Conventional and molecular characterization of Trichophyton rubrum

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

1Department of Medical Mycology and Parasitology, School of Medicine, Kurdistan University of Medical Sciences,

Sanandaj, Iran. 2Department of Biology, Faculty of Science; Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

3Department 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

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. 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). Molecular method

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

Aala et al. 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 DNA PCR products were purified according to the QIAquick PCR Purification Kit (Qiagen, Germany) and send for sequencing (1

st

Laboratories, Seri Kembangan, Malaysia).

RESULTS AND DISCUSSION Morphological characteristics of colonies T. rubrum 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

http://en.wikipedia.org/wiki/Trichophyton_rubrum

http://en.wikipedia.org/wiki/Trichophyton_rubrum

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 molecular characterization of ITS1 of T. rubrum 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 19

th 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 Afr. J. Microbiol. Res.

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 ATCC-

10218. Nucleotide sequence numbering is shown on the left.

Aala et al. 6507

> 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.


> T. rubrum (2970)

50 ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACT

100 TCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACC

150 TCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTC

200 CGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA







550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG

600 CGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCT 650 TTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATA

660 TCAAAAGCGG

> T. rubrum (ATCC-10218)

50 NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACTT

100 CGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCT

150 CACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCC

200 GGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA







550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCG

600 CCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATATATCTT 633 GGCAGGTTGACCTCGGATCAGGTAGGGATACGT

Figure 3. Cotnd.

T. rubrum (1138) --NNNNGGGAGAGCGTAAGTGGGCTGCCA-CTAT-AGAGGAC-CGGACAT 50

T. rubrum (1164) ---CNNNNNAGACCGTACGTTGGCTGCGC-ATATCAGATAAC-CGGACAT 50

T. rubrum (1208) ----TNNGCAGA-CGTACGTGGGCTGCGA-ATATCAGGAAGC---GACAT 50

T. rubrum (1044) NNNANCGGGACAGCCGTAGTGGGCTGCGC-ATATCAGATAACGCGGAGAT 50

T. rubrum (ATCC-10218) -----NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG 50

T. rubrum (2970) ----ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG 50

T. rubrum (1059) ----NCCAGTAACCGTAGGTGACCTGCGC-ATATCAATAAGC----GGAG 50

T. rubrum (1160) --NNNNNAAGAATCGTAAGTGACCTGCGC-ATATCAATAAGC---G-GAG 50

T. rubrum (1298) --NNACNNAGTATCGTAGGTGACCTGCGC-ATATCAATAAGC---G-GAG 50

T. rubrum (1008) ---NACNAAGAGCCGTAGGTGACCTGCGC-ATATCAATAAGC---GAGAG 50

* ** **** *** * * *

T. rubrum (1138) TCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC 100

T. rubrum (1164) GACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC 100

T. rubrum (1208) GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC 100

T. rubrum (1044) TACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC 100

T. rubrum (ATCC-10218) GACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC 100

T. rubrum (2970) GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC 100

T. rubrum (1059) GACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC 100

T. rubrum (1160) GAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGGACATTCTTGTC 100

T. rubrum (1298) GATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGGATTCCGTTGGT 100

T. rubrum (1008) GACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCCACATTCTTGTC 100

* ***** * ** * * ***

T. rubrum (1138) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150




T. rubrum (ATCC-10218) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150




T. rubrum (1298) TACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGC 150


****** ******************************************

T. rubrum (1138) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200




T. rubrum (ATCC-10218) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 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. 6509

T. rubrum (1138) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250




T. rubrum (ATCC-10218) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250






**************************************************

T. rubrum (1138) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300




T. rubrum (ATCC-10218) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300






**************************************************

T. rubrum (1138) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350




T. rubrum (ATCC-10218) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350






**************************************************

T. rubrum (1138) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400




T. rubrum (ATCC-10218) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400





************************************************** T. rubrum (1138) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450

T. rubrum (1164) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450



T. rubrum (ATCC-10218) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450





**************************************************

T. rubrum (1138) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500




T. rubrum (ATCC-10218) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500






**************************************************

T. rubrum (1138) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550




T. rubrum (ATCC-10218) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550






**************************************************

T. rubrum (1138) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600




T. rubrum (ATCC-10218) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATAT 600






****************************************** ****

T. rubrum (1138) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650




T. rubrum (ATCC-10218) ATCTTGGCAGGTTGACCTCGGATCAGGTAGGGATACGT------------ 650






***** ***************************** Figure 4. Contd.


T. rubrum (1138) CATATCAAAAG--------------------------------------- 700

T. rubrum (1164) CATATCAAAAGGGGGGAGGAAGAGGGGGGCCCCCCATAGGGGCCCCCCCC 700

T. rubrum (1208) CATATCAATAAGCCGGGAGGAAGGGGGGGCCCCCCA-AAATGCCCCCCCC 700

T. rubrum (1044) CATATCAAAAG--------------------------------------- 700

T. rubrum (ATCC-10218) -------------------------------------------------- 700

T. rubrum (2970) CATATCAAAAGCGG------------------------------------ 700

T. rubrum (1059) CATATCAATAAGCCGG-AGGAAGGGGGGGCCCCCCATAGGGCCCCCCCGC 700

T. rubrum (1160) CATATCAATAAGCGGGGAGGAA---------------------------- 700

T. rubrum (1298) CATATCAATAAGCGGAGGAA------------------------------ 700

T. rubrum (1008) CATATCAATAAGCCGGAGGAAGGGGGCCCCGAAGAGGAGCCACCCCCCTC 700

T. rubrum (1138) --------------------------- 727

T. rubrum (1164) TTTTTTTTTGGGGTAGCGAGAAGGGGG 727

T. rubrum (1208) TCTCTTTTTGGGGGGGAGAGCGGGG-- 727

T. rubrum (1044) --------------------------- 727

T. rubrum (ATCC-10218) --------------------------- 727

T. rubrum (2970) --------------------------- 727

T. rubrum (1059) TCTCTTTTTGGGGAAGCAAAATGGG-- 727

T. rubrum (1160) --------------------------- 727

T. rubrum (1298) --------------------------- 727

T. rubrum (1008) AGGGTGTGTGAAACAAACGGCGGGCC- 727 Figure 4. Contd.

T. raubitschekii strain NOMH 789 CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC 50

T. megninii strain ATCC 12106 CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC 50

T. megninii strain ATCC 12106 CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC 50

T. rubrum strain UAMH 8547 CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC 50

T. rubrum strain ATCC 28188 CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC 50

T. kanei CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC 50

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 CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG 100

T. megninii strain ATCC 12106 CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG 100

T. saudanese UAMH 8548 CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG 100

T. rubrum strain UAMH 8547 CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG 100

T. rubrum strain ATCC 28188 CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG 100

T. kanei CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG 100

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 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. megninii strain ATCC 12106 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. saudanese UAMH 8548 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. rubrum strain UAMH 8547 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. rubrum strain ATCC 28188 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. kanei GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. rubrum 5.8S rRNA gene --------------------------ACAAGGTTTCCGTAGGTGAACCTG 24


T. rubrum strain NCPF 295 ----------------------------------TCCGTAGGTGAACCTG 16

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 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. saudanese UAMH 8548 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. megninii strain ATCC 12106 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. rubrum strain UAMH 8547 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. rubrum strain ATCC 28188 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. kanei CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. rubrum 5.8S rRNA gene CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G 72

T. rubrum strain WM 06.348 ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 42

T. rubrum strain NCPF 295 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 64

T. rubrum strain 05-287-3929 ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 42

T. rubrum (1138) -------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG 35

T. rubrum (1164) -------------NNNNNAAGAATCGTAAGTGACCTGCG-CATATCA-AT 35

T. rubrum (1298) -------------NNACNNAGTATCGTAGGTGACCTGCG-CATATCA-AT 35

T. rubrum (1008) --------------NACNAAGAGCCGTAGGTGACCTGCG-CATATCA-AT 34

T. rubrum (1059) ---------------NCCAGTAACCGTAGGTGACCTGCG-CATATCA-AT 33

T. rubrum (1208) ----------------TNNGCAGACGTACGTGGGCTGCG-AATATCA-GG 32

T. rubrum (1264) --------------CNNNNNAGACCGTACGTTGGCTGCG-CATATCAGAT 35

T. rubrum (2970) --------------ANCGGACAGCCGTA-GTGGCCTGCGACATATCAGAT 35

T. rubrum (ATCC-10218) ---------------NGGGACCGCCGTA-GTGGCCTGCGACATATCAGAT 34

T. rubrum (1044) ----------NNNANCGGGACAGCCGTA-GTGGGCTGCG-CATATCAGAT 38

** * * * *

Figure 5. Comparison of nucleotide sequence between T. rubrum ITS1

orthologues. Nucleotide sequences that are present in all ITS1 are shaded in black colour. Nucleotide sequence numbering is shown on the right.

Aala et al. 6511

T.raubitschekii strain NOMH 789 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. megninii strain ATCC 12106 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. saudanese UAMH 8548 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. rubrum strain UAMH 8547 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. rubrum strain ATCC 28188 GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. kanei GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG 150

T. rubrum 5.8S rRNA gene --------------------------ACAAGGTTTCCGTAGGTGAACCTG 24


T. rubrum strain NCPF 295 ----------------------------------TCCGTAGGTGAACCTG 16

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 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. saudanese UAMH 8548 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. megninii strain ATCC 12106 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. rubrum strain UAMH 8547 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. rubrum strain ATCC 28188 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. kanei CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198

T. rubrum 5.8S rRNA gene CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G 72

T. rubrum strain WM 06.348 ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 42

T. rubrum strain NCPF 295 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 64

T. rubrum strain 05-287-3929 ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 42

T. rubrum (1138) -------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG 35

T. rubrum (1164) -------------NNNNNAAGAATCGTAAGTGACCTGCG-CATATCA-AT 35

T. rubrum (1298) -------------NNACNNAGTATCGTAGGTGACCTGCG-CATATCA-AT 35

T. rubrum (1008) --------------NACNAAGAGCCGTAGGTGACCTGCG-CATATCA-AT 34

T. rubrum (1059) ---------------NCCAGTAACCGTAGGTGACCTGCG-CATATCA-AT 33

T. rubrum (1208) ----------------TNNGCAGACGTACGTGGGCTGCG-AATATCA-GG 32

T. rubrum (1264) --------------CNNNNNAGACCGTACGTTGGCTGCG-CATATCAGAT 35

T. rubrum (2970) --------------ANCGGACAGCCGTA-GTGGCCTGCGACATATCAGAT 35

T. rubrum (ATCC-10218) ---------------NGGGACCGCCGTA-GTGGCCTGCGACATATCAGAT 34

T. rubrum (1044) ----------NNNANCGGGACAGCCGTA-GTGGGCTGCG-CATATCAGAT 38

** * * * *

T. raubitschekii strain NOMH 789 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 246

T. megninii strain ATCC 12106 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 246

T. saudanese UAMH 8548 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 246

T. rubrum strain UAMH 8547 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 246

T. rubrum strain ATCC 28188 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 246

T. kanei GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 246

T. rubrum 5.8S rRNA gene GA-CCG-ACGTTC-ATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 119

T. rubrum strain WM 06.348 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 90

T. rubrum strain NCPF 295 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 112

T. rubrum strain 05-287-3929 GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 90

T. rubrum (1138) GA-CCGGACATTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 84

T. rubrum (1164) AA-GCG--GAGGAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGG 81

T. rubrum (1298) AA-GCG--GAGGATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGG 82

T. rubrum (1008) AA-GCGA-GAGGACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCC 82

T. rubrum (1059) AA-GCG--GAGGACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCC 80

T. rubrum (1208) AA-GCGA-CATGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 80

T. rubrum (1264) AA-CCGGACATGACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 84

T. rubrum (2970) AACGCGGAGAGGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 85

T. rubrum (ATCC-10218) AACGCGGAGAGGACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCC 84

T. rubrum (1044) AACGCGGAGATTACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC 88

* ** * ***** * ** * *

T. raubitschekii strain NOMH 789 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. megninii strain ATCC 12106 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. saudanese UAMH 8548 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. rubrum strain UAMH 8547 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. rubrum strain ATCC 28188 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. kanei CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. rubrum 5.8S rRNA gene C-ATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 168

T. rubrum strain WM 06.348 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 140

T. rubrum strain NCPF 295 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 162

T. rubrum strain 05-287-3929 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 140

T. rubrum (1138) CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 134

T. rubrum (1164) ACATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 131

T. rubrum (1298) ATTCCGTTGGTTACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTG 132






T. rubrum (ATCC-10218) CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 134


*** ****** ****************** *************

T. raubitschekii strain NOMH 789 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. megninii strain ATCC 12106 CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG 346

T. saudanese UAMH 8548 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG 346

T. rubrum strain UAMH 8547 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. rubrum strain ATCC 28188 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. kanei CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. rubrum 5.8S rRNA gene CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 218

T. rubrum strain WM 06.348 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 190

T. rubrum strain NCPF 295 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 212

T. rubrum strain 05-287-3929 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 190

T. rubrum (1138) CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 184








T. rubrum (ATCC-10218) CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 184


**** ******************* **** *** ****************

T. raubitschekii strain NOMH 789 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. megninii strain ATCC 12106 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. saudanese UAMH 8548 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. rubrum strain UAMH 8547 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. rubrum strain ATCC 28188 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. kanei CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. rubrum 5.8S rRNA gene CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 268

T. rubrum strain WM 06.348 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 240

T. rubrum strain NCPF 295 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 262

T. rubrum strain 05-287-3929 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 240

T. rubrum (1138) CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 234








T. rubrum (ATCC-10218) CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 234


**************************************************

Figure 5. Contd.


T. raubitschekii strain NOMH 789 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. megninii strain ATCC 12106 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. saudanese UAMH 8548 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. rubrum strain UAMH 8547 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. rubrum strain ATCC 28188 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. kanei CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 296

T. rubrum 5.8S rRNA gene C-ATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 168

T. rubrum strain WM 06.348 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 140

T. rubrum strain NCPF 295 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 162

T. rubrum strain 05-287-3929 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 140



T. rubrum (1298) ATTCCGTTGGTTACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTG 132






T. rubrum (ATCC-10218) CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 134


*** ****** ****************** *************

T. raubitschekii strain NOMH 789 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. megninii strain ATCC 12106 CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG 346

T. saudanese UAMH 8548 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG 346

T. rubrum strain UAMH 8547 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. rubrum strain ATCC 28188 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. kanei CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 346

T. rubrum 5.8S rRNA gene CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 218

T. rubrum strain WM 06.348 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 190

T. rubrum strain NCPF 295 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 212

T. rubrum strain 05-287-3929 CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 190









T. rubrum (ATCC-10218) CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG 184


**** ******************* **** *** ****************

T. raubitschekii strain NOMH 789 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. megninii strain ATCC 12106 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. saudanese UAMH 8548 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. rubrum strain UAMH 8547 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. rubrum strain ATCC 28188 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. kanei CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 396

T. rubrum 5.8S rRNA gene CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 268

T. rubrum strain WM 06.348 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 240

T. rubrum strain NCPF 295 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 262

T. rubrum strain 05-287-3929 CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 240









T. rubrum (ATCC-10218) CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG 234


**************************************************

T. raubitschekii strain NOMH 789 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. megninii strain ATCC 12106 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. saudanese UAMH 8548 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. rubrum strain UAMH 8547 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. rubrum strain ATCC 28188 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. kanei TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. rubrum 5.8S rRNA gene TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 318

T. rubrum strain WM 06.348 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 290

T. rubrum strain NCPF 295 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 312

T. rubrum strain 05-287-3929 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 290

T. rubrum (1138) TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 284








T. rubrum (ATCC-10218) TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 284


**************************************************

T.raubitschekii strain NOMH 789 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. megninii strain ATCC 12106 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. saudanese UAMH 8548 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. rubrum strain UAMH 8547 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. rubrum strain ATCC 28188 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. kanei TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. rubrum 5.8S rRNA gene TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 368

T. rubrum strain WM 06.348 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 340

T. rubrum strain NCPF 295 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 362

T. rubrum strain 05-287-3929 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 340

T. rubrum (1138) TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 334








T. rubrum (ATCC-10218) TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 334


**************************************************

T. raubitschekii strain NOMH789 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. megninii strain ATCC 12106 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. saudanese UAMH 8548 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. rubrum strain UAMH 8547 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. rubrum strain ATCC 28188 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. kanei ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. rubrum 5.8S rRNA gene ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 418

T. rubrum strain WM 06.348 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 390

T. rubrum strain NCPF 295 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 412

T. rubrum strain 05-287-3929 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 390

T. rubrum (1138) ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 384








T. rubrum (ATCC-10218) ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 384


**************************************************

Figure 5. Contd.

Aala et al. 6513

T. raubitschekii strain NOMH 789 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. megninii strain ATCC 12106 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. saudanese UAMH 8548 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. rubrum strain UAMH 8547 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. rubrum strain ATCC 28188 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. kanei TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 446

T. rubrum 5.8S rRNA gene TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 318

T. rubrum strain WM 06.348 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 290

T. rubrum strain NCPF 295 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 312

T. rubrum strain 05-287-3929 TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 290









T. rubrum (ATCC-10218) TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC 284


**************************************************

T.raubitschekii strain NOMH 789 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. megninii strain ATCC 12106 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. saudanese UAMH 8548 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. rubrum strain UAMH 8547 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. rubrum strain ATCC 28188 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. kanei TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 496

T. rubrum 5.8S rRNA gene TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 368

T. rubrum strain WM 06.348 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 340

T. rubrum strain NCPF 295 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 362

T. rubrum strain 05-287-3929 TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 340









T. rubrum (ATCC-10218) TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA 334


**************************************************

T. raubitschekii strain NOMH789 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. megninii strain ATCC 12106 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. saudanese UAMH 8548 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. rubrum strain UAMH 8547 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. rubrum strain ATCC 28188 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. kanei ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 546

T. rubrum 5.8S rRNA gene ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 418

T. rubrum strain WM 06.348 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 390

T. rubrum strain NCPF 295 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 412

T. rubrum strain 05-287-3929 ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 390









T. rubrum (ATCC-10218) ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC 384


**************************************************

T. raubitschekii strain NOMH789 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. megninii strain ATCC 12106 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. saudanese UAMH 8548 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. rubrum strain UAMH 8547 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. rubrum strain ATCC 28188 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. kanei TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. rubrum 5.8S rRNA gene TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 468

T. rubrum strain WM 06.348 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 440

T. rubrum strain NCPF 295 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 462

T. rubrum strain 05-287-3929 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 440

T. rubrum (1138) TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 434








T. rubrum (ATCC-10218) TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 434


**************************************************

T. raubitschekii strain NOMH789 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. megninii strain ATCC 12106 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. saudanese UAMH 8548 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. rubrum strain UAMH 8547 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. rubrum strain ATCC 28188 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. kanei CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. rubrum 5.8S rRNA gene CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTC--TTCGGGGGCGGGAC 516

T. rubrum strain WM 06.348 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 490

T. rubrum strain NCPF 295 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 512

T. rubrum strain 05-287-3929 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 490

T. rubrum (1138) CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 484








T. rubrum (ATCC-10218) CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 484


********************************** **************

Figure 5. Contd.


T. raubitschekii strain NOMH789 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. megninii strain ATCC 12106 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. saudanese UAMH 8548 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. rubrum strain UAMH 8547 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. rubrum strain ATCC 28188 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. kanei TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 596

T. rubrum 5.8S rRNA gene TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 468

T. rubrum strain WM 06.348 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 440

T. rubrum strain NCPF 295 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 462

T. rubrum strain 05-287-3929 TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 440









T. rubrum (ATCC-10218) TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC 434


**************************************************

T. raubitschekii strain NOMH789 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. megninii strain ATCC 12106 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. saudanese UAMH 8548 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. rubrum strain UAMH 8547 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. rubrum strain ATCC 28188 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. kanei CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 646

T. rubrum 5.8S rRNA gene CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTC--TTCGGGGGCGGGAC 516

T. rubrum strain WM 06.348 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 490

T. rubrum strain NCPF 295 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 512

T. rubrum strain 05-287-3929 CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 490









T. rubrum (ATCC-10218) CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC 484


********************************** **************

T. raubitschekii strain NOMH789 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 696

T. megninii strain ATCC 12106 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG 696

T. saudanese UAMH 8548 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG 696

T. rubrum strain UAMH 8547 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 696

T. rubrum strain ATCC 28188 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 696

T. kanei GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 696

T. rubrum 5.8S rRNA gene GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 566

T. rubrum strain WM 06.348 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 540

T. rubrum strain NCPF 295 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 562

T. rubrum strain 05-287-3929 GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 540

T. rubrum (1138) GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 534








T. rubrum (ATCC-10218) GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG 534


***************************************** ********

T. raubitschekii strain NOMH789 GGCAGCCAATTCAGCGCCCTCAGG-------------------------- 720

T. megninii strain ATCC 12106 GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 746

T. saudanese UAMH 8548 GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 746

T. rubrum strain UAMH 8547 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 746

T. rubrum strain ATCC 28188 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 746

T. kanei GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 746

T. rubrum 5.8S rRNA gene GGCAGCCAATTCAGCGCCCTCAGGACCGGCNGCCCTGGCCCCAATCTTTA 616

T. rubrum strain WM 06.348 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 590

T. rubrum strain NCPF 295 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 612

T. rubrum strain 05-287-3929 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 590

T. rubrum (1138) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 584








T. rubrum (ATCC-10218) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA 584


********* *************

Figure 5. Contd.

Aala et al. 6515

T. raubitschekii strain NOMH789 --------------------------------------------------

T. megninii strain ATCC 12106 TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT 796

T. saudanese UAMH 8548 TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT 796

T. rubrum strain UAMH 8547 TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 796

T. rubrum strain ATCC 28188 TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 796

T. kanei TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 796

T. rubrum 5.8S rRNA gene TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 666

T. rubrum strain WM 06.348 TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 640

T. rubrum strain NCPF 295 TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 662

T. rubrum strain 05-287-3929 TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 640

T. rubrum (1138) TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG 634








T. rubrum (ATCC-10218) TATCGCGATATATCTTGGCAGGTTGACCTCGGATCAG------------- 621


T.raubitschekii strain NOMH 789 --------------------------------------------------

T. megninii strain ATCC 12106 GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC 846

T. saudanese UAMH 8548 GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC 846

T. rubrum strain UAMH 8547 CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG 846

T. rubrum strain ATCC 28188 CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG 846

T. kanei CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG 846

T. rubrum 5.8S rRNA gene CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG 716

T. rubrum strain WM 06.348 CTGAACTTAA---------------------------------------- 650

T. rubrum strain NCPF 295 CTGAACTTAAGCATATCAAT------------------------------ 682

T. rubrum strain 05-287-3929 CTGAACTTAAGCATATCAATAAGCGG------------------------ 666

T. rubrum (1138) CTGAACTTAAGCATATCAAAAG---------------------------- 656

T. rubrum (1164) CTGAACTTAAGCATATCAATAAGCGGGGAGGAA----------------- 664

T. rubrum (2970) -------------------------------------------------

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

T. rubrum (1044) ------------------------------------------------- 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

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Conventional and molecular characterization of Trichophyton rubrum

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