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© 2005 Plant Management Network.
Accepted for publication 8 July 2005. Published 28 July 2005.


Phytophthora hibernalis: A New Pathogen on Rhododendron and Evidence of Cross Amplification with Two PCR Detection Assays for Phytophthora ramorum


Cheryl Blomquist, Senior Plant Pathologist, and Terra Irving, Agricultural Biotechnologist, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento 95832-1448; Nancy Osterbauer, Senior Plant Pathologist, Oregon Department of Agriculture, 635 Capitol Street NE, Salem 97301-2532; and Paul Reeser, Department of Botany & Plant Pathology, Oregon State University, Corvallis 97331


Corresponding author: Cheryl Blomquist. cblomquist@cdfa.ca.gov


Blomquist, C., Irving, T., Osterbauer, N., and Reeser, P. 2005. Phytophthora hibernalis: A new pathogen on Rhododendron and evidence of cross amplification with two PCR detection assays for Phytophthora ramorum. Online. Plant Health Progress doi:10.1094/PHP-2005-0728-01-HN.


During the surveys for the quarantine pathogen Phytophthora ramorum, large necrotic spots with water-soaked margins and rust-colored centers were observed on rhododendrons in nurseries in Del Norte County, California (Fig. 1), and Yamhill and Curry counties, Oregon, in 2002 and 2003 (5). Symptomatic rhododendrons were collected and DNA was extracted from the margin of the necrotic spots and then amplified using a multiplex PCR assay (5,6). The resultant 738-bp amplicon was purified and sequenced (5). When compared to published sequences in GenBank, no exact match was found. The closest matches were P. ramorum and P. lateralis (GenBank Accession Nos. AF429774 and AF287256, respectively) which were 96% similar to the 738 bp amplicon (5). In 2004, rhododendron leaves with similar symptoms were collected and tested with a federally validated nested PCR protocol for detection of P. ramorum (1). These tests produced a 291-bp amplicon, typical of P. ramorum (1). Tissue taken from the margin of the same necrotic spots was plated onto a selective medium PARP agar (2). Phytophthora isolates were recovered that were homothallic and produced amphigynous and paragynous antheridia and deciduous, semipapillate, ellipsoid-ovoid to obovoid sporangia (Fig. 2). DNA was extracted from the pure culture and the ITS1 and ITS2 regions of the nrDNA were amplified and sequenced. The ITS2 sequence was 100% homologous with a published sequence for P. hibernalis (GenBank Accession No. AF339442). These isolates were identified as P. hibernalis based on morphological characteristics and DNA sequences.


 

Fig. 1. Rhododendron ‘Rocket’ infected with Phytophthora hibernalis from a Del Norte County, California nursery.

 

Fig. 2. Sporangia and zoospores of Phytophthora hibernalis from cultures grown on dilute V8 medium and then incubated in soil water for 2 to 3 days at 18°C with a 16-h light cycle (4).


Pathogenicity tests were performed on Rhododendron ‘Catawbiense Album’ and on Rhododendron ‘Rocket’ in both detached leaf and whole plant assays. For the detached leaf assay, 10 young and 10 mature leaves of Rhododendron ‘Catawbiense Album’ were removed and placed in sterile glass Petri dishes lined with moistened filter paper. The leaves were inoculated with 6-mm plugs of P. hibernalis taken from the margin of 7- to 10-day-old cultures grown on sterile dilute V8 medium. Five young and five mature leaves were wounded once with a sterile pushpin and then a single plug was placed on each wound. An additional five young and five mature leaves were also inoculated but without wounding. Five young and five mature leaves treated similarly and inoculated with 6-mm sterile dilute V8 medium plugs were included as non-inoculated controls. The leaves were then incubated for 21 days (18°C, ambient light). For the whole plant assays, five 1-gal potted plants of Rhododendron ‘Rocket’ were inoculated with P. hibernalis inoculum as described above. Six leaves on each plant were wounded as described above and two P. hibernalis agar plugs were covered with a freezer tube cap filled with sterile distilled H20 and clipped to the underside of the leaves with a sterile pin-curl clip. The inoculated plants were sprayed with water, covered with plastic bags and incubated for 14 days (18°C, 16-h photoperiod). An equal number of leaves were inoculated with 6-mm sterile dilute V8 medium plugs as non-inoculated controls. In both experiments, samples were taken from the lesion margin and plated onto PARP. The PARP plates were incubated for 14 days (18°C) and then examined under light microscopy for characteristic morphological features. The inoculation experiments were repeated with similar results. DNA was extracted from the inoculated leaves, avoiding the agar plugs, diluted, and amplified with nested PCR as described above.

Fig. 3. Lesion on Rhododendron ‘Rocket’ inoculated with Phytophthora hibernalis.

 

In the detached leaf assay, small lesions (£ 1-cm diameter) grew on the P. hibernalis-inoculated leaves. No lesions grew on the control leaves. P. hibernalis produced lesions on and was re-isolated from both young and mature leaves. Symptom production and rate of isolation was higher from wounded leaves than from non-wounded leaves (80% wounded compared to 60% non-wounded for young leaves and 40% wounded compared to 20% non-wounded for mature leaves). P. hibernalis was not isolated from control leaves. In the potted plant assay, lesions ranging in size from 1 to 25 mm in diameter (Fig. 3) grew on the inoculated leaves. Leaf necrosis was observed on one control leaf. P. hibernalis was isolated from 21 of the 30 inoculated leaves. Significant plant-to-plant variation was observed with a 100% isolation rate from inoculated leaves on three of the plants, and 33% and 17% isolation rates from the remaining two plants. P. hibernalis was not isolated from any of the control leaves. A 291-bp amplicon was produced with the nested PCR protocol from the DNA extracted from the inoculated leaves (Fig. 4).


 

Fig. 4. Amplicons from the second amplification reaction of the nested PCR protocol for the detection of Phytophthora ramorum separated on a 3% Nusieve 3:1 agarose gel. Lane 1, size standards; Lane 2, negative control, without DNA; Lane 3, DNA extract of P. ramorum culture; Lanes 4 to 9, DNA extracts of P. hibernalis-inoculated rhododendron. Extracts of even numbered lanes from the first amplification reaction are diluted 1:10 and odd numbered lanes 1:100 before adding to the second amplification reaction. Lanes 4 and 5, 6 and 7, and 8 and 9 represent three individual inoculated leaves.

 

This study completes Koch’s postulates for P. hibernalis on rhododendron. P. hibernalis has previously been described as causing brown rot of citrus (2). This report expands the pathogen’s known natural host range. P. hibernalis does not appear to be widespread in the California and Oregon nursery industries, but was recovered in both states for three consecutive years. The fact that both the multiplex PCR and nested PCR diagnostic protocols for P. ramorum can cross amplify with P. hibernalis DNA in planta is of great concern. A false PCR positive during an official P. ramorum survey could lead to inappropriate regulatory action if no additional tests are performed. The ITS sequence of P. hibernalis is closely related to both P. lateralis and P. ramorum (3), so the issue of cross amplification is unsurprising. Further analysis showed inconsistent cross amplification with nested PCR at greater dilutions of the P. hibernalis DNA (Fig. 4). However, our results show that enough P. hibernalis DNA is present in naturally- and artificially-infected leaves for cross amplification to occur. Because this creates the potential for false positives in the official P. ramorum survey, we suggest using two independent techniques when diagnosing P. ramorum. For example, P. ramorum and P. hibernalis can be readily distinguished in culture by morphological features and by mitochondrial-based PCR assays (4).


Literature Cited

1. Davidson, J. M., Werres, S., Garbelotto, M., Hansen, E. M., and Rizzo, D. M. 2003. Sudden oak death and associated diseases caused by Phytophthora ramorum. Online. Plant Health Progress doi:10.1094/PHP-2003-0707-01-DG.

2. Erwin, D. C., and Robeiro, O. K 1996. Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN.

3. Ivors, K. L., Hayden, K. J., Bonants, P. J. M., Rizzo, D. M., and Garbelotto, M. 2004. AFLP and Phylogenetic analyses of North American and European populations of Phytophthora ramorum. Mycolog. Res. 108:378-392.

4. Martin, F. N., Tooley, P. W., and Blomquist, C. 2004. Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material. Phytopathology 94:621-631.

5. Osterbauer, N. K., Griesbach, J. A., and Hedberg, J. 2004. Surveying for and eradicating Phytophthora ramorum in agricultural commodities. Online. Plant Health Progress doi:10.1094/PHP-2004-0309-02-RS.

6. Winton, L. M., and Hansen, E. M. 2001. Molecular diagnosis of Phytophthora lateralis in trees, water, and foliage baits using multiplex polymerase chain reaction. Forest Pathol. 31:275-283.