Genetic Diversity and Dye-Decolorizing Spectrum of Schizophyllum commune Population
Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of KoreaCorrespondence to:
J. Microbiol. Biotechnol. 2020; 30(10): 1525-1535
Published October 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Synthetic materials such as dyes, polymers and chlorinated hydrocarbons have been extensively used in many modern industries and their wastes are causing severe environmental pollution while being harmful to human health [1, 2]. There is a pressing need to develop eco-friendly treatment techniques to degrade these wastes. Biological treatments serve as a cost-effective and eco-friendly solution for this pollution [1, 3]. White-rot fungi (WRF) are attractive for this purpose because of their capacity to degrade such materials by virtue of their extracellular enzymes [3-5]. WRF are natural decomposers of all components of wood, including the recalcitrant, aromatic, and heterogeneous lignin polymers. Especially, lignin modification is possible because of a wide array of extracellular oxidoreductases produced by WRF [6, 7].
Among these WRF is the split-gill fungus
With such diversity and widespread distribution,
In this study, we extracted ITS sequences from 81
Materials and Methods
Fungal Strains and Culture Media
All 81 strains of
Fungal DNA Extraction
To extract genomic DNA from
ITSs in eukaryotes are two nucleotide regions existing between the 18S, 5.8S and 26S ribosomal RNA encoding sequence, together forming the rRNA cistron which is transcribed as a unit by RNA polymerase I. The rRNA sequences can be split and the ITS removed in the post-transcriptional step. However, for sequence analysis in phylogenetics, the sequence of ITS used consists of both 2 ITS regions and the 5.8S rRNA encoding sequence. The ITS sequence has been found to be a useful genetic marker for distinguishing fungal species (Smith
To amplify the ITS region with DNA product size around 500-800 bps, 50 ng of gDNA template of obtained mushroom and 2 primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-CCTCCGCTTATTGATATGC-3’) were used. The reaction occurred in the presence of 1 unit of Phire Hot Start II DNA Polymerase (Thermo Scientific). PCR reaction was performed with 3 steps: initial denaturation at 98°C, 30 sec, 1 cycle; denaturation at 98°C, 5 sec; annealing at 53~55°C, 5 sec; extension speed of 10-15 s/kb, 35 cycles; final extension at 72°C, 1 min, 1 cycle; holding sample at 4°C.
PCR products were purified according to the protocol of AccuPrep’s PCR/Gel DNA Purification Kit. The precise DNA sequence was confirmed in both directions of ITS based on standard sequencing by Macrogen Co. One final sequence was obtained based on the peak quality obtained by electrophoresis of the bidirectional sequence.
Phylogenetic Analysis of Korean Domestic and Foreign White-Rot Fungi
In addition to the 81 ITS sequences obtained from the CCM of Incheon National University, 137 ITS sequences of
Analysis of Synthetic Dye Decolorization Ability of
S. commune Strains
Four types of dye used in the screening test were classified into 4 chromophore types: diazo-based Congo Red, anthraquinone-based Remazol Brilliant Blue R, thiazine-based Methylene Blue, and triphenylmethane-based Crystal Violet.
Fungal culture medium (PDA) was supplemented with 20 mg/l dye, sterilized, then aliquoted into 6 cm petri dishes to be ready for screening test. A single 4.4 cm diameter cork borer was used to inoculate each strain on each dye-containing medium with the same size of inoculum. The inoculation was repeated twice, placed in a sealed box to maintain humidity, and cultured at 27°C in dark condition. Samples were observed every day and the bottom surface of the medium was scanned every 3 days for decolorization ability and activity comparison. The inoculum dishes were compared with original dye-containing media, and the level of dye-decolorization was determined as (+), for ‘has ability to decolorize,’ or (-) for ‘has no ability.’ In addition, high decolorization ability was recorded as around 6 cm, and low, around 3 cm.
Liquid medium (Potato Dextrose Broth) was prepared similar to solid media without agar. Each 10 ml of dye-containing liquid media was transferred to a 50 ml conical tube. Then, the same amount of inoculum was inoculated into each medium using a 4.4 mm diameter cork borer. The inoculated tubes were incubated at 27°C and 150 rpm in dark condition. The fungal cultures containing dyes were filtered through Whatman filter papers 42 (cat. no. 1442-090) at 12 days post inoculation, then the culture was secured. The filtered culture was transferred to a 1 mL cuvette and the absorbance was measured by using a NanoDrop 2000c instrument. The absorbance wavelengths for each dye, Congo Red, Remazol Brilliant Blue R, Methylene Blue and Crystal Violet, were 485, 595, 620, and 560 nm, respectively. The decolorization rate was calculated based on the absorbance in the control dye-containing medium without strains and the absorbance in medium in which strains were cultured. To standardize the degree of bleaching, various concentrations of control dye media were prepared to measure the absorbance.
Species Identification of White-Rot Fungal Strains Isolated from CCM Bank
The genomic DNA of 81
In order to determine the genetic relation among these strains, four other closely related species in
The phylogeneic analysis ofA total of 81 strains from the CCM bank were used. S. communepopulation. S. communeH4-8 strain (*) and two outgroup strains, S. amplum(**) and S. radiatum(black arrow), were added. The broken lines indicate the shortening of the S. amplumbranch. Maximum likelihood tree was generated by RAxML using the CAT model and automatic bootstrapping options.
Genetic Diversity of
S. commune Population
To investigate the genetic diversity of
To compare the genetic diversity of domestic and foreign
Moreover, the haplotype network of 138 foreign strains was constructed and the correlation between haplotype and its distribution was examined (Fig. S2). The largest haplotype (LV) included strains from all regions, but the major strains in this haplotype were from Europe (23/35 strains). South Asian strains composed the main part (14 strains/total 14 strains) in the 2nd largest haplotype (XXVIII,
To determine how the diversity of the Korean
Haplotype network analysis ofA total of 217 S. communestrains. S. communestrains including domestic and foreign groups were analyzed. Total haplotype number is 113 but only the haplotypes containing more than two strains are displayed.
To investigate the relation between regional distribution and genetic composition of total
Correlation between the maximum likelihood tree and isolated regions in the world population ofSelected residues of ITS sequence were 501 ~ 505 bp. Maximum likelihood tree was generated by RAxML using the CAT model and automatic bootstrapping options. S. commune.
Diverse Dye-Decolorization Ability of
S. commune Population
Several studies have found that some
As mycelia grew on each plate, the degree of dye-decolorization was visually confirmed and divided into three stages (Fig. 4A): ++ strong decolorization, the dye color almost disappeared from media; + has decolorization, dye color faded from media; - no decolorization, dye color remained intact on media. In case of Congo Red and Remazol Brilliant Blue R, the distinction was not clear, so it was divided into only two stages (++ and -). The strains that have the same level of decolorization ability for each dye have been counted and presented in distribution charts (Fig. 4B, when grown on Methylene Blue-containing media, in total 81 strains tested, 60 strains (74%) exhibited decolorization ability (++ and +), implying that MB contained the easiest chromophore type to be decolorized by these
The diverse pattern of dye-decolorization of( S. communestrains. A) Selection standards of dye decolorization ability. -: no decolorization ability, +: has decolorization ability, ++: has strong decolorization ability. Decolorization was determined at 9 dpi. CR: Congo Red, RBBR : Remazol Brilliant Blue R, MB: Methylene Blue, CV: Crystal Violet. ( B) Distribution on pie charts of the dye decolorization ability. Different intensity of colors indicates levels of decolorization. Number indicates the number of strains for the specific level.
Dye-decolorization spectrum of 81Maximum likelihood tree was generated by RAxML using the CAT model and automatic bootstrapping options. In the heatmap, strong intensity indicates strong decolorizing ability. Five selected strains are colored in red. CR: Congo Red, RBBR: Remazol Brilliant Blue R, MB: Methylene Blue, CV: Crystal Violet. S. communestrains.
In addition, the above decolorization results needed to be confirmed by quantitative method which can be performed using liquid culture media. To do that, five strains showing excellent dye decolorization abilities (decolorized at least 2 types of dye) were selected as listed in Fig. 5A. These 4 domestic strains, IUM 1114, IUM 1800, IUM 1870, IUM 2813, and one foreign strain, IUM 4203 from Italy, were grown on liquid media containing different dyes. The light absorbance of media containing dye before and after fungus inoculation was measured and the decolorization ratio was calculated. As shown in Fig. 5B, the dye-decolorization of 5 selected strains on Congo Red, Methylene Blue, Crystal Violet has been confirmed in liquid media. The absence of Remazol dye in this test is explained below. Among them, strain IUM1114 which showed strong decolorization of 4 dyes on solid media, was again confirmed to be an excellent strain in decolorization ability on liquid media with ability to decolorize Congo Red (77.6%), Methylene Blue (41.1%), and Crystal Violet (50.6%). A similar result was seen in case of IUM 2813, which showed decolorization values of 54.8% in Congo Red, 52.5% in Methylene Blue and 24.6% in Crystal Violet containing liquid media. In case of Crystal Violet, IUM1114 and IUM 2813 were found to have the same Crystal Violet degrading ability in solid media ++, whereas liquid medium showed two times better dye degradability in IUM 1114 strain. Congo Red decolorization ability was not found in the solid medium of IUM 1870, but was 30% in liquid dye culture. Therefore, the exact degree of decolorization could be distinguished through the culture of the liquid medium. However, although the same method was applied on media containing Remazol Brilliant Blue R dye, there was no difference in absorbance of the dye in media with or without fungus. It seems like these fungus strains did not decolorize this dye in the liquid media.
Decolorization of the selected( S. communestrains. A) List of five excellent strains that showed strong decolorization ability on dye-containing solid media. ( B) The decolorization rate of the five strains on dye-containing liquid media including Congo Red (CR), Methylene Blue (MB), and Crystal Violet (CV).
Comparison of Phylogenetic Relation and Dye-Decolorization Ability in CCM Population
In order to investigate whether dye decolorization ability correlates with the phylogenetic identification, the decolorization spectrum of 81
In this study, the use of a large population of
To study the DNA sequence variation at the intraspecific level, the combination of two types of graph phylogenetic trees and haplotype networks is useful for investigating the genetic diversity . In this study, the phylogenetic tree showed the distance among strains (Figs. 1 and 3). Meanwhile, the haplotype network showed additional quantitative information such as: size of the population and the regional distribution of these
We also found that ITS is still a good barcode for molecular phylogenetic analysis in
In our study, we examined 75 Korean and 6 foreign strains of
Through this study, we presented that the Korean white-rot mushroom has genetic diversity and a wide spectrum of decolorization for different dyes. The finding of dye decolorization ability in more than 60 strains is impressive. There was no clear correlation between the spectrum and the ITS-based phylogenetic tree (Fig. 6). This phenotypic diversity might be explained by genotypic variation of some oxidase genes. Such diverse phenotypic spectrum implied that diverse genotypes would be good resources for further study to explore genomes of
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
This work was supported by the Incheon National University Research Grant in 2015 and by the National Research Foundation of Korea (NRF) grant (2017R1D1A1B0403587914).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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