Acta Sci. Agron. Maringá, v. 30, n. 3, p. 333-337, 2008 Mycelial growth of two Mycelial growth of two Mycelial growth of two Mycelial growth of two Lentinula edodesLentinula edodesLentinula edodesLentinula edodes strains in culture media strains in culture media strains in culture media strains in culture media prepared with sawdust extracts from seven eucalyptus species and prepared with sawdust extracts from seven eucalyptus species and prepared with sawdust extracts from seven eucalyptus species and prepared with sawdust extracts from seven eucalyptus species and three eucalyptus clonesthree eucalyptus clonesthree eucalyptus clonesthree eucalyptus clones Meire Cristina Nogueira de Andrade1*, Juliano Hojah da Silva2, Marli Teixeira de Almeida Minhoni2 and Diego Cunha Zied2 1Instituto Nacional de Pesquisas da Amazônia, Av. André Araújo, 2936, Cx. Postal 478, 69060-001, Aleixo, Manaus, Brazil. 2Departamento de Produção Vegetal, Faculdade de Ciências Agrárias, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil. *Author for correspondence. E-mail: meire@inpa.gov.br ABSTRACT. The in vitro mycelial growth of Lentinula edodes strains LE-95/01 and LE- 96/18 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. Key words: shiitake, in vitro, mushroom. RESUMO. Crescimento micelial de duas linhagens de Lentinula edodes em meios de cultura à base de extrato de serragem de sete espécies e três clones de eucalipto. Avaliou-se o crescimento micelial in vitro das linhagens LE-95/01 e LE-96/18 de Lentinula edodes, em meios de cultura sólidos à base de extrato de serragem de sete espécies (Eucalyptus saligna, E. grandis, E. urophylla, E. pellita, E. paniculata, E. citriodora e E. camaldulensis) e três clones (híbridos de E. grandis x E. urophylla) de eucalipto. As avaliações foram realizadas por meio de medições do diâmetro das colônias (média de quatro medidas diametralmente opostas), a cada 48 horas, durante dez dias de incubação, no escuro a 25ºC ±1°C. O delineamento experimental foi o de blocos casualizados, com uso do teste de Tukey para a comparação das médias. O meio de cultura à base de extrato de serragem de E. citriodora foi o mais promissor no crescimento das linhagens LE-96/18 e LE-95/01 de L. edodes. A linhagem LE-96/18 de L. edodes foi a que apresentou o crescimento micelial mais rápido após dez dias de incubação, independentemente do tipo de extrato de serragem utilizado no meio de cultura. Palavras-chave: shiitake, in vitro, cogumelo. IntroductionIntroductionIntroductionIntroduction The mushroom Lentinula edodes (Berk.) Pegler, popularly known as Shiitake, is a lignolytic, aerobic basidiomycete (Chen, 2005; Minhoni et al., 2007). 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). 334 Andrade et al. 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 methodsMaterial and methodsMaterial and methodsMaterial and methods The experiment was conducted at the Microbiology Laboratory of the Departamento de Produção Vegetal (Plant Production Department), Faculdade de Ciências Agronômicas (College of Agronomic Sciences), Unesp, Botucatu, São Paulo State, Brazil. Lentinula edodes strainsLentinula edodes strainsLentinula edodes strainsLentinula edodes strains Two Lentinula edodes strains, LE-95/01 and LE-96/18 were used. The strains were stored at the Microbiology Laboratory’s Fungus Culture Collection (DPV - FCA/Unesp). Strain LE-95/01 was isolated from mushrooms of a producer from Londrina, Paraná State, in 1995. Strain LE- 96/18 was isolated from mushrooms of producers from Cooperativa dos Produtores de Cogumelos de Rio Claro (COPCO) in Rio Claro, São Paulo State. Extracts used in culture media preparationExtracts used in culture media preparationExtracts used in culture media preparationExtracts used in culture media preparation The 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). CulCulCulCulture media preparationture media preparationture media preparationture media preparation 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, colonizationInoculation, colonizationInoculation, colonizationInoculation, 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 analysisExperimental design and statistical analysisExperimental design and statistical analysisExperimental 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 Mycelial growth of Lentinula edodes strains 335 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 discussionResults and discussionResults and discussionResults 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. Table 1. In vitro mycelial growth (mm) 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), after a ten-day development period at 25ºC ± 1°C. L. edodes strains Sawdust Extract LE-95/01 LE-96/18 E. saligna 50.33 B b(1) 58.10 A bc E. grandis 48.78 B bcd 53.43 A e Clone 23 46.85 B ef 56.48 A cd Clone 25 45.13 B f 58.23 A bc Clone 24 47.30 B de 59.08 A b E. urophylla 47.33 B de 53.45 A e E. pellita 46.63 B ef 55.75 A d E paniculata 49.33 B bc 57.30 A bcd E. citriodora 53.23 B a 61.40 A a E. camaldulensis 47.58 B cde 55.48 A d (1) Means followed by different uppercase letters in each row and lowercase letters in each column are different (Tukey, 1%). 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 (potato- dextrose-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 for 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. Figure 1 displays the mycelial growth of both L. edodes strains (LE-96/18 and LE-95/01), inside each culture medium, during ten days of in vitro development. Strain LE-96/18 had significantly higher growth in eight culture media prepared with sawdust extracts from E. saligna, clone 23, clone 25, clone 24, E. pellita, E. paniculata, E. citriodora and E. camaldulensis, than strain LE-95/01 during the entire evaluation period (Figure 1). These mycelial growth differences between L. edodes strains have already been reported by many researchers (Andrade et al., 2007; Andrade and Graciolli, 2005; Boyle, 1998; Maki et al., 2001; Silva et al., 2005). 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. 336 Andrade et al. Acta Sci. Agron. Maringá, v. 30, n. 3, p. 333-337, 2008 ---y = - 3.470 + 5.360x (R2 = 0.998**) y = - 4.378 + 6.181x (R2 = 0.998**) ---- y = - 1.853 + 5.049x (R2 = 0.998**) y = - 4.208 + 5.694x (R2 = 0.997**) y = - 5.318 + 6.094x (R2 = 0.995**) ---- y = - 6.337 + 5.086x (R2 = 0.973*) y = - 5.675 + 6.287x (R2 = 0.995**) ---- y = - 6.452 + 4.884x (R2 = 0.962*) ---- y = - 6.370 + 5.132x (R2 = 0.971*) y = - 4.618 + 6.246x (R2 = 0.995**) y = - 3.495 + 5.630x (R2 = 0.998**) ---- y = - 1.397 + 4.871x (R2 = 0.999**) y = - 4.688 + 5.969x (R2 = 0.998**) ---- y = - 6.225 + 5.053x (R2 = 0.972*) y = - 4.790 + 6.143x (R2 = 0.998**) ---- y = - 6.475 + 5.370x (R2 = 0.978**) y = - 5.808 + 6.606x (R2 = 0.996**) ---- y = - 4.160 + 5.708x (R2 = 0.998**) y = - 3.325 + 5.815x (R2 = 0.998**) ---- y = - 2.753 + 5.009x (R2 = 0.998**) 60 40 20 20 40 60 20 40 60 20 40 60 40 60 (a) (b) (c) (d) (e) (f) (g) (h) (j) Days after inoculation 2 4 6 8 10 10 8 6 4 2 20 M yc el ia l g ro w th , m m (i) Figure 1. Mycelial growth of two L. edodes strains, LE-96/18 (￭) and LE-95/01 (♦), in culture media prepared with sawdust extracts from (a) E. saligna, (b) E. grandis, (c) Clone(1) 23, (d) Clone 25, (e) Clone 24, (f) E. urophylla, (g) E. pellita, (h) E. paniculata, (i) E. citriodora, (j) E. camaldulensis, at 25ºC. * and ** are significant at 5 and 1%, respectively. (1)Clones 23, 24, and 25 are E. grandis × E. urophylla hybrids. M yc el ia l g ro w th ( m m ) —ŷ=4.378+6.181x(R2=0.998**) ---ŷ=3.470+5.360x(R2=0.998**) —ŷ=4.208+5.694x(R2=0.997**) ---ŷ=1.853+5.049x(R2=0.998**) —ŷ=5.318+6.094x(R2=0.995**) ---ŷ=6.337+5.086x(R2=0.973**) —ŷ=5.675+6.287x(R2=0.995**) ---ŷ=6.452+4.884x(R2=0.962**) —ŷ=4.618+6.246x(R2=0.995**) ---ŷ=6.370+5.132x(R2=0.971**) —ŷ=3.495+5.630x(R2=0.998**) ---ŷ=1.397+4.871x(R2=0.999**) —ŷ=4.688+5.969x(R2=0.998**) ---ŷ=6.225+5.053x(R2=0.972**) —ŷ=4.790+6.143x(R2=0.998**) ---ŷ=6.475+5.370x(R2=0.978**) —ŷ=5.808+6.606x(R2=0.996**) ---ŷ=4.160+5.708x(R2=0.998**) —ŷ=3.325+5.815x(R2=0.998**) ---ŷ=2.753+5.009x(R2=0.998**) Mycelial growth of Lentinula edodes strains 337 Acta Sci. Agron. Maringá, v. 30, n. 3, p. 333-337, 2008 ConclusionConclusionConclusionConclusion 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. 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Accepted on August 03, 2007.