Advanced Journal of Microbiology Research

Advanced Journal of Microbiology Research Vol. 2010

Available online at http://internationalscholarsjournals.org/journal/ajmr

© 2010 International Scholars Journals

Full Length Research Paper

Identification of Candida glabrata and Candida parapsilosis strains by polymerase chain reaction assay using RPS0 gene fragment

Emira Noumi^1,2*, Mejdi Snoussi^1,3, Maria del Pilar Vercher², Eulogio Valentin², Lucas Del Castillo² and Amina Bakhrouf¹

¹Laboratoire d’Analyse, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Département de

Microbiologie, Faculté de Pharmacie, Monastir, Tunisia.

²Departamento de Microbiología y Ecología, Facultad de Farmacia, Universidad de Valencia, Burjassot, Valencia, Spain.

³Laboratoire de Traitement et de Recyclage des Eaux. Centre de Recherches et des Technologies des eaux, Technopôle de Borj-Cédria, BP 901, 2050 Hammam-Lif, Tunisia.

Accepted 02 April, 2010

Abstract

Two Candida species were identified by the amplification of the RPS0 gene intron fragment. For this, two pairs of primers were used in PCR analysis performed with genomic DNA of clinical isolates of Candida. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. For Candida glabrata, the size of the amplicon was 406 pb and 150 bp for C. parapsilosis. The designed primers were able to amplify all C. glabrata isolates. One of three C. parapsilosis strains was confirmed as C. orthopsilosis, when we used the designed oligonucleotides. The used primers cannot amplify the other Candida species such as C. albicans. These results indicate that sequences of intron genes can be useful to specifically identify Candida strains by PCR. This molecular identification will be considered as an early identification of Candida species responsible for all candidiasis.

Key words: Candida glabrata, C. parapsilosis, C. orthopsilosis, PCR- identification, RPS0 intron.

Advanced Journal of Microbiology Research Vol. 2010

Available online at http://internationalscholarsjournals.org/journal/ajmr

© 2010 International Scholars Journals

Full Length Research Paper

Effect of some alternative medicine and biological factors on Candida albicans in Saudi Arabia

Amany H. Aboellil^1* and Majdah M. Y. Al-Tuwaijri²

Botany Department (Microbiology), Faculty of Applied Science (Girl Branch), Umm Al Qura University, Makkah Almukarramah, Saudi Arabia.

Accepted 09 March, 2010

Abstract

Yeast of the genus Candida has been recognized as important microorganisms responsible for Nosocomial fungemia. Six isolates of clinical Candida albicans isolated from patients and studied by electrophoretic karyotyping of chromosomal DNA by pulsed field gel electrophoresis. Six chromosal DNA profiles were more or less identical. Natural fungicide nystatin is more effective on C. albicans growth than the other synthesitic miconazole nitrate and clotrimazole. The minimum inhibitory concentration for 90% growth of C. albicans (MIC90) of nystatin is1500 unit/ml. The antimicrobial activities of alternative and traditional medicine in Saudi Arabia were evaluated against C. albicans growth. Natural acetic acid, plant extract, charcoal and Elshab are used as natural drug for Candidiasis. The percent of 36% Concentration of acetic acid in media causes maximum inhibition of C. albicans growth. There are direct relation between acetic acid concentrations and inhibition zones. Increase or decrease of pH inhibits the growth of C. albicans. The ideal pH for inhibiting C. albicans growth is pH 10.  The combination of Salvadora persica extract (Meswak) and Elshab (KAl(SO₄)₂) in concentration of (28.5 + 12.75 mg/ml, respectively), have an antimicrobial effect on C. albicans growth (0.01 g/10ml of media). Charcoal in 20 mg/ml concentration or Lawsonia alba extract (Henna) in 250 mg/ml is the ideal concentration used for stop growth of C. albicans. The susceptibility of yeast to the previous alternative medicine shows that, it could be used in treating candidiasis resulting from different strains of C. albicans instead of commercial antibiotics used, without any adverse side effects. Also, it is an attempt to stop development of multidrug resistance organism. Aspergillus fumigatus performed a good tool as biocontrol for C. albicans which caused infections mainly candidiasis. Both are isolated as clinical isolates of nosocomial of infected patients.

Key words: Candida albicans, chromosomal DNA, Lawsonia alba, Salvadora persica, antibiotics, biocontrol.

Advanced Journal of Microbiology Research Vol. 2010

Available online at http://internationalscholarsjournals.org/journal/ajmr

© 2010 International Scholars Journals

Full Length Research Paper

Antagonism of Trichoderma farzianum isolates on soil borne plant pathogenic fungi from Embu District, Kenya

E. N. Siameto¹, S. Okoth^2*, N. O. Amugune², and N. C. Chege²

¹School of Sciences, Narok University College, P. O. Box 861, Narok, Kenya.

²School of Biological Sciences, University of Nairobi, P. O. Box 30197, Nairobi.

Accepted 22 January, 2010

Abstract

Species in the genus Trichoderma are important as commercial source of several enzymes and as biofungicides/growth promoters. The most common biological control agents of the genus are strains of T. harzianum, T. viride and T. viriens. In this study, sixteen selected isolates of T. harzianum from different land use types in Embu, Kenya were tested for anatognism against five soil borne phytopathogenic fungi (Rhizoctonia solani, Pythium sp, Fusarium graminearum, F. oxysporum f. sp phaseoli and F. oxysporum f. sp Lycopersici) using dual culture assay and through production of non-volatile inhibitors. Seven isolates were further characterized using RAPD-PCR procedure to determine genetic variability. All T. harzianum isolates had considerable antagonistic effect on mycelial growth of the pathogens in dual cultures compared to the controls. Maximum inhibitions occurred in Pythium sp-055E interactions (73%).The culture filtrates obtained from Czapek’s liquid medium reduced the dry weight (mg) of the mycelia significantly while those from the potato dextrose broth showed minimum inhibition growth. Pythium sp was inhibited the most compared to other pathogens. Genetic similarities generated using Jacquard’s coefficient of similarity ranged from 0.231 between isolates 055E and 011E to 0.857 between isolates 010E and 015E. The technique of RAPD was efficient in demonstrating the DNA polymorphism in the isolates of T. harzianum tested showing intraspecific genetic variability. Since all T. harzianum isolates evaluated were effective in controlling colony growth of the soil borne pathogens both in dual cultures and in culture filtrates they could be tried as a broad spectrum biological control agent in the green house and under field conditions.

Key words: Trichoderma harzianum, growth antagonism, genetic similarity, RAPDs.

Advanced Journal of Microbiology Research Vol. 2010

Available online at http://internationalscholarsjournals.org/journal/ajmr

© 2010 International Scholars Journals

Full Length Research Paper

Molecular cloning and characterization of STL1 gene of Debaryomyces hansenii

Juan Carlos González-Hernández

Laboratorio de Bioquímica del Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Morelia; Avenida

Tecnológico 1500. C. P. 58120. Morelia, Michoacán, México. E-mail: [email protected]. Tel: (+52-433) 3121570 Ext. 240. Fax: (+52-433) 3121570 Ext. 211.

Accepted 26 April, 2010

Abstract

Debaryomyces hansenii is often found in salty environments. This yeast species is not only halotolerant, but also halophilic. Its genome sequence is known completely, but the mechanisms behind its halotolerance are poorly understood. It was compared to the STL1 protein sequence of Saccharomyces cerevisiae against the translated sequence from the D. hansenii genome sequence database released by Génolevure. An ORF (DEHA0E01122g) was found with 54% homology and 39% identity with Stl1p from S. cerevisiae. DhSTL1 was heterologously expressed successfully in a S. cerevisiae (BY4741) wild type and in another strain lacking its own system for the glycerol transport (STL1) gene. The DhSTL1 gene in transformed S. cerevisiae strains showed a slight but significant difference in the doubling times in growth curves obtained in liquid YNB-ura medium, with glycerol as carbon source. DhSTL1 gene in transformed stl1 yeast strain showed phenotype growth at pH 7.5 under salt stress conditions (glucose as carbon source). The kinetic parameters of transport and glycerol accumulation conferred by DhSTL1 in the S. cerevisiae transformant strains did not show significant differences. An increase in the transcript level of DhSTL1 gene in the presence of saline stress at pH 5.6; whereas, at 7.5 pH, it was expressed in all evaluated conditions.

Key words: Debaryomyces hansenii, glycerol, transport, salt tolerance.

Advanced Journal of Microbiology Research Vol. 2010

Available online at http://internationalscholarsjournals.org/journal/ajmr

© 2010 International Scholars Journals

Full Length Research Paper

Study on combined antimicrobial activity of some biologically active constituents from wild Moringa peregrina Forssk

M. A. Abdel-Rahman Tahany¹*, A. K. Hegazy¹, A. Mohsen Sayed¹, H. F. Kabiel¹, T. El-Alfy² and S. M. El-Komy²

¹Cairo University, Faculty of Science, Cairo, Egypt.

²Cairo University, Faculty of Pharmacy, Cairo, Egypt.

Accepted 31 July, 2010

Abstract

Three plants were used in this study: Moringa pregrina, Achillea fragrantissima and Coleome droserifolia. Six active constituents were separated from n-hexane and ethyl acetate fractions of Moringa pregrina. These active constituents were lupeol acetate, -amyrin, -amyrin, -sitosterol, - sitosterol-3-O- -D-glucoside and apignin were assayed individually and in combination against pathogenic bacteria and fungi. All constituents were proved to be more antibacterial than antifungal agents. Aspergillus flavus and Fusarium solani were completely resistant to all constituents. -amyrin was the most effective antibacterial compound. The least relative activity was achieved by -sitosterol against Bacillus subtilis compared to ampicillin. Reasonable antifungal activity was recorded in case of lupeol acetate, -amyrin and -amyrin, while -sitosterol and -sitosterol-3-O- -D-glucoside, revealed no antifungal activity. Apignin missed both antifungal and antibacterial activities. Low MICs were detected by -amyrin, -amyrin and -sitosterol-3-O- -D-glucoside against all tested bacteria. Concerning fungi, -sitosterol and - sitosterol- 3-O- -D-glucoside showed no antimycotic activity. Lupeol acetate, -amyrin and -amyrin, however, have slightly high MICs for all tested dermatophytic fungi compared to that of fluconazole. Among ninety nine assayed combination mixtures, thirty seven synergistic combination mixtures were detected which exerts 37 synergisms against different pathogens with FICI less than 0.5, which indicates high efficacy of combination mixtures over monotherapy treatments.

Key words: Combined antimicrobial therapy, active constituents, Moringa peregrina, Achillea fragrantissima, Coleome droserifolia.

Advanced Journal of Microbiology Research Vol. 2010

Available online at http://internationalscholarsjournals.org/journal/ajmr

© 2010 International Scholars Journals

Full Length Research Paper

Isolation and characterization of a fungal isolate for phosphate solubilization and plant growth promoting activity

Varsha Nenwani, Pratima Doshi, Tithi Saha and Shalini Rajkumar*

Department of Biochemistry and Biotechnology, Institute of Science, Nirma University, Sarkhej – Gandhinagar Highway, Ahmedabad Gujarat 382 481, India.

Accepted 21 July, 2010

Abstract

A fungal species namely F1 was isolated from the rhizosphere on the basis of its ability to form halos (zone of solubilization) on Pikovskaya’s agar. F1 was assessed for phosphate solubilization, titratable acidity (TA), gluconate concentration and change in pH over incubation period of 21 days and other plant growth promoting traits. F1 solubilized maximum inorganic phosphorus (662 g P ml^-1) from tricalcium phosphate present in the Pikovskaya’s broth on 18th day. The TA followed a similar trend as that of P solubilized, except on day 21 when the value that for TA was highest. A similar pattern was also observed with production of gluconic acid, for which a constant value of 8.96 × 10^-4 g% was observed till 18th day of incubation. However, gluconic acid was not the only organic acid produced in the culture broth, because the amount of gluconic acid produced did not relate to the high values observed for P solubilized and TA. Efficiency of the F1 to solubilize phosphate from organic reserves was determined by performing assays of phosphatases and phytases. The culture F1 produced 1.86 and 1.90 EU of enzymes alkaline and acid phosphatase, respectively and phytase activity was 28 mU. The concentration of catechol and hydroxymate type siderophores produced by F1 was 4.50 and 4.55 µgml^-1 respectively and it also produced 11.45 µgml^-1 of IAA which is significantly high. Some fungi may possess traits associated with biocontrol of plant pathogens such as production of enzyme chitinase which was 0.037 EU for F1. On the basis of cultural and microscopic features, the isolate F1 could be Absidia spp. and has potential of being a competent bioinoculant.

Key words: Phosphate solubilization, bioinoculant, plant growth promoting fungus (PGPF).