Conventional and molecular characterization of ... - Academic Journals

African Journal of Microbiology Research Vol. 6(36), pp. 6502-6516, 20 September, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR10.736 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Conventional and molecular characterization of Trichophyton rubrum Farzad Aala1*, Rosimah Nulit2, Umi Kalsom Yusuf2 and Sassan Rezaie3 1

Department of Medical Mycology and Parasitology, School of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran. 2 Department of Biology, Faculty of Science; Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 3 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. Accepted 10 September, 2012

Different studies illustrated that Trichophyton rubrum, among all species of Trichophyton, is the most prevalent and consequently the most important genus. T. rubrum as a worldwide filamentous pathogen fungus can infect human keratinized tissue (skin, nails and rarely hair), and causes dermatophytosis. Researchers use two general methods for the identification of dermatophytes namely, conventional methods on the basis of phenotype variations and molecular methods on the basis of molecular differences. Due to some limitations in traditional methods, in the recent years, molecular biological methods are regarded as useful in the exact and rapid recognition of dermatophytes. The present study identified nine clinical isolates and one ATCC as a control strain of T. rubrum by using both conventional and molecular methods. The molecular systematics method was used to elucidate genetic diversity among strains of T. rubrum and within Trichophyton species. Morphological characteristics of all colonies T. rubrum quite varies among each other; we revealed that that conventional methods are generally prolonged and may be indecisive. However, molecular studies based on internal transcribed spacer (ITS) sequencing provides a very accurate result, which is more than 96% the similarity of T. rubrum among all isolates, and more than 90% similarity within Trichophyton spp. Key words: Trichophyton rubrum, conventional method, internal transcribed spacer (ITS) regions, identification, dermatophytes. INTRODUCTION Trichophyton rubrum is one of the most commonly encountered dermatophytes that infect human keratinized tissue such as skin, nails and possibly hair. This pathogen causes well-characterized superficial infections, and also produces skin infections in unusual parts of the body in immunodepressed patients (Cervelatti et al., 2004). Nearly 80% of onychomycosis due to T. rubrum and 90% of the chronic dermatophyte infections are caused mostly by T. rubrum, this pathogen developed mechanisms to avoid or suppress cell- mediated immunity ((Baeza et al., 2006; Baeza et al., 2007).

*Corresponding author. E-mail: [email protected]. Tel: +98-9197544944.

Researchers use two general methods for the laboratory identification of various species of dermatophytes: a) identification on the basis of phenotype differences (conventional methods) and b) Identification on the basis of molecular differences. Faggi et al. (2001) mentioned that identification of dermatophyte species by conventional methods requires the examination of colony, particularly with the method of slide culture and microscopic morphological structures. Morphological and physiological features are dynamic. As a matter of fact, outside factors such as temperature variation, medium and chemotherapy, can greatly influence the phenotypic characteristics and consequently can make the identification more difficult. Molecular biological methods, in the recent years, are regarded as useful in the exact and rapid recognition of

Aala et al.

dermatophytes. Sequencing of the Internal Transcribed Spacer (ITS) region of the ribosomal DNA, Sequencing of protein-encoding genes, Restriction Fragment Lenght Polymorphism (PCR-RFLP) analysis of mitochondrial DNA, Polymerase Chain Reaction (PCR); Random Amplification of Polymorphic DNA (RAPD), Arbitrarily Primed PCR [AP-PCR], and PCR fingerprinting are all instances of molecular techniques which have brought prominent advance in differentiating between species and strains (Faggi et al., 2001; Kanbe et al., 2003; Girgis et al., 2006; Yoshida et al., 2006; Li et al., 2007). In the recent years, quite a few molecular studies have been conducted on the internal transcribed spacer (ITS) region of the rRNA gene. Sequencing analysis of the ITS regions is considered as a useful tool for phylogenetic delineation and the identification of dermatophytes (Yoshida et al., 2006; Li et al., 2007). Even though about 80 to 90% of all isolated are T. rubrum (Brasch and Hipler, 2008), has been isolated to identify the morphological similarity and the variability among this species, but only a few study has been done about the genetic relationship of Trichophyton. The aim of this work is to identify ten clinical isolates of T. rubrum by using both conventional methods and molecular method based on universal fungal primers which are internal transcribed spacer 1 (ITS1). T. rubrum (ATCC-10218) was used as a control strain. The molecular systematics method was used to elucidate genetic diversity among strains of T. rubrum and within Trichophyton species. MATERIALS AND METHODS Isolates Nine isolates of T. rubrum which are T. rubrum (1138), T. rubrum (1059), T. rubrum (1164), T. rubrum (1208), T. rubrum (1160), T. rubrum (1008), T. rubrum (1298), T. rubrum (1044) and T. rubrum (2970), were obtained from the culture collection of clinical isolates preserved at the laboratory of Medical Mycology Department in Tehran University of Medical Sciences, Iran for study; and T. rubrum (ATCC-10218) was used as a control strain. All clinical isolates were kept in sterile saline (0.85%) v/v NaCl at 4°C until required for bioassays.

6503

(PBS) and finally stored at −70°C.

DNA extraction Fungal genomic DNA from T. rubrum was isolated according to Rezaie et al. (2000) with slight modification. 200 to 300 mg of mycelia was ground with liquid nitrogen to powder form. 500 μl of DNA extraction buffer (50 mM Tris-HCl pH 8.0), 50 mM EDTA, 25 μl 20% SDS, and 10 μl of proteinase-K, was added and mixed gently. Then, incubated at 65°C for 60 min and centrifuged at 3000×g for 15 min. 25 μl Rnase H (10 mg/ml) was added to supernatant and incubated again at 37°C for 30 min. Then mixed with 500 μl of phenol:chloroform:isoamyl alcohol (25:24:1) and and centrifuge at 10000×g for 10 min and the supernatant were collected and transferred to new steril eppendorff tubes. Then mixed again with 500 μl of chloroform:isoamyl alcohol (24:1) and centrifuge at 10000×g for 10 min, and the supernatant were collected and transferred to new steril eppendorff tubes. DNA was precipitated by adding 500 μl isopropanol and 30 μl 3 M sodium acetate followed by centrifugation at 15000×g for 30 min and the supernatant was discarded. DNA pellet was rinsed twice or more with 200 μl of 70% cold ethanol and centrifuged at 10000×g for 10 min. The pellet was air-dry and resuspended DNA pellet in 30 μl of distilled water at 37°C for 60 min and stored at -20°C.

PCR amplification Internal transcribed spacer 1 and 4 (ITS1 and ITS4) (AIT-Biotech, Singapore) were designed as ITS1 forward primer is 5’-TCC GTA GGT GAA CCT GC-3’ and the ITS4 reverse primer 5’-TCC TCC GCT TAT TGA TAT G-3’ (Shehata et al., 2008; Yang et al ., 2008 ). PCR reaction mixtures were prepared in a 25 μl volume containing 2.5 μl of 10× reaction buffer, 1.5 μl of 25 mM MgCl2, 0.5 μl of 10 mM dNTPs, 0.5 μl of 0.2 mM of each ITS 1 primer and ITS 4 primer, 0.5 μl of genomic DNA and 0.5 μl of 1 U Go Taq DNA polymerase (Promega Corporation, USA), and 18.5 μl of distiled water. PCR reactions were carried out on a thermal cycler (MJ Research. Inc. USA) with the following conditions: 1 cycle in an initial step of 94°C for 5 min and then subjected to 30 cycles consisting of denaturation at 94°C for 30 s, annealing at 55°C for 40 s, and extention at 72°C for 40 s. After the last cycle, this was followed by a final extention step at 72°C for 10 min. Then, 5 μl of PCR product was loaded on 1% agarose in 1X Tris–Acetic Acid– EDTA buffer and stained with 0.5 mg/ml ethidium bromide at 80 V for 40 min and visualised with UV transilluminator (Alpha Innotech, USA), compared with a standard DNA size marker; 100 bp DNA ladder (Fermenats, USA), and photographed in UV light.

PCR purification Conventional method All isolates of T. rubrum were cultured on Sabouraud dextrose agar media (Difco Laboratories, Detroit, Michigan) at 28°C for 14 days. Then, slide cultures of isolates were prepared and identified under light microscope (Carl Zeiss, Germany).

DNA PCR products were purified according to the QIAquick PCR Purification Kit (Qiagen, Germany) and send for sequencing (1st Laboratories, Seri Kembangan, Malaysia).

RESULTS AND DISCUSSION

Molecular method

Morphological characteristics of colonies T. rubrum

All isolates of T. rubrum maintained on Sabouraud’s dextrose agar medium and stored at 4°C. Then fungus was cultured in Sabouraud dextrose broth, and incubated at 28°C for 14 days. 200 to 300 mg of mycelia was harvested and centrifuged at 1600×g for 10 min, then washed twice with ice-cold sterile phosphate buffered saline

This study used both conventional and molecular methods to diagnose ten isolates of T. rubrum. Studies revealed that colonies characterization of all isolates quite varies among each other. Of these isolates, isolate

6504

Afr. J. Microbiol. Res.

numbers 1138 and 1059 are white and cottony or fluffy but isolates number 1164, 1160, 1008, and 1298 are cream, flat and downy, but the others are cream with a carmine and woolly or granular type (isolates numbers 1208, 1044, 2970 and 10218) (Figure 1). The microscopic features of the isolates also varies, which are macroconidia and microconidia of isolates numbers, 1138, 1008, 1298, 1044, 2970 and 10218 more abundant than isolates number 1059, 1164, 1208, and 1160. However, the shape of the macroconidia and microconidia of all isolates are almost similar, which is cyclindrical to cigar shaped (Figure 1). Isolation, identification and characterization of ITS1 of T. rubrum

molecular

Figure 2 showed that ITS1 of all isolates T. rubrum had been amplifed and then were isolated and sequenced. The length of nucleotides sequence of all isolates are not similar which is T. rubrum (1138) 658 bp, T. rubrum (1059) 715 bp, T. rubrum (1164) 722 bp, T. rubrum (1208) 713 bp, T. rubrum (1160) 614 bp, T. rubrum (1160) 614 bp, T. rubrum (1008) 719 bp, T. rubrum (1298) 668 bp, T. rubrum (1044) 658 bp, T. rubrum (2970) 660 bp and T. rubrum (ATCC-10218) 633 bp. Nucleotide sequence of all isolates of T. rubrum and ATCC-10218 are shown in Figure 3. Previous studies by Rakeman et al. (2005) and Shehata et al. (2008) also revealed that the universal fungal primers amplified the ITS regions (ITS1-5.8S-ITS2) of the ribosomal DNA nearly 690 bp for T. rubrum isolates. Nucleotide sequence of ten isolates of T. rubrum shown in Figure 3. All nucleotide sequences of T. rubrum isolates were analyzed using online software CLUSTALW (www.Pir.geogetown.edu/pirwww/search/multialn.shtm) to reveal the similarities among isolates. Figure 4 showed that the similarities among nine isolates of T. rubrum are higher, which is more than 96% identities. Nucleotide sequence of isolates T. rubrum were analyzed using online software CLUSTALW (www. Pir.geogetown.edu/pirwww/search/multialn.shtm) to reveal the similarities among isolates T. rubrum and other species of Trichophyton which are Trichophyton raubitschekii strain NOMH 789 (GenBank accession no. AF170469), T. rubrum strain UAMH 8547 (GenBank accession no. AF170471), T. kanei (GenBank accession no. AF170460), T. rubrum strain WM 06.348 (GenBank accession no. EF568093), T. rubrum strain 05-287-3929 (GenBank accession no. EU200395), T. rubrum 5.8S rRNA (GenBank accession no. AJ270808), T. soudanense strain UAMH 8548 (GenBank accession no. AF170474), T. rubrum strain NCPF 295 (GenBank accession no. EU181449), T. megninii strain ATCC 12106 (GenBank accession no. AF170464), and T. rubrum strain ATCC 28188 (GenBank accession no. AF 170472). The similarities of all isolates of T. rubrum and

other species of Trichopthyon is also higher than 90% as shown in Figure 5, CLUSTAL 2.0.12 multiple sequence alignment. DISCUSSION Traditional method such as investigation of macroscopic and microscopic features of cultures of fungi had been applied since early 19th century. However, these methods seem to be difficult to amplify due to the polymorphic feature of these characters, besides increased by differences in media compounds, temperature variations, and other variables of cultivation. Furthermore, in some cases, the dermatophytes fail to make reproductive organization in culture (sterile mycelia) that makes it impossible for final identification (Malinovschi et al., 2009). Besides that, conventional method is often difficult due to abnormal microscopic or macroscopic morphology (Li et al., 2008). Currently, molecular studies become crucial and necessary for identification of pathogenic fungi (Borman et al., 2008; Malinovschi et al., 2009). The internal transcribed spacer (ITS) regions of the fungal ribosomal DNA (rDNA) had been used as one of techniques for species identification becuase it is faster, accurate species determination, specific, and are less feasible to be affected by exterior effects such as temperature changes and chemotherapy (Girgis et al., 2006; Kong et al., 2008). Studies revealed that morphological characteristics of colonies of all isolates T. rubrum are similar to T. rubrum isolated from tinea cruris, tinea pedis, and tinea capitis of human (Graser et al., 2000). Colonies of T. rubrum are fluffy to cotonny and white to cream in colour. Macroconidia are sparse or abundant and microconidia are present in all isolates. In this studies, the length of ITS1 of all isolates is about 690 bp, 10 clinical isolates of T. rubrum were collected from the Clinical Mycology Laboratory at Westmead Hospital, Sydney, and the Women’s and Children’s Hospital, Adelaide, Australia also have almost the same length of ITS1, which is 666 bp (Kong et al., 2008). Consequently, the results of our study are in agreement with these studies and showed that molecular method based on ITS sequencing is a reliable and useful method for the identification of dermatophytes as well as for confirmation of diagnosis of the conventional methods. In this study, the molecular method was also used to clarify genetic diversity among strains of T. rubrum and within Trichophyton species. The results of this study regarding nucleotide sequence of isolates of T. rubrum demonstrated that the similarities among ten isolates of T. rubrum are more than 96% identities. It also showed the similarities among ten isolates of T. rubrum and ten isolates of other genus of Trichophyton are higher than 90%. The results of this study are in agreement with Graser et al. (2000) who showed that the Trichophyton species are supported by high similarities with value of

Aala et al.

6505

Figure 1. The colonies and microscopy of 10 isolates of T. rubrum with (macroconidia and microconidia) × 400.

more than 86% among isolates of T. rubrum and isolates of other genus of Trichophyton. Our results are also in

agreement with Li et al. (2008), who revealed that percentage identity of Trichophyton species with

6506


Figure 2. PCR amplification of isolates of T. rubrum on 1% agarose gel electrophoresis. T. rubrum ATCC-10218 as positive control strain also showed DNA amplification at 690 bp.

> T. rubrum (1138) 50 NNNNGGGAGAGCGTAAGTGGGCTGCCACTATAGAGGACCGGACATTCCAT 100 CAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACC 150 TCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTC 200 CGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGA 250 CAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGC 300 AAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATC 350 GATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCG 400 TGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGG 450 GCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGAT 500 GGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCA 550 GTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG 600 CGCCCTCAGGACCGGCCGCCTGGCCCCAATCTTTATATATATATATATC 650 TTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCAT 658 ATCAAAAG

> T. rubrum (1059) 50 NCCAGTAACCGTAGGTGACCTGCGCATATCAATAAGCGGAGGACTCCGTG 100 GGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCTCACC 150 CGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGCG 200 GGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGAC 250 ACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCACAGA 300 CAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGA 350 AGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAAT 400 CATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATG 450 CCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACG 500 ACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGC 550 CAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCC 600 TCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTCA 650 GGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAT 700 AAGCCGGAGGAAGGGGGGGCCCCCCATAGGGCCCCCCCGCTCTCTTTTTG 715 GGGAAGCAAAATGGG > T. rubrum (1164) 50 CNNNNNAGACCGTACGTTGGCTGCGCATATCAGATAACCGGACATGACAT 100 CGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCT 150 CACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCC 200 GGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA 250 GACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAA 300 GCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGA 350 TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTG 400 AATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGC 450 ATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGG 500 ACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGT 550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCG 600 CCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTT 650 TTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATAT 700 CAAAAGGGGGGAGGAAGAGGGGGGCCCCCCATAGGGGCCCCCCCCTTTTT 722 TTTTGGGGTAGCGAGAAGGGGG

Figure 3. Nucleotide sequences of 9 isolates of T. rubrum and ATCC10218. Nucleotide sequence numbering is shown on the left.

Aala et al.

> T. rubrum (1208) 50 TNNGCAGACGTACGTGGGCTGCGAATATCAGGAAGCGACATGACTTCGGG 100 GGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCTCACC 150 CGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGCG 200 GGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGACA 250 CCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGCAC 300 AATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAA 350 GAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAATC 400 ATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATGC 450 CTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACGA 500 CCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGCC 550 AGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCCTC 600 AGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTCAGG 650 TTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAA 700 GCCGGGAGGAAGGGGGGGCCCCCCAAAATGCCCCCCCCTCTCTTTTTGGG 713 GGGGAGAGCGGGG > T. rubrum (1160) 50 NNNNNAAGAATCGTAAGTGACCTGCGCATATCAATAAGCGGAGGATCCGT 100 AGGTGAACCTGCGCGTATCAATAAGCGGAGGACATTCTTGTCTACCTCAC 150 CCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGC 200 GGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGAC 250 ACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGCA 300 CAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGA 350 AGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAAT 400 CATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATG 450 CCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACG 500 ACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGC 550 CAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCC 600 TCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTC 650 AGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAA 614 TAAGCGGGGAGGAA

> T. rubrum (1008) 50 NACNAAGAGCCGTAGGTGACCTGCGCATATCAATAAGCGAGAGGACTCCG 100 TAGGTGAACCTGCGTGTATCGGCCGTACGCCCACATTCTTGTCTACCTCA 150 CCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGG 200 CGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGA 250 CACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGC 300 ACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATG 350 AAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAA 400 TCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCAT 450 GCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGAC 500 GACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGG 550 CCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCC 600 CTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTT 650 CAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCA 700 ATAAGCCGGAGGAAGGGGGCCCCGAAGAGGAGCCACCCCCCTCAGGGTGT 719 GTGAAACAAACGGCGGGCC > T. rubrum (1298) 50 NNACNNAGTATCGTAGGTGACCTGCGCATATCAATAAGCGGAGGATTCCG 100 TAGGTGAACCTGCGCATATCAATAAGCGGAGGATTCCGTTGGTTACCTCG 150 CCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGG 200 CGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGA 250 CACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGC 300 ACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATG 350 AAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAA 400 TCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCAT 450 GCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGAC 500 GACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGG 550 CCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCC 600 CTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTT 650 CAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCA 668 ATAAGCGGAGGAA > T. rubrum (1044) 50 NNNANCGGGACAGCCGTAGTGGGCTGCGCATATCAGATAACGCGGAGATT 100 ACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCT 150 ACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCC 200 GTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGG 250 ACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAG 300 CAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCAT 350 CGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCC 400 GTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGG 450 GGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGA 500 TGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCA 550 GTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG 600 CGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATC 650 TTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCAT 658 ATCAAAAG

Figure 3. Cotnd.

6507

6508


> T. rubrum (2970) 50 ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACT 100 TCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACC 150 TCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTC 200 CGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA 250 GACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAA 300 GCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGA 350 TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTG 400 AATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGC 450 ATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGG 500 ACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGT 550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG 600 CGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCT 650 TTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATA 660 TCAAAAGCGG > T. rubrum (ATCC-10218) 50 NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACTT 100 CGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCT 150 CACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCC 200 GGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA 250 GACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAA 300 GCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGA 350 TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTG 400 AATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGC 450 ATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGG 500 ACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGT 550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCG 600 CCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATATATCTT 633 GGCAGGTTGACCTCGGATCAGGTAGGGATACGT

Figure 3. Cotnd.

T. T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

--NNNNGGGAGAGCGTAAGTGGGCTGCCA-CTAT-AGAGGAC-CGGACAT ---CNNNNNAGACCGTACGTTGGCTGCGC-ATATCAGATAAC-CGGACAT ----TNNGCAGA-CGTACGTGGGCTGCGA-ATATCAGGAAGC---GACAT NNNANCGGGACAGCCGTAGTGGGCTGCGC-ATATCAGATAACGCGGAGAT -----NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG ----ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG ----NCCAGTAACCGTAGGTGACCTGCGC-ATATCAATAAGC----GGAG --NNNNNAAGAATCGTAAGTGACCTGCGC-ATATCAATAAGC---G-GAG --NNACNNAGTATCGTAGGTGACCTGCGC-ATATCAATAAGC---G-GAG ---NACNAAGAGCCGTAGGTGACCTGCGC-ATATCAATAAGC---GAGAG * ** **** *** * * *

50 50 50 50 50 50 50 50 50 50

T. T. T. T. T. T. T. T. T. T.


(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

TCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC TACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGGACATTCTTGTC GATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGGATTCCGTTGGT GACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCCACATTCTTGTC * ***** * ** * * ***

100 100 100 100 100 100 100 100 100 100

T. T. T. T. T. T. T. T. T. T.


(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC ****** ****************************************** T. rubrum (1138) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1164) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1208) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1044) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (ATCC-10218) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (2970) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1059) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1160) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1298) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1008) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA **************************************************

150 150 150 150 150 150 150 150 150 150 200 200 200 200 200 200 200 200 200 200

Figure 4. Comparison of nucleotide sequence between T. rubrum ITS1 orthologues. Nucleotide sequences that are present in all ITS1 are shaded in blue colour. Nucleotide sequence numbering is shown on the right.

Aala et al.

T. T. T. T. T. T. T. T. T. T.


GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT ************************************************** T. rubrum (1138) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1164) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1208) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1044) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (ATCC-10218) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (2970) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1059) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1160) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1298) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1008) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC **************************************************

250 250 250 250 250 250 250 250 250 250

T. T. T. T. T. T. T. T. T. T.

350 350 350 350 350 350 350 350 350 350

ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ************************************************** T. rubrum (1138) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1164) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1208) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1044) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (ATCC-10218) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (2970) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1059) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1298) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1008) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG **************************************************

T. T. T. T. T. T. T. T. T.


(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

400 400 400 400 400 400 400 400 400

GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT ************************************************** T. rubrum (1138) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1164) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1208) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1044) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (ATCC-10218) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (2970) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1059) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1160) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1298) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1008) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA **************************************************

450 450 450 450 450 450 450 450 450

T. T. T. T. T. T. T. T. T. T.

GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT ************************************************** T. rubrum (1138) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1164) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1208) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1044) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (ATCC-10218) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATAT T. rubrum (2970) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1059) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1160) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1298) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1008) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT ****************************************** ****

550 550 550 550 550 550 550 550 550 550

T. T. T. T. T. T. T. T. T. T.

650 650 650 650 650 650 650 650 650 650



(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1008)

300 300 300 300 300 300 300 300 300 300

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

Figure 4. Contd.

ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTGGCAGGTTGACCTCGGATCAGGTAGGGATACGT-----------ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ***** *****************************

500 500 500 500 500 500 500 500 500 500

600 600 600 600 600 600 600 600 600 600

6509

6510


T. T. T. T. T. T. T. T. T. T.


(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

CATATCAAAAG--------------------------------------CATATCAAAAGGGGGGAGGAAGAGGGGGGCCCCCCATAGGGGCCCCCCCC CATATCAATAAGCCGGGAGGAAGGGGGGGCCCCCCA-AAATGCCCCCCCC CATATCAAAAG---------------------------------------------------------------------------------------CATATCAAAAGCGG-----------------------------------CATATCAATAAGCCGG-AGGAAGGGGGGGCCCCCCATAGGGCCCCCCCGC CATATCAATAAGCGGGGAGGAA---------------------------CATATCAATAAGCGGAGGAA-----------------------------CATATCAATAAGCCGGAGGAAGGGGGCCCCGAAGAGGAGCCACCCCCCTC

T. T. T. T. T. T. T. T. T. T.


(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

--------------------------TTTTTTTTTGGGGTAGCGAGAAGGGGG TCTCTTTTTGGGGGGGAGAGCGGGG-------------------------------------------------------------------------------TCTCTTTTTGGGGAAGCAAAATGGG-----------------------------------------------------AGGGTGTGTGAAACAAACGGCGGGCC-

700 700 700 700 700 700 700 700 700 700

727 727 727 727 727 727 727 727 727 727

Figure 4. Contd.

T. raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. megninii strain ATCC 12106 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T.rubrum 5.8S rRNA gene T.rubrum strain WM 06.348 T.rubrum strain NCPF 295 T.rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

50 50 50 50 50 50

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

100 100 100 100 100 100

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG --------------------------ACAAGGTTTCCGTAGGTGAACCTG -----------------------------------------------------------------------------------TCCGTAGGTGAACCTG ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

150 150 150 150 150 150 24 16

Figure 5. Comparison sequence between T. rubrum ITS1 T. raubitschekii strain NOMHof789nucleotide CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198 T. saudanese UAMH 8548 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G orthologues. Nucleotide sequences that are present in all ITS1 are shaded198in T. megninii strain ATCC 12106 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198 black colour. Nucleotide numbering is shown on the right. T. rubrum strain UAMH 8547sequence CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198 T. T. T. T. T. T. T.

rubrum kanei rubrum rubrum rubrum rubrum rubrum

strain ATCC 28188 5.8S rRNA gene strain WM 06.348 strain NCPF 295 strain 05-287-3929 (1138)

CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G -------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG

198 198 72 42 64 42 35

T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum

(1059) (1208) (1264) (2970) (ATCC-10218) (1044)

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------


raubitschekii strain NOMH 789 saudanese UAMH 8548 megninii strain ATCC 12106 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G -------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG -------------NNNNNAAGAATCGTAAGTGACCTGCG-CATATCA-AT -------------NNACNNAGTATCGTAGGTGACCTGCG-CATATCA-AT --------------NACNAAGAGCCGTAGGTGACCTGCG-CATATCA-AT ---------------NCCAGTAACCGTAGGTGACCTGCG-CATATCA-AT ----------------TNNGCAGACGTACGTGGGCTGCG-AATATCA-GG --------------CNNNNNAGACCGTACGTTGGCTGCG-CATATCAGAT --------------ANCGGACAGCCGTA-GTGGCCTGCGACATATCAGAT ---------------NGGGACCGCCGTA-GTGGCCTGCGACATATCAGAT ----------NNNANCGGGACAGCCGTA-GTGGGCTGCG-CATATCAGAT ** * * * *

198 198 198 198 198 198 72 42 64 42 35 35 35 34 33 32 35 35 34 38



GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTC-ATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCGGACATTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AA-GCG--GAGGAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGG AA-GCG--GAGGATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGG AA-GCGA-GAGGACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCC AA-GCG--GAGGACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCC AA-GCGA-CATGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AA-CCGGACATGACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AACGCGGAGAGGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AACGCGGAGAGGACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCC AACGCGGAGATTACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC * ** * ***** * ** * *

246 246 246 246 246 246 119 90 112 90 84 81 82 82 80 80 84 85 84 88



CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG C-ATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG ACATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG ATTCCGTTGGTTACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTG ACATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG *** ****** ****************** *************

296 296 296 296 296 296 168 140 162 140 134 131 132 132 130 130 134 135 134 138

CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG

346 346 346 346 346

Aala et al.

Figure 5. Contd. T. T. T. T. T.

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188

6511

T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

6512


CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 135 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 134 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 138 *** ****** ****************** *************



CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG **** ******************* **** *** ****************

346 346 346 346 346 346 218 190 212 190 184 181 182 182 180 180 184 185 184 188



CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG **************************************************

396 396 396 396 396 396 268 240 262 240 234 231 232 232 230 230 234 235 234 238



TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC **************************************************

446 446 446 446 446 446 318 290 312 290 284 281 282 282 280 280 284 285 284 288

TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA

496 496 496 496 496

Figure 5. Contd. T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188

T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum

(1059) (1208) (1264) (2970) (ATCC-10218) (1044)

TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC **************************************************

280 280 284 285 284 288

T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA **************************************************

496 496 496 496 496 496 368 340 362 340 334 331 332 332 330 330 334 335 334 338


raubitschekii strain NOMH789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC **************************************************

546 546 546 546 546 546 418 390 412 390 384 381 382 382 380 380 384 385 384 388



TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC **************************************************

596 596 596 596 596 596 468 440 462 440 434 431 432 432 430 430 434 435 434 438

Figure 5. Contd.strain NOMH789 T. raubitschekii T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei

CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC

646 646 646 646 646 646

Aala et al.

6513

6514

T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) Afr. J. Microbiol. Res. T. rubrum (ATCC-10218) T. rubrum (1044)

TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC **************************************************

430 430 434 435 434 438

CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTC--TTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC ********************************** **************

646 646 646 646 646 646 516 490 512 490 484 481 482 482 480 480 484 485 484 488

GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG ***************************************** ******** raubitschekii strain NOMH789 GGCAGCCAATTCAGCGCCCTCAGG-------------------------megninii strain ATCC 12106 GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA saudanese UAMH 8548 GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain UAMH 8547 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain ATCC 28188 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA kanei GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum 5.8S rRNA gene GGCAGCCAATTCAGCGCCCTCAGGACCGGCNGCCCTGGCCCCAATCTTTA rubrum strain WM 06.348 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain NCPF 295 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain 05-287-3929 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1138) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1164) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1298) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1008) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1059) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1208) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1264) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (2970) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (ATCC-10218) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1044) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA ********* *************

696 696 696 696 696 696 566 540 562 540 534 531 532 532 530 530 534 535 534 538






Figure 5. Contd.

720 746 746 746 746 746 616 590 612 590 584 581 582 582 580 580 584 585 584 588

Aala et al.

T. raubitschekii strain NOMH789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044) T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

-------------------------------------------------TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATCGCGATATATCTTGGCAGGTTGACCTCGGATCAG------------TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG -------------------------------------------------GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAA---------------------------------------CTGAACTTAAGCATATCAAT-----------------------------CTGAACTTAAGCATATCAATAAGCGG-----------------------CTGAACTTAAGCATATCAAAAG---------------------------CTGAACTTAAGCATATCAATAAGCGGGGAGGAA-----------------------------------------------------------------------------------------------------------------------------------------------------------------

6515

796 796 796 796 796 666 640 662 640 634 631 632 632 630 630 634 635 621 638 846 846 846 846 846 716 650 682 666 656 664

Figure 5. Contd.

reference sequence in GenBank (BLAST search) ranged from 85.9 to 100%. Conclusion By conventional characterization, colonies of all isolates quite varies; however, the shape of macroconidia and microconidia are similar. Beside that, molecular characterization also revealed that all isolates of T. rubrum show high similarity among them and with other Trichophyton species.

ACKNOWLEDGEMENT This study was supported by the Research University Grants Scheme (RUGS) from University Putra Malaysia. REFERENCES Baeza LC, Matsumoto MT, Almeida AMF, Mendes-Giannini MJS (2006). Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer. J. Med. Microbiol. 55:429-436. Baeza LC, Bailao AM, Borges CL, Pereira M, Soares CM, Mendes Gianni MJ (2007). cDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin. Microbes. Infect. 9:1415-1421.

Borman AM, Linton CJ, Miles SJ, Johnson EM (2008). Molecular identification of pathogenic fungi. J. Antimicrob. Chemother. 61: S1, i7-i12, dio: 10.1093/jac/dkm425. Brasch J, Hipler UC (2008). Clinical Aspects of Dermatophyte Infections. Human and Animal Relationships. 2nd Edition. The Mycota. VI. 263-286. Cervelatti EP, Ferreira-Nozawa MS, Aquino-Ferreira R, Fachin AL, Martinez-Rossi NM (2004). Electrophoretic molecular karyotype of the dermatophyte Trichophyton rubrum. Genetics Mol. Biol. 27(1): 99-102. Faggi E, Pini G, Campisi E, Bertellini C, Difonzo E, Mancianti F (2001). Application of PCR to distinguish common species of dermatophytes. J. Clin. Microbiol. 39: 3382-3385. Girgis S, Fakkar N, Badr H, Shaker O, Metwally F, Bassim H (2006). Genotypic identification and antifungal susceptibility pattern of dermatophytes isolated from clinical specimens of dermatophytosis in Egyptian patients. J. Egyptian Dermatol. 2(2): 1-23. Graser Y, Kuijpers AFA, Presber W, De hoog GS (2000). Molecular Taxonomy of the Trichophyton rubrum Complex. J. clin. microbiol. 38(9): 3329-3336. Kanbe T, Suzuki Y, Kamiya A, Mochizuki T, Kawasaki M, Fujihiro M, Kikuchi A (2003). Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCRRFLP targeting of the DNA topoisomerase II genes. J. Dermatol. Sci. 33: 41-54. Kong F, Tong Z, Chen X, Sorrell T, Wang B, Wu Q, Ellis D, Chen S (2008). Rapid Identification and Differentiation of Trichophyton Species, Based on Sequence Polymorphisms of the Ribosomal Internal Transcribed Spacer Regions, by Rolling-Circle Amplification. J. Clin. Microbiol. 46(4): 1192-1199. Li HC, Bouchara JP, Hsu MML, Barton R, Su S, Chang TS (2008). Identification of dermatophytes by sequence analysis of the rRNA gene internal transcribed spacer regions. J. Med. Microbiol. 57: 592600. Li HC, Bouchara JP, Hsu MML, Barton R, Chang TS (2007).

6516


Identification of Dermatophytes by an Oligonucleotide Array. J. Clin. Microbiol. 45(10): 3160-3166. Malinovschi G, Kocsube S, Galgoczy L, Somogyvari F, Vagvolgyi C (2009). Rapid PCR based identification of two medically important dermatophyte fungi, Microsporum canis and Trichophyton tonsurans. Acta. Biol. Szegediensis 53(1): 51-54. Rakeman JL, Bui U, LaFe K, Chen YC, Honeycutt RJ, Cookson BT (2005). Multilocus DNA sequence comparisons rapidly identify pathogenic molds. J. Clin. Microbiol. 43(7):3324-3333. Rezaie S, Ban J, Mildner M, Poitschek C, Brna C (2000). Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum. Gene. 241: 27-33. Shehata AS, Mukherjee PK, Aboulatta HN, El Akhras AI, Abbadi SH, Ghannoum MA (2008). Single-Step PCR Using (GACA) 4 Primer: Utility for Rapid Identification of Dermatophyte Species and Strains. J. Clin. Microbiol. 46(8): 2641-2645.

Yang G, Zhang M, Li W, An L (2008). Direct species identification of common pathogenic dermatophyte fungi in clinical specimens by semi-nested PCR and restriction fragment length polymorphism. Mycopathologia. 166: 203-208. Yoshida E, Makimura K, Mirhendi H, Kaneko T, Hiruma M, Kasai T, Uchida K, Yamaguchi H, Tsuboi R (2006). Rapid identification of Trichophyton tonsurans by specific PCR based on DNA sequences of nuclear ribosomal internal transcribed spacer (ITS) 1 region. J. Dermatolog. Sci. 42: 225-230.