First report of Phytophthora pluvialis causing needle lesions on Pinus pinea in New Zealand

Abstract

During routine surveillance in October 2020, needle lesions and necrosis were observed in two 42-year-old stone pines (Pinus pinea) situated in an amenity planting within an infected Pinus radiata plantation in Nelson, New Zealand. Symptoms were widespread across the foliage of infected trees and samples were sent to Scion's Forest Health Reference Laboratory. Approximately fifteen needles displaying yellow and red bands (Fig. 1) tested positive for Phytophthora using a Phytophthora ImmunoStrip® (Agdia, USA), and PCR and subsequent DNA sequencing with Ypap2F/Ypap2R primers (McDougal et al., 2021), indicated Phytophthora pluvialis. A Phytophthora species was isolated from diseased tissue. Morphological characteristics were consistent with those described by Reeser et al. (2013) for P. pluvialis. This identification was confirmed by sequencing the internal transcribed spacer region (White et al., 1990). The isolate was deposited in the National Forest Culture Collection (NZFS 5280) and symptomatic needles were lodged into the National Forest Mycological Herbarium (NZFRI 5931). Foliage symptoms caused by P. pluvialis on other conifers, including Pinus radiata, Pseudotsuga menziesii and Tsuga heterophylla, typically express as distinct khaki/red lesions and black resinous bands (Dick et al., 2014), rather than the yellow and red bands observed on the stone pines (Fig. 2). Pathogenicity testing was done by inoculating stone pine needles with P. pluvialis zoospores using a needle dip assay according to Dick et al. (2014). Stone pine needles from young healthy seedlings were placed in sterile pond water inoculated with P. pluvialis zoospores and incubated in darkness at 17°C for 24 hours. Control needles were placed in sterile pond water. Needles were then lain on moist paper towels in a sealed tray and incubated at 17°C at a 14-hour photoperiod. After ten days, olive/yellow lesions were observed on needles (Fig. 3). These lesions were lighter than those observed on field samples, however, this is common in detached needle assays of other species, such as P. pluvialis infection of Pinus radiata (Dick et al., 2014). Sporangia were observed near the lesion margin of needles (Fig. 4). Sections of symptomatic needles were surface sterilised and plated onto a Phytophthora-selective media (10% carrot agar + ampicillin, pimaricin, rifampicin, nystatin and hymexazol). The resultant cultures displayed morphological features of P. pluvialis, fulfilling Koch's postulates (Fig. 5). Control needles remained healthy (Fig. 3). To our knowledge, this is the first report of P. pluvialis causing needle disease on stone pine. Stone pine is an economically important species due to the production of high value pine nuts and timber. Although stone pine accounts for a small proportion (<1%) of New Zealand's forests, continued monitoring is recommended to assess the impact of disease on the growth and yield of these plantations. Forest Growers Levy Trust funded surveillance and diagnosis through the Forest Biosecurity Surveillance system.