An efficient method for DNA extraction from Cladosporioid fungi.

We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A(260)/A(280) ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses.

Molecular detection of ochratoxigenic Aspergillus species isolated from coffee beans in Saudi Arabia.

Ten fungal isolates from coffee beans were morphologically identified as Aspergillus niger, A. ochraceus and A. carbonari-us (N = 5, 3, and 2, respectively). Only one isolate, morphologically identified as A. niger, was unable to produce ochratoxin A (OTA). This may be a new species in the Aspergillus section Nigri. OTA levels in all the other isolates were above the limit of detection (0.15 mg/kg). Based on microsatellite-primed PCR (MP-PCR) profiles, using three microsatellite primers, three main groups were obtained by UPGMA cluster analysis: A. niger, A. ochraceus and A. carbonarius. A clear-cut association was found between the MP-PCR genotype and the ability to produce OTA. Using the primer pairs OCRA1/OCRA2, a single fragment of about 400 bp was amplified only when genomic DNA from the A. ochraceus isolates was used.

Application of RAPD for molecular characterization of plant species of medicinal value from an arid environment.

The use of highly discriminatory methods for the identification and characterization of genotypes is essential for plant protection and appropriate use. We utilized the RAPD method for the genetic fingerprinting of 11 plant species of desert origin (seven with known medicinal value). Andrachne telephioides, Zilla spinosa, Caylusea hexagyna, Achillea fragrantissima, Lycium shawii, Moricandia sinaica, Rumex vesicarius, Bassia eriophora, Zygophyllum propinquum subsp migahidii, Withania somnifera, and Sonchus oleraceus were collected from various areas of Saudi Arabia. The five primers used were able to amplify the DNA from all the plant species. The amplified products of the RAPD profiles ranged from 307 to 1772 bp. A total of 164 bands were observed for 11 plant species, using five primers. The number of well-defined and major bands for a single plant species for a single primer ranged from 1 to 10. The highest pair-wise similarities (0.32) were observed between A. fragrantissima and L. shawii, when five primers were combined. The lowest similarities (0) were observed between A. telephioides and Z. spinosa; Z. spinosa and B. eriophora; B. eriophora and Z. propinquum. In conclusion, the RAPD method successfully discriminates among all the plant species, therefore providing an easy and rapid tool for identification, conservation and sustainable use of these plants.

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil.

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.

Interpretation of electrophoretograms of seven microsatellite loci to determine the genetic diversity of the Arabian Oryx.

Microsatellite markers are commonly used for examining population structure, especially inbreeding, outbreeding and gene flow. An array of microsatellite loci, preferably with multiallelic presentation, is preferable for ensuring accurate results. However, artifact peaks or stutters in the electrophoretograms significantly hamper the reliable interpretation of genotypes. We interpreted electrophoretograms of seven microsatellite loci to determine the genetic diversity of the Arabian Oryx. All the alleles of different loci exhibited good peak resolutions and hence were clearly identified. Moreover, none of the stutter peaks impaired the recognition or differentiation between homozygote and heterozygote. Our findings suggest that correct identification of alleles in the presence of co-amplified nonspecific fragments is important for reliable interpretation of microsatellite data.

Airborne viable fungi in Riyadh and allergenic response of their extracts.

The allergenicity and antigenicity of various airborne fungi isolated from the atmosphere of Riyadh were studied. Protein nitrogen contents were estimated and found to range from 0.9 mg ml(-1) for Cladosporium to 2.1 mg ml for Aspergillus extracts. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis for those extracts exhibited a number of protein bands of higher molecular weight between 13 and 80 kDa for Alternaria, Ulocladium, Penicillium, Aspergillus and Cladosporium. Extracts in both aqueous and lyophilized forms were sterilized and tested for diagnostic skin prick test in 100 consecutive patients having bronchial asthma and allergic rhinitis. Overall, 13% of patients reacted positively to fungal extracts, revealing allergic sensitization to these fungi. These findings necessitate further investigation as regards the purification and characterization of these local extracts for better diagnostic use in patients in Saudi Arabia.

Seasonal incidence of airborne fungal allergens in Riyadh, Saudi Arabia.

Aerobiological studies to evaluate allergenic fungal spores in the atmosphere of Riyadh were conducted. Burkard personal volumetric sampler were operated as volumetric 'viable' spore traps at two different sites (Al-Batha, a more developed area in the south and Al-Ulia, a less developed area in the north) in Riyadh. Twice a week samplings were carried out over a period of 12 month. The seasonal fluctuations of the most frequent fungi were plotted as 'major' components. The dominant species at the two sites were members of the genera Alternaria, Aspergillus, Cladosporium, Penicillium and Ulocladium. Drechslera, Fusarium, Rhizopus and Stachybotrytis species were minor components or sporadic. Seasonal variations of the total colonies were significantly (p < 0.05) different. They showed higher concentrations in the winter season and the lowest in summer. The Al-Batha site was always higher in spore concentrations than the Al-Ulia site. The results provide valuable information for the diagnosis and prophylaxis of allergic diseases due to airborne fungi.

Fungal flora in house dust in Riyadh, Saudi Arabia.

Fungi in the house-dust of houses in the less populated and densely populated areas of Riyadh, Saudi Arabia, were screened. The screened area included drawing rooms, living rooms, kitchens, bedrooms and bathrooms. A total of 74 fungal species were isolated. The highest number of fungal colonies were found in the living rooms followed by bedrooms. The number of fungal colonies isolated were higher in densely populated areas compared with less populated areas. Aspergillus was the predominant genus represented by highest number of species followed by Penicillium.

In vitro production of pectolytic and cellulolytic enzymes byColletotrichum lindemuthianum isolated from soybean grown in Saudi Arabia.

Colletotrichum lindemuthianum isolated from soybean in Saudi Arabia produced polygalacturonase, pectin methylesterase, pectin trans-eliminase and carboxymethylcellulasein vitro. Polygalacturonase showed maximaum activity at 30 to 35°C and pH 4.0 to 5.0. The absorption maximum for pectin trans-eliminase reaction products was at approximately 548 nm. The polygalacturonase and pectin trans-eliminase activities increased with culture age. The degradation of carboxymethylcellulose was also demonstrated.

Production of xylanase by Verticillium dahliae.

Xylan induced the production of xylanase by Verticillium dahliae. Other cellulolytic enzymes such as glucanase and beta-glucosidase were synthesized in smaller quantities. The process of degradation indicated that xylanase behaved like a typical endo-enzyme causing first production of high mol, wt. products, and indicated that V. dahliae produced at least three enzymes which degrade xylan.