AJMR- 20 September, 2012 Issue

Volume 6 Number 36 20 September, 2012

African Journal of

Microbiology Research

ISSN 1996-0808

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Dept. of Environmental Health Sciences,

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OMU Medical School,

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International Journal of Medicine and Medical Sciences

African Journal of Microbiology Research

ARTICLES

Research Articles

Conventional and molecular characterization of Trichophyton rubrum 6502

Farzad Aala, Rosimah Nulit, Umi Kalsom Yusuf and Sassan Rezaie

Inhibitory effect of some plant extracts on clinical isolates of Staphylococcus

aureus 6517

Rajaa Milyani and Nahed Ashy

Role of CSE1034 in bacterial lipids and polysaccharides involved in biofilm

formation: a comparison with other drugs 6525

Chaudhary Manu and Anurag Payasi

Analytical specificity and sensitivity determination of 16SrRNA gene based

diagnostic polymerase chain reaction (PCR) for molecular detection of

Coxiella burnetii 6532

Mohammad Soleimani, Keivan Majidzadeh A., Amirhossein Mohseni and

Mohammad Khalili

Effect of Bacillus cereus Br on bacterial community and gossypol content

during fermentation in cottonseed meal 6537

Xin Wang, Jiang-wu Tang, Xiao-hong Yao, Yi-fei Wu, Hong Sun and Yao-xing Xu

Identification and characterization of a fungal strain with lignin and cellulose

hydrolysis activities 6545

Ran Jin, Hongdong Liao, Xuanming Liu, Mang Zheng, Xianqiu Xiong, Xinwu Liu,

Liyong Zhang and Yonghua Zhu

Table of Contents: Volume 6 Number 36 20 September, 2012

ARTICLES

Influence of ciprofloxacin on glioma cell line GL26: A new application for

an old antibiotic

Abdolreza Esmaeilzadeh, Massoumeh Ebtekar, Alireza Biglari and

Zuhair Mohammad Hassan 4891

Identification of microbial diversity in caecal content of broiler chicken

S. Nathiya, G. Dhinakar Raj, A. Rajasekar, D. Vijayalakshmi and T. Devasena 4897

Microbial quality of some non-sterile pharmaceutical products sourced

from some retail pharmacies in Lagos, Nigeria

Adeola Anifowoshe R., Opara Morrison I. and Adeleye Isaac A. 4903

Molecular detection of adhesins genes and biofilm formation in methicillin

resistant Staphylococcus aureus

Karima BEKIR, Omayma HADDAD, Mohammed GRISSA, Kamel CHAIEB,

Amina BAKHROUF and Salem IBRAHIM ELGARSSDI 4908

Amylase production by moderately halophilic Bacillus cereus in solid

state fermentation

P. Vijayabaskar, D. Jayalakshmi and T. Shankar 4918

Networking clusters and sequence characteristics of clustered regularly

interspaced short palindromic repeats (CRISPR) direct repeats and their

evolutionary comparison with cas1 genes in lactic acid bacteria

Kaibo Deng, Fei Liu, Chuntao Gu and Guicheng Huo 4927

Antibacterial screening of the root, stem and leaf extracts of Terminalia albida sc.

elliot on selected pathogenic bacteria

S. M. Ayodele, G. Alpheus and O. M. Iruaga

1457

Table of Content: Volume 6 Number 23 21 June, 2012

ARTICLES

DNA viral infections and transient bone marrow failure in southern Iran 6551

Kambiz Bagheri, Mohammad Hossein Karimi, Ramin Yaghobi,

Behnam Mohammadi, Mehdi Dehghani and Padideh Ebadi

Survival of microorganisms in high pressure treated minced meat during

chilled storage and at pH and temperature mimicking gastrointestinal tract 6558

Sami Bulut

Efficacy and toxicity of neutralizers against disinfectants and antiseptics

used in vaccine production facility 6565

Norhan S. Sheraba, Aymen S. Yassin, Aly Fahmy and Magdy A. Amin

Effects of essential oil extracted from Citrullus colocynthis (CCT) seeds on

growth of phytopathogenic bacteria 6572

Zahra Setayesh Mehr, Nima Sanadgol and LeylaVafadar Ghasemi

Development and evaluation of a novel TaqMan fluorescence probe-based

real-time reverse transcriptase polymerase chain reaction assay for

detection and quantification of West Nile virus 6576

Lijun Shi, Huiqiong Yin, Jingang Zhang, Zhan-zhong Zhao and Gang Li

Microbial water quality in the upper Olifants River catchment: Implications

for health 6580

W. J. le Roux, L. M. Schaefer and B. Genthe

Partial characterization of a bacteriocin produced by Lactobacillus alivarius

isolated from oral cavity of desert foxes 6589

Aly E. Abo-Amer and Mohammed Y. Shobrak

Table of Contents: Volume 6 Number 36 20 September, 2012

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 Aala

1

*, Rosimah Nulit

2

, Umi Kalsom Yusuf

2

and Sassan Rezaie

3

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

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 (1st 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

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

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.

6508 Afr. J. Microbiol. Res.

> 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. 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 (1164) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150 T. rubrum (1208) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150 T. rubrum (1044) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150 T. rubrum (ATCC-10218) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150

T. rubrum (2970) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150 T. rubrum (1059) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150 T. rubrum (1160) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150 T. rubrum (1298) TACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGC 150 T. rubrum (1008) TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC 150 ****** ****************************************** T. rubrum (1138) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200 T. rubrum (1164) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200 T. rubrum (1208) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200 T. rubrum (1044) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200

T. rubrum (ATCC-10218) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200

T. rubrum (2970) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200 T. rubrum (1059) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200 T. rubrum (1160) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200 T. rubrum (1298) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA 200 T. rubrum (1008) 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 (1164) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (1208) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (1044) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (ATCC-10218) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (2970) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (1059) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (1160) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (1298) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 T. rubrum (1008) GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT 250 ************************************************** T. rubrum (1138) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (1164) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (1208) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (1044) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (ATCC-10218) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (2970) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (1059) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (1160) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (1298) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 T. rubrum (1008) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC 300 ************************************************** T. rubrum (1138) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (1164) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (1208) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (1044) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (ATCC-10218) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (2970) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (1059) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (1160) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (1298) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 T. rubrum (1008) ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT 350 ************************************************** T. rubrum (1138) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (1164) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (1208) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (1044) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (ATCC-10218) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (2970) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (1059) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (1298) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 T. rubrum (1008) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG 400 ************************************************** T. rubrum (1138) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (1164) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (1208) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (1044) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (ATCC-10218) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (2970) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (1059) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (1160) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 T. rubrum (1008) GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT 450 ************************************************** T. rubrum (1138) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (1164) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (1208) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (1044) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (ATCC-10218) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (2970) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (1059) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (1160) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (1298) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 T. rubrum (1008) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA 500 ************************************************** T. rubrum (1138) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (1164) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (1208) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (1044) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (ATCC-10218) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (2970) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (1059) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (1160) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (1298) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 T. rubrum (1008) GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT 550 ************************************************** T. rubrum (1138) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (1164) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (1208) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (1044) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (ATCC-10218) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATAT 600 T. rubrum (2970) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (1059) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (1160) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (1298) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 T. rubrum (1008) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT 600 ****************************************** **** T. rubrum (1138) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (1164) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (1208) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (1044) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (ATCC-10218) ATCTTGGCAGGTTGACCTCGGATCAGGTAGGGATACGT--- 650 T. rubrum (2970) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (1059) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (1160) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (1298) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 T. rubrum (1008) ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG 650 ***** ***************************** Figure 4. Contd.

6510 Afr. J. Microbiol. Res.

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 WM 06.348 --- 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.