Bioinformatic tools and guideline for PCR primer design

Bioinformatics has become an essential tool not only for basic research but also for applied research in biotechnology and biomedical sciences. Optimal primer sequence and appropriate primer concentration are essential for maximal specificity and efficiency of PCR. A poorly designed primer can result in little or no product due to non-specific amplification and/or primer-dimer formation, which can become competitive enough to suppress product formation. There are several online tools devoted to serving molecular biologist design effective PCR primers. This review intends to provide a guide to choosing the most efficient way to design a new specific-primer by applying current publicly available links and Web services. Also, the purpose here is to provide general recommendations for the design and use of PCR primers. (African Journal of Biotechnology: 2003 2(5): 91-95

Web-based bioinformatic resources for protein and nucleic acids sequence alignment

DNA sequencing is the deciphering of hereditary information. It is an indispensable prerequisite for many biotechnical applications and technologies and the continual acquisition of genomic information is very important. This opens the door not only for further research and better understanding of the architectural plan of life, but also for future clinical diagnosis based on the genetic data of individuals. Bioinformatics can be broadly defined as the creation and development of advanced information and computational techniques for problems in biology. More narrowly, bioinformatics is the set of computing techniques used to manage and extract useful information from the DNA/RNA/protein sequence data being generated (at high volumes) by automated techniques (e.g., DNA sequencers, DNA microarrays) and stored in large public databases (e.g., GenBank, Protein DataBank). Certain method for analyzing genetic/protein data has been found to be extremely computationally intensive, providing motivation for the use of powerful computers. The advent of the Internet and the World Wide Web (WWW) has substantially increased the availability of information and computational resources available to experimental biologists. This review will describe the current on-line resources available, including protein and nucleic acids sequence alignment. Key words: Sequence alignment, DNA, Protein, ClustalW, FASTA. African Journal of Biotechnology Vol. 2 (12), pp. 714-718, December 200

Organic and Inorganic Salts as Postharvest Alternative Control Means of Citrus

Several postharvest disease control means alternative to conventional chemical fungicides, such as organic and inorganic salts, will be highlighted in the proposed chapter. In particular, it will comprehensively cover different aspects of the use of salts against postharvest Penicillium decay of citrus. It will be an essential resource for the graduate and postgraduate students, researchers, professionals, supply chain players, citrus industries, and retailers. Organic and inorganic salts have a broad spectrum of activity against a wide range of fungi. In addition, they are easy to apply, inexpensive, safe for humans and the environment, and suitable for commercial postharvest handling practices. Different application strategies of salts, before and after harvest, and combined application (with wax, natural compounds, and fungicides, etc.) will be also discussed. The present chapter attempts to highlight how the use of organic and inorganic salts as alternative postharvest disease management technologies has developed from the laboratory to the market

Molecular phylogeny of Fusarium species by AFLP fingerprint

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships within and between natural populations of five Fusarium spp. AFLP templates were prepared by the digestion of Fusarium DNA with EcoRI and MseI restriction endonucleases and subsequent ligation of corresponding site-specific adapters. An average of 44 loci was assayed simultaneously with each primer pair and DNA markers in the range 100 to 500 bp were considered for analysis. A total of 80 AFLP polymorphic markers were obtained using four primer combinations, with an average of 20 polymorphic markers observed per primer pair. UPGMA analyses indicated 5 distinct clusters at the phenon line of 30% on the genetic similarity scale corresponding to the 5 taxa. The similarity percent of each group oscillated between 87 and 97%. The phenetic dendrogram generated by UPGMA as well as principal coordinate analysis (PCA) grouped all of the Fusarium spp. isolates into five major clusters. No clear trend was detected between clustering in the AFLP dendrogram and geographic origin, host genotype of the tested isolates with a few exceptions. The results of the present study provide evidence of the high discriminatory power of AFLP analysis, suggesting the possible applicability of this method to the molecular characterization of Fusarium. (African Journal of Biotechnology: 2003 2(3): 51-55

Genetic affinities of Fusarim spp. and their correlation with origin and pathogenicity

Random amplified polymorphic DNA (RAPD) analyses was used in combination with pathogenicity assays to study the taxonomic kinships among five Fusarium species. A total of 46 isolates of Fusarium spp. obtained from diseased cotton seedlings showing typical root rot and dampping-off symptoms were characterized. Of 10 primers tested, four primers produced polymorphic amplification patterns with taxon-specific bands, in addition to individual-specific bands. Genetic analysis indicated into 2 main clusters, with the minor cluster included all F. moniliforme and F. solani at the genetic similarity of GS=57.82%. The major cluster consisted of all F. oxysporum, F. avenaceum and F. chlamydosporum clustered at 71% similarity. There was no clear-cut relationship between clustering in the RAPD dendrogram, pathogenicity test and geographic origin of tested isolates. The results suggest that RAPD-PCR is a useful method for analysing genetic variation within and between Fusarium spp. (African Journal of Biotechnology: 2003 2(5): 109-113

PCR identification of Fusarium genus based on nuclear ribosomal-DNA sequence data

We have developed two taxon-selective primers for quick identification of the Fusarium genus. These primers, ITS-Fu-f and ITS-Fu-r were designed by comparing the aligned sequences of internal transcribed spacer regions (ITS) of a range of Fusarium species. The primers showed good specificity for the genus Fusarium, and the approximately 389-bp product was amplified exclusively. PCR sensitivity ranged from 100 fg to 10 ng for DNA extracted from Fusarium oxysporum mycelium. No amplification products were detected with PCR of DNA from Rhizoctonia solani and Macrophomina phaseolina isolates using these primers. The assay is useful for rapid identification of Fusarium spp. cultures. The application of these PCR methods for early diagnosis of the seedling and wilt disease of cotton needs to be studied further. (African Journal of Biotechnology: 2003 2(4): 82-85

Characterization of Novel Di-, Tri-, and Tetranucleotide Microsatellite Primers Suitable for Genotyping Various Plant Pathogenic Fungi with Special Emphasis on Fusaria and Mycospherella graminicola

The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence of Mycospherlla graminicola and Fusarium graminearum. A total of 20 new microsatellite primers as easy-to-score markers were developed. Microsatellite primer PCR (MP-PCR) yielded highly reproducible and complex genomic fingerprints, with several bands ranging in size from 200 to 3000 bp. Of the 20 primers tested, only (TAGG)4, (TCC)5 and (CA)7T produced a high number of polymorphic bands from either F. graminearum or F. culmorum. (ATG)5 led to successful amplifications in M. graminicola isolates collected from Germany. Percentage of polymorphic bands among Fusarium species ranged from 9 to 100%. Cluster analysis of banding patterns of the isolates corresponded well to the established species delineations based on morphology and other methods of phylogenetic analysis. The current research demonstrates that the newly designed microsatellite primers are reliable, sensitive and technically simple tools for assaying genetic variability in plant pathogenic fungi

The Genomes of the Fungal Plant Pathogens Cladosporium fulvum and Dothistroma septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogensCladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu \u3e61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation

Special Issue: Microbial Nanotechnology

Microbial nanotechnology (MN), or microbial nanobiotechnology, is a rapidly expanding research area with the potential to transform various fields, including bioremediation, energy production, medicine, and agriculture [...

Special Issue: Agricultural Nanotechnology

Agricultural nanotechnology has considerable promise for addressing global agricultural production/security, biodiversity, and global warming issues. Current trends in publications and patents demonstrate that biotechnology technologies, particularly for crops, are being developed to improve agricultural productivity and disease management. In the current issue, we strongly advocate for the use of biosynthesized nanoparticles from a variety of sources, including plants, agricultural waste, and microbes, as a prerequisite for significant and in-depth study. Nanomaterials offer a wide range of practical uses in agriculture, including nanofertilizers, nanopesticides, nanoherbicides, nanosensors, and smart delivery systems for controlled agrochemical release. Additionally, nano-tools are employed for plant breeding and genetic manipulation. A thorough examination of the physicochemical soil properties of the agricultural fields where nanoparticles will be used will aid in minimizing their impact on plant and soil biota. Finally, and most importantly, we strongly recommend the inclusion of nanotoxicity, legislation, biosafety, and risk assessment as the top priorities when developing regulatory policies to address biosafety concerns. Starting today, thorough efforts must be carried out to advance and develop futuristic work based on recognized knowledge shortages