The common split-gilled mushroom, Schizophyllum commune is found throughout the world on woody plants. This study was initiated to evaluate conditions for favorable vegetative growth and to determine molecular phylogenetic relationship in twelve different strains of S. commune. A suitable temperature for mycelial growth was obtained at 30℃. This mushroom grew well in acidic conditions and pH 5 was the most favorable. Hamada, glucose peptone, Hennerberg, potato dextrose agar and yeast malt extract were favorable media for growing mycelia, while Lilly and glucose tryptone were unfavorable. Dextrin was the best and lactose was the less effective carbon source. The most suitable nitrogen sources were calcium nitrate, glycine, and potassium nitrate, whereas ammonium phosphate and histidine were the least effective for the mycelial growth of S. commune. The genetic diversity of each strain was investigated in order to identify them. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The size of the ITS1 and ITS2 regions of rDNA from the different strains varied from 129 to 143 bp and 241 to 243 bp, respectively. The sequence of ITS1 was more variable than that of ITS2, while the 5.8S sequences were identical. A phylogenetic tree of the ITS region sequences indicated that the selected strains were classified into three clusters. The reciprocal homologies of the ITS region sequences ranged from 99 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) with 20 arbitrary primers. Twelve primers efficiently amplified the genomic DNA. The number of amplified bands varied depending on the primers used or the strains tested. The average number of polymorphic bands observed per primer was 4.5. The size of polymorphic fragments was obtained in the range of 0.2 to 2.3 kb. These results indicate that the RAPD technique is well suited for detecting the genetic diversity in the S. commune strains tested.
The common split-gilled mushroom, Schizophyllum commune is found throughout the world on woody plants. This study was initiated to evaluate conditions for favorable vegetative growth and to determine molecular phylogenetic relationship in twelve different strains of S. commune. A suitable temperature for mycelial growth was obtained at 30℃. This mushroom grew well in acidic conditions and pH 5 was the most favorable. Hamada, glucose peptone, Hennerberg, potato dextrose agar and yeast malt extract were favorable media for growing mycelia, while Lilly and glucose tryptone were unfavorable. Dextrin was the best and lactose was the less effective carbon source. The most suitable nitrogen sources were calcium nitrate, glycine, and potassium nitrate, whereas ammonium phosphate and histidine were the least effective for the mycelial growth of S. commune. The genetic diversity of each strain was investigated in order to identify them. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The size of the ITS1 and ITS2 regions of rDNA from the different strains varied from 129 to 143 bp and 241 to 243 bp, respectively. The sequence of ITS1 was more variable than that of ITS2, while the 5.8S sequences were identical. A phylogenetic tree of the ITS region sequences indicated that the selected strains were classified into three clusters. The reciprocal homologies of the ITS region sequences ranged from 99 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) with 20 arbitrary primers. Twelve primers efficiently amplified the genomic DNA. The number of amplified bands varied depending on the primers used or the strains tested. The average number of polymorphic bands observed per primer was 4.5. The size of polymorphic fragments was obtained in the range of 0.2 to 2.3 kb. These results indicate that the RAPD technique is well suited for detecting the genetic diversity in the S. commune strains tested.
Schizophyllum commune one of the most common mushrooms, is widely distributed worldwide and usually grows abundantly during the rainy season. This species frequently appears on dead wood and is a known wood decomposer of over 150 genera of flowering plants [1]. In addition it is a well documented wound parasite of trees and human pathogen of minor but increasing importance [2].In S. commune, single meiospores germinate to produce haploid, monokaryotic mycelia. Two complex mating-type factors control sexual compatibility in the monokaryons and regulate the maintenance of the dikaryotic state [3]. Fusion of sexually compatible haploid monokaryotic mycelia results in the formation of the dikaryotic mycelium. The hyphae of S. commune dikaryons develop clamp connections at each septum, while the hyphae of monokaryons do not. The dikaryon is the predominant vegetative structure in S. commune. Under appropriate conditions, the dikaryon produces the fruiting bodies within which meiosis occurs. The monokaryotic and dikaryotic mycelia are capable of indefinite growth, allowing for the maintenance and duplication of the genotype of each ploidy state [4]. It is generally recognized that growing mycelia is a defined medium is a rapid and alternative method to obtain fungal biomass for further use [5].S. commune is a model organism for transmission genetics, developmental biology, and population genetics and has also been used in several different quantitative genetic studies on growth rate [6]. To identify S. commune strains and other traits of interest, assessment of genetic and phenotypic diversity is necessary. Recent molecular phylogenetic studies have demonstrated that the internal transcribed spacer (ITS) region of genomic DNA is very useful for assessing phylogenetic relationships at lower taxonomic levels. The ITS of rDNA is considered as a variable region among the species and even among the strains [7]. Random amplification of polymorphic DNA (RAPD) markers has been widely used for assessing genetic diversity, genome mapping, and molecular diagnostics of many fungal species [8]. RAPD was particularly successful when used to verify mushroom strains from various hosts with a wide range of geographical origins [9]. The present study was aimed to assess the genetic diversity and physicochemical requirements for the vegetative growth in different strains of S. commune.
Materials and Methods
Mushroom strains
Twelve wild strains of S. commune were collected from the different ecological regions of China (IUM-1763 and IUM-1768) and Korea (IUM-0137, IUM-0157, IUM-0202, IUM-0395, IUM-0548, IUM-2324, IUM-2650, IUM-2659, IUM-3353, and IUM-3566). Pure cultures were deposited in the Culture Collection and DNA Bank of Mushrooms (CCDBM) and acquired the accession number of Incheon University Mushroom (IUM). Seven strains of S. commune, AF-249389, AF-249390, AY-573544, AY-636062, EF-488416, FJ-372682, and FJ-372683 were also used as control strains for the phylogenetic comparison with our selected IUM strains. Sequencing data of the control strains were collected from the National Center for Biotechnology Information (NCBI) gene bank data base.
Temperature and pH
Five different temperatures, 15, 20, 25, 30, and 35℃, were used to find the optimum temperature for the mycelial growth of S. commune. A 5 mm diameter agar plug was removed from 10 day old cultures and placed in the center of a potato dextrose agar (PDA) plate. The medium was adjusted to pH 6 and incubated for 10 days at 15, 20, 25, 30, and 35℃. To determine the optimum pH, the medium was adjusted to pH 5, 6, 7, 8, and 9 by adding 1 N NaOH or HCl before autoclaving. Samples were incubated for 10 days at 25℃. The measurement of mycelial growth was performed according to the described method [10].
Culture media
Ten different types of culture media, Czapek dox, glucose peptone, glucose tryptone, Hamada, Hennerberg, Hoppkins, Lilly, mushroom complete, potato dextrose agar, and yeast malt extract, were used to investigate the mycelial growth of S. commune. The different types of culture media were prepared according to the described method [11]. The media were adjusted to pH 6 before being autoclaved.
Carbon and nitrogen sources
The experiments were performed on basal medium (0.05 g MgSO4, 0.46 g KH2PO4, 1.0 g K2HPO4, 120 µg thiamine-HCl, 20 g agar, and 1 litter of distilled water) supplemented with one of 10 carbon sources (dextrin, fructose, galactose, glucose, lactose, maltose, mannose, sorbitol, sucrose, and xylose) and ten nitrogen sources (alanine, ammonium acetate, ammonium phosphate, arginine, calcium nitrate, glycine, histidine, methionine, potassium nitrate, and urea). To evaluate the most favorable carbon and nitrogen sources for mycelial growth, each carbon source with 5 g of peptone was added to the basal medium separately at the concentration of 0.1M and mixed thoroughly. Each nitrogen source along with 20 g of glucose was supplemented to the basal medium at the concentration of 0.02M [12]. In both cases, the basal medium was adjusted to pH 6 before autoclaving.
DNA extraction
Genomic DNA was extracted according to the described procedure [13] with some modifications as follows. Fresh mycelia were collected from the 10 day old cultures grown on PDA medium and frozen with liquid nitrogen. Frozen mycelia were ground with a sterilized mortar-pestle and kept in 1.5 mL micro-tubes. 500 µL extraction buffer (equal volumes of 50 mM Tris-HCl [pH 7.5], 50 mM EDTA [pH 8], and 1% sarkosyl) was added to each of the micro-tubes and incubated at 65℃ for 30 min. After incubation, the same volume of PCI (25 mL phenol: 24 mL chloroform: 1 mL isoamyl-alcohol) was added, and the samples were vortexed and then centrifuged at 4℃, 10 minutes, 12,000 rpm. Afterwards, the upper phase was transferred to a 1.5 mL micro-tube, 1,000 µL of 99.9% alcohol was added and then it was centrifuged at 4℃, 5 min, 12,000 rpm. Subsequently, the supernatant was removed, 500 µL of 70% alcohol was added to the precipitated DNA, and then it was vortexed and centrifuged at 4℃, 5 min, 12,000 rpm. Again the supernatant was the removed and the residual alcohol evaporated. The DNA pellet was resuspended in 500 µL of sterilized distilled water. The DNA concentration was measured using spectrophotometer [14].
Amplification of the ITS region and sequence analysis
The ITS region of the rDNA in selected strains of S. commune was amplified by polymerase chain reaction (PCR) using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). Amplification reactions were performed in a total volume of 20 µL containing 2 µL 10 × PCR buffer, 1.6 µL dNTP, 0.5 µL of each primer, 0.2 µL Taq polymerase, 1 µL of genomic DNA, and 14.2 µL of sterilized distilled water. The PCR was performed using a thermal cycler (Veriti thermal cycler, Applied Biosystems, Foster City, CA, USA) with an initial denaturation stage of 5 min at 95℃, followed by 35 cycles of denaturation for 30 sec at 94℃, annealing for 30 sec at 52℃, extension for 1 min at 72℃, and a final 10 min extension at 72℃. Amplification products were analyzed by gel electrophoreses on a 1.5% agarose gel with a 1 kb DNA ladder as a marker. ITS sequences were aligned for phylogenetic analysis using the program Cluster W [15]. The phylogenetic tree was constructed by neighbor-joining method using CLC free Workbench program. Bootstrap analysis was repeated 1,000 times to examine the reliability of the interior branches and the validity of the trees obtained [16, 17].
RAPD analysis
Genomic DNA was amplified by the RAPD technique [8] in which 20 arbitrary 10-base oligonucleotide primers (Operon Technologies Inc., Alameda, CA, USA) were used to produce amplified fragments. The primer sequences are listed in Table 1. RAPD-PCR reaction was performed using a thermal cycler with an initial denaturation stage of 5 min at 94℃, followed by 35 cycles of denaturation for 1 min at 94℃, annealing for 1min at 36℃, extension for 2 min at 72℃, and a final 7 min extension at 72℃. RAPD products were run on a 1.4% agarose gel in 1 × Tris-acetate-EDTA buffer for 1.15 hr at 100 V along with a 1 kb DNA ladder as a size marker. The gel was then stained with an ethidium bromide (EtBr) solution (0.5% µg/mL). The stained gels were visualized and photographed using a UV transilluminator. RAPD bands were recorded as present (1) or absent (0) to generate the data matrix. The similarity coefficients (S) were calculated between isolates across bands for all primers using the formula S = 2Nxy/(Nx + Ny), where Nx and Ny are the number of bands shared by the two strains [18].
Table 1
Random amplification of polymorphic DNA primers used in this study
Results and Discussion
Effect of temperature and pH
To determine the most suitable temperature for mycelial growth of S. commune, a range from 15~35℃ was considered. The highest average mycelial growth was found in 30℃, followed by 35, 25, 20, and 15℃. Among the selected strains, maximum mycelial growth (87.0 mm) was recorded in IUM-0137, 0157, 0395, 1763, and 3353 at 30℃ (Table 2). These findings are comparable to the previous studies on Macrolepiota procera [19], which was shown to have optimum mycelial growth at 30℃.
Table 2
Effect of temperature on the mycelial growth in different strains of Schizophyllum commune
aMean of 3 replications.
pH values ranging from 5~9 were considered to determine the most favorable culture conditions for the mycelial growth of S. commune. The highest and lowest level of radial growth of mycelium was found at pH 5 and 9, respectively (Table 3). Mycelial growth decreased with an increase in pH values, thus acidic pH was better for the mycelial growth of S. commune. This result agrees with the data from the study of Pholiota adiposa, as it grew well in acidic conditions and pH 5 was the most favorable for vegetative growth [20].
Table 3
Effect of pH on the mycelial growth in different strains of Schizophyllum commune
aMean of 3 replications.
Effect of culture media
Ten different types of culture media were used to determine the optimum medium for mycelial growth of the selected S. commune strains. Based on mycelial growth, Hamada, glucose peptone, Hennerberg, potato dextrose agar and yeast malt extract were the most favorable, while Lilly and glucose tryptone were the least favorable for the vegetative growth of S. commune. The highest and lowest mycelial growth was recorded in Hamada and Lilly, respectively (Table 4). This result is analogous to that of P. sinclairii and P. fumosoroseus [21], in which mycelial growth was more favorable on Hamada medium.
Table 4
Effect of culture media on the mycelial growth in different strains of Schizophyllum commune
Ten different carbon sources were used to monitor mycelial growth. Dextrin was found to be the best for mycelial growth of S. commune, followed by fructose, sucrose, and glucose. The lowest growth of mycelium was obtained in lactose (Table 5). This result indicates that mannitol, sorbitol, glucose, and fructose were the best utilized carbon sources having a significant effect on the vegetative growth of S. commune [22].
Table 5
Effect of carbon sources on the mycelial growth in different strains of Schizophyllum commune
Ten different nitrogen sources were assayed to determine their effects on the vegetative growth of S. commune. Among the nitrogen sources, calcium nitrate was found to be the best, followed by glycine and potassium nitrate. The lowest growth was recorded in ammonium phosphate and histidine (Table 6). The most favorable and unfavorable nitrogen sources for the vegetative growth of M. procera were glycine and histidine, which is partially similar to our findings [19]. In general, organic nitrogen sources are more effective than inorganic nitrogen sources.
Table 6
Effect of nitrogen sources on the mycelial growth in different strains of Schizophyllum commune
To investigate the genetic diversity of S. commune, twelve wild strains were collected from various ecological regions of China and Korea at different times. The ITS region was amplified using ITS1 and ITS4 primers and then sequenced. Our results revealed that the length of the sequences among the selected strains ranged from 539 to 561 bp. The size of the ITS1 and ITS2 regions varied among the strains from 129 to 143 bp and 241 to 243 bp, respectively. The total G + C and A + T content of the ITS region varied from 46.99 to 47.68% and 52.32 to 53.01%, respectively (Table 7). Sequence analysis indicated that the 5.8S of rDNA sequence was identical (151 bp) for all of the strains tested. The size variation was caused by differences in nucleotide sequence, revealing that these strains are clearly distinguishable from each other based on the ecological distribution, substitution, and insertion or deletion polymorphisms of the base position [23].
Table 7
Nucleotide distribution, internal transcribed spacer (ITS)1, 5.8S, and ITS2 of rDNA sequences in twelve different strains of Schizophyllum commune
A, adenine; C, cytosine; G, guanine; T, thymine.
The phylogenetic tree based on the nucleotide sequence of the ITS region in twelve different strains of S. commune was obtained by the neighbor-joining method. Reciprocal homologies of the ITS region sequences ranged from 99 to 100%. The phylogenetic tree out separated into three groups (Fig. 1). The maximum difference was observed between IUM-2659 (Korea) and FJ-372682 (NCBI gene bank strain). IUM-0157 (Korea) and IUM-1763 (China) were similar to all NCBI gene bank strains tested and belongs to the same cluster. The ITS sequences are genetically constant or show little variation within species [24]. The genetic distance exhibited a high level of similarity with identical ITS sequences. The base sequences of the ITS region of rDNA were variable among the strains tested. The genetic variation within groups was greater than that observation between groups. The high genetic diversity detected within groups is probably due to an efficient gene flow and a high genetic compatibility within the strains tested [25].
Fig. 1
Phylogenetic tree in nineteen strains of Schizophyllum commune based on the nucleotide sequence of the internal transcribed spacer region using neighbor-joining method with 1,000 bootstrapping trails.
Twenty arbitrary 10 base oligonucleotide primers were used to amplify segments of DNA for selected IUM strains of S. commune. Twelve primers, OPA-01, 02, 03, 05, 08, 09, 10, 13, 15, 18, 19, and 20 were found to be efficient for amplifying the genomic DNA (Table 8). These primers showed significant band profiles on the strains tested and high possibilities for screening of each strain (Figs 2~4). RAPD-PCR generated distinct multiple products showing considerable variability among the strains tested. The number of amplified bands varied depending on the primers used or the strains tested. The average number of polymorphic bands observed per primer was 4.5. The size of these polymorphic fragments was obtained in the range from 0.2 to 2.7 kb. The DNA polymorphisms showed the same characteristics in the replication tests. Therefore, if same primers are used for screening DNA polymorphisms, it will be possible to distinguished genetically different strains of S. commune (Table 8). To maximize the specificity of the polymorphic patterns, a combined dendrogram was constructed using the RAPD-PCR amplified bands obtained from the twelve RAPD primers. Four putative groups among the 12 strains of S. commune were obtained by cluster analysis based on banding patterns and size of amplified products (Fig. 5). Among the twelve strains, 90~100% similarities were found between IUM-3353 (Korea) and IUM-3566 (Korea), IUM-0137 (Korea) and IUM-0157 (Korea), and IUM-0548 (Korea) and IUM-0763 (China), which belong to three different groups. In most of the cases, IUM-0202, IUM-2324, and IUM-0395 showed different band patterns compared to all of the other strains, which was collected from three different ecological regions of Korea. Genetic variability was observed among the tested strains of S. commune [11], which could be due to the adaptation of the strains to different environments [23]. Therefore, RAPD is a useful tool for clarifying the genetic relationships among strains.
Table 8
DNA bands in different strains of Schizophyllum commune by random amplification of polymorphic DNA assay on 10 base OPA primers
lane 1, IUM-0137; lane 2, IUM-0157; lane 3, IUM-0202; lane 4, IUM-0395; lane 5, IUM-0548; lane 6, IUM-1763; lane 7, IUM-1768; lane 8, IUM-2324; lane 9, IUM-2650; lane 10, IUM-2659; lane 11, IUM-3353; lane 12, IUM-3566.
Fig. 2
Random amplification of polymorphic DNA profiles in different strains of Schizophyllum commune with primer OPA-1. M, molecular size marker (1 kb DNA ladder); lane 1, IUM-0137; lane 2, IUM-0157; lane 3, IUM-0202; lane 4, IUM-0395; lane 5, IUM-0548; lane 6, IUM-1763; lane 7, IUM-1768; lane 8, IUM-2324; lane 9, IUM-2650; lane 10, IUM-2659; lane 11, IUM-3353; lane 12, IUM-3566.
Fig. 4
Random amplification of polymorphic DNA profiles in different strains of Schizophyllum commune with primer OPA-13. M, molecular size marker (1 kb DNA ladder); lane 1, IUM-0137; lane 2, IUM-0157; lane 3, IUM-0202; lane 4, IUM-0395; lane 5, IUM-0548; lane 6, IUM-1763; lane 7, IUM-1768; lane 8, IUM-2324; lane 9, IUM-2650; lane 10, IUM-2659; lane 11, IUM-3353; lane 12, IUM-3566.
Fig. 5
Dendrogram constructed based on the random amplification of polymorphic DNA markers of Schizophyllum commune strains determined by the average linkage cluster. 1, IUM-0137; 2, IUM-0157; 3, IUM-0202; 4, IUM-0395; 5, IUM-0548; 6, IUM-1763; 7, IUM-1768; 8, IUM-2324; 9, IUM-2650; 10, IUM-2659; 11, IUM-3353; 12, IUM-3566.
Authors: Sung Mi Shim; Yun Hee Oh; Kyung Rim Lee; Seong Hwan Kim; Kyung Hoan Im; Jung Wan Kim; U Youn Lee; Jae Ouk Shim; Mi Ja Shim; Min Woong Lee; Hyeon Su Ro; Hyun Sook Lee; Tae Soo Lee Journal: Mycobiology Date: 2005-03-31 Impact factor: 1.858
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