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Phytophthora stricta Isolated from Rhododendron maximum in Pennsylvania

    Authors and Affiliations
    • T. L. Widmer
    • M. B. McMahon
    • R. D. Frederick , USDA/ARS/Foreign Disease-Weed Science Research Unit, Fort Detrick, MD.

      During a survey in October 2013 in the Michaux State Forest in Pennsylvania (Pennsylvania Department of Conservation and Natural Resources permit number SFRA-1351), mature plants of Rhododendron maximum were noticed alongside a stream with nondescript necrotic lesions at the tips of some mature leaves that had been in contact with the stream water. Several leaves were collected and taken back to the laboratory for processing. Segments from 10 leaves were removed at the interface of the lesion area, surface-sterilized for 20 s in 70% ethanol, and rinsed three times in sterile water. The segments were plated on PARPH+V8 Phytophthora selective medium (Ferguson and Jeffers 1999). The plates were kept in the dark at 20°C. After approximately 3 days, colonies resembling a Phytophthora sp. were observed growing from the segments of two leaves. Mycelial tips were transferred to 20% V8 agar (isolates MSF-C and MSF-F). The isolates were identified as P. stricta based on sequences from the internal transcribed spacer (ITS) region and cytochrome oxidase I (cox1) gene and their morphological features produced on V8 agar. Genomic DNA was extracted from mycelium of isolates growing in 20% V8 broth for 5 days. The ITS and cox1 gene products were generated by PCR using the primer sets ITS4 and ITS5, and CoxF4N and CoxR4N, respectively. Cloned PCR fragments were sequenced and analyzed using BLAST. ITS alignments of the 813-bp amplicon of MSF-C (GenBank accession no. MF138883) and MSF-F (MF138884) shared 100% identity to each other and exhibited 99% identity to P. stricta accessions KF192694 and KP749384. The cox1 alignments of the 867-bp amplicon of MSF-C (MF138885) and MSF-F (MF138886) were 100 and 99% identical to P. stricta accessions KF192702, KP749424, and KP749425. Sporangia were formed after 24 h exposure of mycelial plugs to 1% nonsterile soil extract. Sporangia averaged 60.0 ± 10.2 μm in length and 33.9 ± 9.3 μm in width (n = 20), fitting within the range for P. stricta (Yang et al. 2014). No chlamydospores were observed and the isolates were not tested for their mating type. Pathogenicity was confirmed by transferring 3-mm-diameter plugs from the leading edge of an actively growing colony on V8 agar to R. maximum leaves (three for each isolate) that were either first wounded by pricking with a sterile needle or not. Controls using V8 agar only were set up in the same manner. The leaves were placed in Petri plates on a plastic screen with a moist paper towel and kept in the dark at 20°C. After 2 days, necrosis was observed on the leaves regardless if wounded nor not. No necrosis was observed on the controls. Necrotic lesions were surface-sterilized and rinsed as described above and plated on PARPH+V8. Colonies resembling the isolates were recovered after 3 days, while no colonies were recovered from the control leaves. P. stricta has been isolated previously only from irrigation water in Mississippi and stream water from Virginia (Yang et al. 2014). To the authors’ knowledge, this is the first report of P. stricta being isolated from host material collected in a natural environment resulting from a natural infection. Additional research is needed on this species in order to determine its impact on the environment.