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Appl Environ Microbiol, March 1998, p. 948-954, Vol. 64, No. 3
Department of Plant Pathology, North Carolina
State University, Raleigh, North Carolina 27695
Received 7 August 1997/Accepted 15 December 1997
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.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
PCR Amplification of Ribosomal DNA for Species Identification
in the Plant Pathogen Genus Phytophthora
*
Corresponding author. Mailing address: Box 7616, Department of Plant Pathology, North Carolina State University,
Raleigh, NC 27695. Phone: (919) 515-3257. Fax: (919) 515-7716. E-mail: Jean_Ristaino{at}ncsu.edu.
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