SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE‐PRIMED PCR FOR THE IDENTIFICATION OF <i>RHIZOCTONIA SOLANI</i> AND SOME PHYTOFUNGI

Abd-Elsalam, Kamel A.; Guo, Jianrong; Moslem, Mohamed A.; Bahkali, Ali H.; Verreet, Joseph‐Alexander

doi:10.1111/j.1745-4581.2009.00178.x

Cited by 8 publications

(6 citation statements)

References 31 publications

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“…() have used the internally transcribed spacer regions in the ribosomal repeat region (ITS1 and ITS2). Combining the intergenic spacer/ITS‐microsatellite‐primed PCR technique with microsatellite‐detection assay allows the rapid and specific detection of Rhizoctonia solani anastomosis groups and different phytopathogenic fungi (Abd‐Elsalam et al ., ). However, the PCR techniques used so far require the sequencing and analysis of specific amplified genes.…”

Section: Introductionmentioning

confidence: 97%

High-resolution melting analysis allowed fast and accurate closed-tube genotyping of Fusarium oxysporum formae speciales complex

Ganopoulos

Madesis

Zambounis

et al. 2012

FEMS Microbiol Lett

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The fungus Fusarium oxysporum is a highly complex species composed by many strains put together into groups called formae speciales. As it is difficult and laborious to discriminate Fusarium formae specials via biochemical or phenotypic methods, it is very important to develop novel, rapid, and simple to perform identification methods. Herein, real-time PCR assay [using universal internal transcribed spacer (ITS) primers] coupled with high-resolution melting (HRM) analysis was developed for identifying and distinguishing F. oxysporum formae speciales complex. The melting curve analysis of these amplicons specifically classified all isolates into seven F. oxysporum formae speciales and generated seven HRM curve profiles. The smallest DNA sequence difference recognized in this study was one nucleotide. The results presented show that HRM curve analysis of Fusarium ITS sequences is a simple, quick, and reproducible method that allows both the identification of seven F. oxysporum formae speciales and at the same time their screening for variants. Our genotyping assay uses the combined information of simultaneously acquired HRM data from an unlabeled probe and the full-length amplicon. Finally, the completion of both reaction and analysis in a closed tube saves time by eliminating the separate steps and reduces the risk of contamination.

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Section: Introductionmentioning

confidence: 97%

High-resolution melting analysis allowed fast and accurate closed-tube genotyping of Fusarium oxysporum formae speciales complex

Ganopoulos

Madesis

Zambounis

et al. 2012

FEMS Microbiol Lett

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“…isolates [ 23 , 24 ], Phoma tracheiphila [ 25 , 26 ] . Pythium [ 27 , 28 ] and Rhizoctonis solani [ 16 , 29 , 30 ].…”

Section: Introductionmentioning

confidence: 99%

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

Bahkali

Abd-Elsalam

Guo

et al. 2012

IJMS

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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.

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“…Samples were placed into a glass capillary, capped, centrifuged for a few seconds in a micro-centrifuge using appropriate adapters, and then placed into the LightCycler rotor. In addition, the PCR products were recovered from the capillaries and analyzed by agarose gel (1.5%) electrophoresis and stained with ethidium bromide [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer

Abd-Elsalam

Bahkali

Moslem

et al. 2011

IJMS

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Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 μL, as compared to 10 pg/25 μL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, specific, cost-effective and reliable for the identification and quantification of M. graminicola in wheat.

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SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE‐PRIMED PCR FOR THE IDENTIFICATION OF RHIZOCTONIA SOLANI AND SOME PHYTOFUNGI

Cited by 8 publications

References 31 publications

High-resolution melting analysis allowed fast and accurate closed-tube genotyping of Fusarium oxysporum formae speciales complex

High-resolution melting analysis allowed fast and accurate closed-tube genotyping of Fusarium oxysporum formae speciales complex

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

Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer

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