Ribosomal RNA genes (rDNA) possess suitable characteristics for the detection of pathogens at the species level. They occur in multiple copies arranged in tandem repeats with each repeat consisting of the 18S Small SubUnit (SSU), the 5.8S, and the 28S Large SubUnit (LSU) genes, separated by internal spacers (ITS). ITS have proven particularly useful for separation of Phytophthorae at the species or genus level, although P. megasperma still remain one of the most problematic species in the genus. The utility of the region between the LSU and SSU genes, known as the intergenic spacer (IGS), has not been fully exploited for diagnostic purposes in Phytophthora. Thus, to develop a specific molecular tool, the complete sequence of the IGS region in P. megasperma was determined. Primer pairs were designed for amplification of the region spanning a portion of the LSU, the IGS, and the 5' end of SSU. The tandem repeated unit was 5,259 bp long appearing to be the shortest rDNA cluster described so far among the available sequences. A pair of oligonucleotide primers (IGph6 and IGph5), designed on the P. megasperma IGS sequence, were evaluated for specificity and absence of cross reactivity, by using a wide range of Phytophthora species and other fungi, commonly occurring in the soil. The resulting amplicon was of the expected size (176 bp), thus allowing the specific identification of all P. megasperma isolates, but not of those known as the AC form (isolate SCRP448).
Specific PCR detection of Phytophthora megasperma using the intergenic spacer region of ribosomal DNA
NIGRO, Franco;Ippolito A.;
2005-01-01
Abstract
Ribosomal RNA genes (rDNA) possess suitable characteristics for the detection of pathogens at the species level. They occur in multiple copies arranged in tandem repeats with each repeat consisting of the 18S Small SubUnit (SSU), the 5.8S, and the 28S Large SubUnit (LSU) genes, separated by internal spacers (ITS). ITS have proven particularly useful for separation of Phytophthorae at the species or genus level, although P. megasperma still remain one of the most problematic species in the genus. The utility of the region between the LSU and SSU genes, known as the intergenic spacer (IGS), has not been fully exploited for diagnostic purposes in Phytophthora. Thus, to develop a specific molecular tool, the complete sequence of the IGS region in P. megasperma was determined. Primer pairs were designed for amplification of the region spanning a portion of the LSU, the IGS, and the 5' end of SSU. The tandem repeated unit was 5,259 bp long appearing to be the shortest rDNA cluster described so far among the available sequences. A pair of oligonucleotide primers (IGph6 and IGph5), designed on the P. megasperma IGS sequence, were evaluated for specificity and absence of cross reactivity, by using a wide range of Phytophthora species and other fungi, commonly occurring in the soil. The resulting amplicon was of the expected size (176 bp), thus allowing the specific identification of all P. megasperma isolates, but not of those known as the AC form (isolate SCRP448).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
