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

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

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

Molecular detection of <i>Fusarium oxysporum</i> f. sp. <i>vasinfectum</i> in cotton roots by PCR andreal-time PCR assay

A PCR assay based on a pair of oligonucleotide primers targeting the 16S and 23S rRNA genes was used to detect Fusarium oxy­sporum f. sp. vasinfectum (Fov), a fungus causing Fusarium wilt of cotton, in infected cotton seedlings. By using the primer pair Fov1-Eg-f and Fov1-Eg-r, a 438-bp DNA fragment was consistently amplified from 41 Egyptian isolates of Fov (race 3), while no amplification was obtained from template genomic DNAs obtained from other fungal pathogens, namely F. oxysporum, F. solani, F. verticillioides, F. sambucinum, Rhizoctonia solani, and Macrophomina phaseolina. PCR product of the expected size was amplified from as little as 100 fg of purified Fov genomic DNA. The sensitivity of real-time PCR allowed detection of 200 fg of target genomic DNA. The melting temperature (Tm) values of Fov isolates were in the range 86.83 to 87.02. Fov DNA was detected in infected cotton roots, while no amplification was obtained from other plant structures, such as stem and leaf. The real-time PCR assay successfully detected Fov and correlated well with assessments based on conventional PCR techniques. The application of this technique will be useful for identification of Fov in infected cotton roots and detection of Fov in infected plants that lack visible morphological structures or symptoms

Comparison of multi-locus enzyme and protein gel electrophoresis in the discrimination of five Fusarium species isolated from Egyptian cottons

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Fusarium   species recovered from cotton-growing areas in Egypt by numerical taxonomy methods. The obtained data revealed that SDS-PAGE and esterase isozymes are more efficient in grouping isolates in their respective species while peroxidase and malate dehydrogenase isozyme has much limited resolution in organizing all isolates in their respective species-specific clusters. A low correlations was detected between geographical origin of isolates and genetic diversity. Results indicate that the estimated inter-specific variation may be more pronounced with protein markers than with isoyzmes when the two approaches are applied to the same populations. The level of genetic variability detected within and between Fusarium spp. accessions with protein and esterase isoyzmes analysis suggests that it is a reliable, efficient, and effective marker technology for determining genetic relationships in Fusarium genus