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PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora.

ABSTRACT: We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora.

SUBMITTER: Ristaino JB

PROVIDER: S-EPMC106350 | biostudies-literature | 1998 Mar

REPOSITORIES: biostudies-literature

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PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora.

Ristaino J B JB Madritch M M Trout C L CL Parra G G

Applied and environmental microbiology 19980301 3

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar a ...[more]

PMID: 9501434

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Project description:Intragenomic polymorphism of ITS1 and ITS2 of nuclear ribosomal DNA sequences was analysed in 33 samples belonging to the Nitraria species N. schoberi, N. sibirica, and N. komarovii. The nucleotide variability of the ITS region was detected in the Nitraria species as single-nucleotide substitutions (mainly transitions) and single-nucleotide deletion. Information about the nucleotide variability of fragments is given for the first time by us. The ITS1-5.8S-ITS2 region contained 17 phylogenetically informative single-nucleotide polymorphisms. Eleven single-nucleotide substitutions (transitions, C/T) were detected in ITS1. The ITS2 spacer contained 273-274 bp and was more conservative. A total of 5 phylogenetically informative single-nucleotide polymorphisms (4 transitions: C/T, G/A, one transversion: G/C), one single-nucleotide deletion (T/-) were detected in ITS2. The average GC content was 61.5 %. The GC content was lower in N. sibirica (59.2 %) than in N. schoberi and N. komarovii (62.7 %). It has been shown that the shorter ITS2 is a suitable molecular marker separating these species, due to the low interspecific variability and simultaneous available intraspecific variability. Phylogenetic ML and BI trees constructed separately for the ITS1 and ITS2 spacers, as well as separately for the full-size ITS region and the ITS2 spacer, were congruent. The results obtained on the intraspecific differentiation of N. sibirica revealed two main ribotypes among the samples of this species: the main Siberian sibirica-ribotype and the main Kazakh sibiricaribotype. Geographical features of the distribution of N. sibirica ribotypes, as well as the presence of significant differences between the main Siberian and Kazakh sibirica-ribotypes (3 single-nucleotide substitutions) indicated significant inter-population differences and taxonomic heterogeneity of N. sibirica. Most likely, the processes of homogenization of nuclear ribosomal DNA of N. sibirica samples, the origin of which is associated with hybridization and speciation, are currently continuing.

Dataset Information

PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora.

Publications

PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora.

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