxes of polycarbonate that were then introduced into the liquid nitrogen container. Samples remained in the container for 15 days.
After the freezing time had elapsed, recovered samples were evaluated according to the following parameters: contact time (ct), during which the sample (sorghum grain invaded by mycelium) was in contact with the cryoprotectant before being frozen in nitrogen (1, 2 and 3 hours); thawing time (tt), during which the flasks containing samples were in a bain-marie (5, 10 and 15 minutes) after removal from the container; and thawing temperature (tT), at which the bain-marie was maintained (30, 45 and 60ºC). Table 1 illustrates how these parameters combined.
Table 1.Treatments evaluated before being frozen in nitrogen (contact time) and after that the samples were removed from nitrogen container (thawing time, thawing temperature).
The letters a...o indicate the type of treatment used. Example: a=1 hour contact time (ct) and 10 minutes thawing time (tt) at 30ºC (tT).
The flasks were removed from the nitrogen container and immediately submerged in a bain-marie at the temperature being tested. After the required time had passed, each sorghum grain contained in the flasks was placed in a sterile agar medium with malt extract. With 10 grains per dish, they were incubated during 15 days at 27 ± 1ºC in darkness.
We checked the petri dishes daily and recorded the number of samples retrieved, such samples being those that exhibited mycelial growth when examined under a stereoscopic microscope. The data obtained were submitted to an analysis of variance (ANOVA) for a Blocks Factorial Design at a=0.05% level, to determine if significant differences exist between the treatments (ct, tt, tT).
RESULTS AND DISCUSSION Table 2 shows the number of samples recovered using the different treatments. As no mycelia were recovered from samples in treatments e, j and o, they were not included in the statistical analysis. In the other treatments, the mycelia in the samples were recovered between the first and sixth day with at least 85%. The best treatment turned out to be m, with a contact period of 3 hours and a thawing time of 15 minutes at 30ºC, in which the 100% of all samples from all strains were recovered. The other treatments yielded varying numbers of samples, even within strains of the same species. For example, as indicated in Table 3, in the case of P. djamor var. djamor, the recovery rate for strain IE-111 was 100% of frozen samples, and 95% for strain IE-116. Strain 9 of P. ostreatus var. columbinus had a 95% recovery rate, compared to 84.2% for IE-140. This same table shows an increase in recovery figures on the second day, and that by the fourth day, more than 90% of the total samples had been recovered. Strains IE-9, IE-111, IE-116 and IE-129 were recovered between the first and fifth day and strains IE-4 and IE-140 between the first and sixth. These results surpass those of earlier studies for species of the Pleurotus genus in which other techniques for preservation in nitrogen were employed, the recovery rate there being 0-58% (3, 12). Our results are comparable to those cited for other species, with 99.25-100% of recovery (10, 13, 14, 17).
Table 2. Number and percentage of samples recovered per strain in the different treatments.
* see Table 1.
** Percentage of recovery.
Table 3. Total number of samples recovered per day from the different strains studied, for all evaluated treatments*.
* see table 1.
Pleurotus djamor var. djamor (IE-111, IE-116).
P. ostreatus var. columbinus (IE-9, IE-140).
P. ostreatus var. ostreatus (IE-129).
P. pulmonarius (IE-4).
Results of the analysis of variance to Blocks Factorial Design indicated that the tT (thawing temperature) and the s (strains) were significant (P<0.05) for all strains, which means that thawing temperature did influence recovery sample and that after being frozen, there were differences in the way particular recovered strains (Table 4).
Table 4. Analysis of variance for a Blocks Factorial Design at a=0.05 level, using the data obtained of the recovered samples of all strains and treatments, after the 6th day of incubation at 27 ± 1ºC.
S.V.= Source of variation; S.C.= Sum of squares; D.F.= Degrees of freedom; M.S.= Mean squares; F= F ratio; P= Probability; ct= contact time; tt =thawing time; tT= thawing temperature.
* Factors that produce differences.
As mentioned earlier, the strain that showed the highest recovery rate with all treatments was IE-111. Strain IE-4, however, had a faster recovery rate on average, with 19 samples by the second day at 30ºC. This contrasts with strain IE-140, which, at the same temperature, had an average recovery rate of 6.6 samples. Said temperature is that indicated by Chang and Miles (1) for the thawing of samples, although the most commonly used is 37-38º C with times that vary from a few seconds to more than 10 minutes (10, 11, 15, 18).
The variations observed are due to innate properties in the strains themselves, according to Hwang and San Antonio (7) and Hwang et al. (6). These characteristics are influenced either by the way the material is managed during preparation or by unavoidable variations in the composition of material, in this case the sorghum used as spawn, or in the way the mycelia are handled during evaluation (8).
The present study has demonstrated that contact and thawing times have not significative influence in the mycelium recovery, while strain and thawing temperature did make a difference: in general, an increase in the latter yielded less samples. Furthermore, the liquid nitrogen storage method used in this study, in which sorghum grains are the means of support for the micelium and without prefreezing of the samples, is adequate for the preservation of strains of the genus Pleurotus.
ACKNOWLEDGMENTS The authors are grateful to Dr. Gastón Guzmán for his support during this research. The study was financed by CONACyT (1810-N9211).
Estudo da viabilidade de cepas de Pleurotus spp. após congelamento em nitrogênio líquido
A viabilidade de 6 cepas de fungos do gênero Pleurotus (2 de P. djamor var. djamor, 1 de P. ostreatus var. ostreatus, 2 de P. ostreatus var. columbinus e 1 de P. pulmonarius), foram avaliadas após congelamento em nitrogênio líquido (-196ºC). Foram estudados os efeitos do tempo de contato dos micélios com o criopreservativo (glicerol), antes do congelamento (por 1, 2 e 3 h), e o efeito do descongelamento sobre os mesmos, em diferentes tempos (5, 10 e 15 min) e temperaturas (30, 45 e 60ºC). Os resultados mostraram marcada tendência a uma maior recuperação dos micélios quando as amostras eram descongeladas a 30ºC, sem aparente influência dos tempos de contato com o criopreservativo e de descongelamento. A 60ºC não se observou nenhuma recuperação.
Palavras-chave: Pleurotus, viabilidade, nitrogênio líquido, cogumelos.
REFERENCES 1. Chang, S.T.; Miles, P.G. Edible mushroom and their cultivation. CRC Press, Boca Raton, 1989. [ Links ]
2. Elliott, T.J.; Challen, M.P. The storage of mushroom strains in liquid nitrogen.The Glasshouse Crops Res. Inst. Ann. Rep. 194-204, 1979. [ Links ]
3. García-Hinojosa, C.A.; Martínez-Cruz, J. Estudio comparativo de la eficiencia de los métodos de conservación a corto y largo plazo aplicados a hongos filamentosos. IV Congreso Nacional de Micología, Tlaxcala, 1991, p.130. [ Links ]
4. Hwang, S.W. Effects of ultra-low temperature on the viability of selected fungus strain. Mycologia 52: 227-229, 1960. [ Links ]
5. Hwang, S.W. Investigation of ultra-low temperature for fungal cultures. I. An evaluation of liquid nitrogen storage for preservation of selected fungal cultures. Mycologia 60: 613-621, 1968. [ Links ]
6. Hwang, S.W.; Kwolek, W.F.; Haynes, W.C. Investigation of ultra low temperature for fungal cultures. III. Viability and growth rate of mycelial cultures following cryogenic storage. Mycologia 68: 377-387, 1976. [ Links ]
7. Hwang, S.W.; San Antonio, J.P. Stability of spawn stock of the cultivated mushroom after 26 months liquid nitrogen refrigeration (-160ºC to -196ºC). Mushroom Sci. 8: 35-42, 1972. [ Links ]
8. Jodon, M.H.; Royse, D.J.; Jong, S.C. Productivity of Agaricus brunnescens stock cultures following 5-, 7-, and 10-year storage periods in liquid nitrogen. Criobiology 19: 602-606, 1982. [ Links ]
9. Jong, S.C. Conservation of the cultures. In: Chang, S.T.; Hayes, W.A.(eds). The biology and cultivation of edible mushrooms. Academic Press, New York, 1978, p. 119-133. [ Links ]
10. Maekawa, N.; Fukuda, M.; Arita, I.; Komatsu, M. Cryopreservation of edible basidiomycetous fungi in liquid nitrogen. Rep. Tottori Mycol. Inst. 26: 15-28, 1988. [ Links ]
11. Maekawa, N.; Fukuda, M.; Arita, T.; Komatsu, M. Effects of liquid-nitrogen cryopreservation on stock cultures of three cultivated basidiomycetous fungi. Rept. Tottori Mycol. Inst. 28: 227-232, 1990. [ Links ]
12. Mata, G.; Salmones, D.; Pérez, R.; Guzmán, G. Behavior of some strains of the genus Pleurotus after different procedures for freezing in liquid nitrogen. Rev. Microbiol. 25: 192-200, 1994. [ Links ]
13. Morris, G.J.; Smith, D.; Coulson, G.E. A comparative study of the changes in the morphology of hyphae during freezing and viability upon thawing for twenty species of fungi. J. Gen. Microbiol. 134: 2897-2906, 1988. [ Links ]
14. Ortega-Cerda, P.M. Evaluación de la viabilidad de dos cepas del género Lentinus (Fungi: Basidiomycetes) en nitrógeno líquido. Xalapa, 1996, 77 p.(Licentiate Thesis. Universidad Veracruzana. Mexico). [ Links ]
15. San Antonio, J.P.; Hwang, S.C. Liquid nitrogen preservation of Agaricus bisporus (Lange)Sing. spawn stocks. Mush. J. 120: 410-419, 1982. [ Links ]
16. Smith, D. Tolerance to freezing and thawing. In: Jennings, D.H. (ed.). Stress Tolerance in Fungi. Marcel Dekker. New York, 1993, p. 145-171. [ Links ]
17. Smith, D. Culture Collections. In: Chang, S.T.; Buswell, A.; Miles,P.G. (eds). Genetics and Breeding of Edible Mushrooms. Gordon and Breach Science Publishers, Amsterdam, 1993, p.15-33. [ Links ]
18. Suman, B.C.; Jandaik, G.L. Preservation of cultures Agaricus bisporus (Lange)Sing. in liquid nitrogen and its effect on yield and characters of fruiting bodies. Indian J. Mycol. Pl. Pathol. 21: 34-37, 1991.[ Links ]