Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants.

Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 µg (in 100-µL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.

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.

First morphogenetic identification of the fungal pathogen Colletotrichum musae (Phyllachoraceae) from imported bananas in Saudi Arabia.

Colletotrichum musae is the causal agent of anthracnose in banana fruits; infection by this fungal pathogen results in severe post-harvest losses. Eleven C. musae isolates were obtained from infected imported banana fruit samples with anthracnose lesions collected from different markets in Riyadh, Saudi Arabia. The pathogenic, morphological, cultural, and molecular characteristics of these C. musae isolates were evaluated. The cultures had characteristic fast-growing sparse aerial mycelia that were white, with copious cinnamon conidial masses, conidia usually elliptical, and setae absent. An inoculation test was used to determine whether isolates could cause anthracnose symptoms on banana fruits. Necrotic lesions developed and orange-colored spore structures were later observed on these lesions. Microsatellite-primed PCR (MP-PCR) was used to identify genetic variation among the C. musae isolates. The dendrogram obtained from cluster analysis of the MP-PCR fingerprints revealed a great deal of homogeneity among the isolates, shown by the formation of two clusters. Intraspecific similarity among the C. musae isolates ranged from 83 to 100%. This is the first report demonstrating morphological and genetic variation within a population of C. musae in Saudi Arabia.

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.

Rapid and efficient extraction of genomic DNA from different phytopathogenic fungi using DNAzol reagent.

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 microg g(-1) dry mycelia and the A(260)/A(280) ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A(260)/A(280) ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.