Mycelial growth of two Lentinula edodes strains in culture media prepared with sawdust extracts from seven eucalyptus species and three eucalyptus clones

The in vitro mycelial growth of Lentinula edodes strains LE-95/01 and LE96/18 were evaluated in solid culture media prepared with sawdust extracts from seven eucalyptus species (E. saligna, E. grandis, E. urophylla, E. pellita, E. paniculata, E. citriodora, and E. camaldulensis) and three eucalyptus clones (E. grandis × E. urophylla hybrids). Evaluations were made every 48 hours by means of colony diameter measurements (mean of four transversely-oriented measurements), during ten days of incubation in the dark at 25oC ±1°C. The experimental design consisted of randomized blocks, and treatment means were compared by Tukey test. The culture medium prepared from E. citriodora sawdust extract was the most promising to grow L. edodes strains LE-96/18 and LE-95/01. L. edodes strain LE-96/18 presented the fastest mycelial growth after incubation for ten days, regardless of sawdust extract type used in the culture medium.

In its metabolism, the fungus secretes exoenzymes that degrade compounds to obtain carbon, nitrogen, sulfur, and other nutrients (Donini et al., 2005).The in vitro cultivation of this mushroom seeks to elucidate the optimal growth conditions for the fungus with regard to culture media, temperatures, and incubation times (Hatvani, 2001).This knowledge is a pre-requisite for commercial cultivation in formulated substrate.
During a given time period, the fungus mycelial growth can be represented as a typical sigmoidal curve, including several stages with distinctive physiological properties (Montini et al., 2006).Mycelial growth can be measured in different ways, such as through radial growth, vigor, growth velocity, and mycelial mass (Marino, 1997).Under experimental conditions, the use of solid culture medium to evaluate fungal growth is considered adequate, since in nature fungi normally develop on solid substrates, such as plant and animal residues, or in the soil (Griffin, 1994).
Acta Sci.Agron. Maringá, v. 30, n. 3, p. 333-337, 2008 In Brazil, the most frequently used substrate for L. edodes commercial cultivation consists of Eucalyptus spp.logs or sawdust.Hence, based on in vitro L. edodes mycelial growth evaluation in culture media prepared with sawdust extract from different species and clones of eucalyptus, it is possible to determine the most suitable type of eucalyptus for development, by simulating the natural cultivation conditions of the fungus.
Therefore, the objective of this work was to evaluate the in vitro mycelial growth of L. edodes strains LE-95/01 and LE-96/18 in solid culture media prepared with sawdust extract from seven eucalyptus species (E.saligna, E. grandis, E. urophylla, E. pellita, E. paniculata, E. citriodora, and E. camaldulensis) and three eucalyptus clones (E. grandis × E. urophylla hybrids).

Material and methods
The The culture media were prepared with sawdust extracts from the seven eucalyptus species and three eucalyptus clones previously cited.In order to prepare each extract, we used 230 g sawdust, 30 g soybean meal, and 10 g lime.The substrate resulting from this mixture was moistened at 60-70%, arranged in HDPE (high density polyethylene) plastic bags, and autoclaved twice at 121°C for one and a half hour each time, with a 24-hour interval between times.Then, 40 g of each substrate were added to 500 mL distilled water and then fully boiled for five minutes.The extract thus obtained was filtered through a fine mesh screen and through cotton fabric and then placed in Duran ® flasks, and the volume was completed to 500 mL with distilled water.The extract was then autoclaved at 121ºC for 30 minutes; after 24 hours, 8 g agar were added and the extract was autoclaved again for 30 minutes.The medium thus prepared was poured into Petri dishes.

Inoculation, colonization Inoculation, colonization Inoculation, colonization Inoculation, colonization and variable analyzed and variable analyzed and variable analyzed and variable analyzed
Secondary-parent disks (4 mm in diameter) of strains LE-95/01 and LE-96/18 (grown in Petri dishes containing culture medium prepared with Eucalyptus spp.sawdust extract) were inoculated in Petri dishes containing the media previously prepared, comprising the treatments of this experiment.The dishes were distributed in randomized blocks and maintained in an incubator at 25ºC ± 1°C.During this period, colony diameter measurements were made every 48 hours (mean of four transversely-oriented measurements) until the fungus colony in one of the treatments almost reached the edges of the Petri dish, which occurred after ten days of incubation.

Experimental design and statistical analysis Experimental design and statistical analysis Experimental design and statistical analysis Experimental design and statistical analysis
A randomized block experimental design was adopted, organized in a 2 × 10 factorial arrangement, whose treatments corresponded to combinations between the two L. edodes strains and seven eucalyptus species and three eucalyptus Acta Sci.Agron.Maringá, v. 30, n. 3, p. 333-337, 2008 clones, totaling 20 treatments.Each treatment consisted of 10 replicates, each corresponding to a Petri dish, totaling 200 plates.The data were submitted to analysis of variance and the means were compared by Tukey test (5%) (Snedcor and Cochran, 1967).

Results and discussion
The mycelial growth results for L. edodes strains after cultivation for ten days in the culture media are presented in Table 1.There were significant differences for the interaction between culture media and L. edodes strains.Strain LE-96/18 produced the greatest mycelial growth in all culture media (Table 1).Donini et al. (2005) evaluated the mycelial growth velocity of Pleurotus spp.strains on different substrates and also observed significant differences for the interaction between strains, substrates, and days of evaluation.The culture medium prepared from E. citriodora sawdust extract provided the fastest mycelial growth after ten days of incubation at 25 ± 1ºC for both strains studied (Table 1).The influence of physical and chemical characteristics of the substrates on the mycelial growth, productivity, and biological efficiency of L. edodes strains has been emphasized in recent studies (Dias et al., 2003, Donini et al., 2006;Kopytowski Filho, 2006;Özçelik and Peksen, 2006).Menin et al. (2000) evaluated the effect of Eucalyptus citriodora sawdust essential, oil and crude extract on L. edodes mycelial growth and observed that the culture medium prepared with E. citriodora sawdust extract provided faster mycelial growth than PDA medium (potatodextrose-agar).
The poorest mycelial growth performances were observed in the culture media from sawdust extracts of clones 23 and 25 and of the species E. pellita strain LE-95/01, and in the culture media from sawdust extract of E. grandis and E. urophylla for strain LE-96/18. Tarui (1997) and Teixeira (2000) reported that L. edodes cultivation in sawdust from different eucalyptus species also conditioned mycelium development differently.
After 2 days of incubation, strain LE-95/01 produced significantly higher mycelial growth than strain LE-96/18 in the culture medium prepared with E. grandis sawdust.However, after 4 and 6 days from inoculation, strains LE-96/18 and LE-95/01 had similar growth periods.Finally, at 8 and 10 days after inoculation, growth of strain LE-96/18 was significantly higher than in strain LE-95/01 (Figure 1).In the culture medium prepared with E. urophylla sawdust extract, after 2 and 4 days from inoculation, strains LE-96/18 and LE-95/01 had similar mycelial growths.From then on, at 6, 8, and 10 days after inoculation, strain LE-96/18 had greater mycelial growth (Figure 1).Teixeira (2000) compared the mycelial growth velocity of ten L. edodes strains in sawdust from three eucalyptus species and reported that the growth velocities for L. edodes strains were not different in the eucalyptus species E. grandis and E. urophylla, except for strain JAB P. Acta Sci.Agron. Maringá, v. 30, n. 3, p. 333-337, 2008

Conclusion Conclusion Conclusion Conclusion
The culture medium prepared from E. citriodora sawdust extract was the most promising to grow L. edodes strains LE-96/18 and LE-95/01.
Strain LE-96/18 presented the fastest mycelial growth after incubation for ten days, regardless of sawdust extract type used in the culture medium.
Extracts used in culture media preparation Extracts used in culture media preparation Extracts used in culture media preparation Extracts used in culture media preparationThe extracts used to prepare the culture media consisted of sawdust obtained from eucalyptus species E. saligna, E. grandis, E. urophylla, E. pellita, E. paniculata, E. citriodora, and E. camaldulensis, and from clones 23, 24, and 25.The clones are E. grandis × E. urophylla hybrids and were developed by the following companies: VCP, International Paper, and Ripasa.All eucalyptus material was obtained from Estação Experimental de Ciências Florestais (Experimental Station), Esalq -USP, located in the municipality of Itatinga, São Paulo State, whose stands were planted in an area with the same soil type and same planting date (January/1997).
(1) Means followed by different uppercase letters in each row and lowercase letters in each column are different (Tukey, 1%).