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.

FIG. 1

Extracted DNA of P. infestans was amplified with primer pairs ITS 5 and 2, ITS 5 and 4, and ITS 3 and 4. Pythium ultimum DNA amplified with the same primer pairs is shown in the intervening lanes. The no-template control (−) and 100-bp DNA ladder (Lad) are also shown.

FIG. 2

Restriction analysis with RsaI of DNA amplified with primer pair ITS 5 and ITS 4 from P. cactorum 1298 (lane 2), P. capsici B1HB14 (lane 3), P. citrophthora M86 (lane 4), P. citrophthora 34-4-7 (lane 5), P. nicotianae D-1 (lane 6), P. nicotianae Rmt 6 (lane 7), P. nicotianae 1-3A (lane 8), P. palmivora P8 (lane 9), and P. citricola M213 (lane 10). Lanes 1 and 11 contain 100-bp ladders.

FIG. 3

Restriction analysis with RsaI of DNA amplified with primer pair ITS 5 and ITS 4 from P. infestans US-6 NY (lane 2), P. infestans 93-1 (lane 3), P. mirabilis 0S0016 (lane 4), P. fragariae A-8 (lane 5), P. megasperma NY 318 (lane 6), P. megasperma NY 412 (lane 7), P. sojae R1 (lane 8), P. cinnamomi 2302 (lane 9), P. cryptogea PCR-1 (lane 10), and P. erythroseptica 4 (lane 11). Lanes 1 and 12 contain 100-bp ladders.

FIG. 4

Restriction analysis with MspI of DNA amplified with ITS 5 and ITS 4 from P. cactorum 1298 (lane 2), P. infestans US-6 NY (lane 3), P. mirabilis 0S0016 (lane 4), P. capsici B1HB14 (lane 5), and P. citricola M213 (lane 6). Lanes 1 and 7 contain 100-bp ladders.

FIG. 5

Restriction analysis with HaeIII of DNA amplified with ITS 5 and ITS 4 from P. palmivora P8 (lane 2), P. erythroseptica 4 (lane 3), P. cryptogea PCR-1 (lane 4), P. citrophthora M86 (lane 5), P. cinnamomi 2302 (lane 6), P. fragariae A-8 (lane 7), P. megasperma NY 412 (lane 8), P. sojae R1 (lane 9), and P. megasperma NY 318 (lane 10). Lanes 1 and 11 contain 100-bp ladders.

FIG. 6

DNA amplified with the PCAP primer and ITS 1 from P. capsici 17 (lane 2), P. capsici 18 (lane 3), P. capsici 19 (lane 4), P. capsici 20 (lane 5), P. capsici 21 (lane 6), P. capsici 22 (lane 7), P. capsici 23 (lane 8), P. capsici 25 (lane 9), P. citrophthora M86 (lane 10), P. citricola M213 (lane 11), and P. capsici 87 (lane 12). Lanes 1 and 14 contain 100-bp ladders; lane 13 contains a no-template control.

FIG. 7

Restriction digest with MspI of DNA amplified with the PCAP primer and ITS 1 from P. capsici 17 (lane 2), P. capsici 18 (lane 3), P. capsici 19 (lane 4), P. capsici 20 (lane 5), P. capsici 21 (lane 6), P. capsici 22 (lane 7), P. capsici 23 (lane 8), P. capsici 25 (lane 9), P. citrophthora M86 (lane 10), P. citricola M213 (lane 11), and P. capsici 87 (lane 12). Lanes 1 and 14 contain 100-bp ladders; lane 13 contains a no-template control.