First Report of Neofusicoccum parvum Causing Stem Necrosis of ‘Ōhi‘a Trees (Metrosideros polymorpha) on O‘ahu, Hawai‘i, U.S.A.

‘Ōhi‘a lehua (Metrosideros polymorpha) is the most abundant and ecologically important native tree in the Hawaiian Islands (Friday and Herbert 2006). In November 2016, extensive ‘ōhi‘a mortality was reported in the ‘Ewa Forest Reserve in the Ko‘olau Mountains of O‘ahu. A site visit revealed 150+ (≈50% disease incidence) ‘ōhi‘a trees along the Poamoho Trail with branch dieback and defoliated crowns, often with epicormic sprouts emerging below dead branch terminals (157°55′31.98″W, 21°31′58.905″N). Trees showed evidence of prior leaf infection by Austropuccinia psidii (pustules with urediniospores), which is likely associated with the widespread defoliation. Stem sections (>2.5 cm diameter) containing the margin between healthy and necrotic xylem were collected from five symptomatic trees for diagnosis. Woody stems were externally asymptomatic; however, when debarked, an extensive brown to black necrotic vascular discoloration was visible from the branch apex downward several centimeters on stems. Additional isolated lesions were found lower on the stems away from branch terminals. Sections of discolored xylem were surface disinfested in 3% sodium hypochlorite and plated onto potato dextrose agar (PDA). Within 4 days, tissue from four of five trees yielded a similar fungus containing white, aerial mycelium that later turned gray. Single-spore isolates (P17-79 and P17-80) were derived from two of four infected trees. To identify the isolates, the internal transcribed spacer of the rDNA (ITS), partial β-tubulin (tub2), and translation elongation factor 1-α (tef1) gene regions were amplified by polymerase chain reaction (PCR) and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively. Both fungal isolates were identical to each other and were 99% (ITS) and 100% (tub2 and tef1) similar the ex-type isolate (CMW9081 and ICMP 8003) in a BLASTn comparison. Sequences were deposited in GenBank with accessions numbers MF631021 and MF631022 (ITS), MF631023 and MF631024 (tub2), and MF631025 and MF631026 (tef1). Pathogenicity tests were conducted by inoculating 1-year-old ‘ōhi‘a seedlings with PDA plugs containing 7-day-old mycelium of Neofusicoccum parvum (P17-79 and P17-80) into wounds made by a 5-mm cork borer and wrapped in Parafilm. Each N. parvum isolate was inoculated into five plants. Five negative control plants were inoculated with sterile PDA plugs. Plants were kept in a greenhouse (21.8 ± 1.0°C) with daily mist irrigation. After 4 months, inoculated stems were debarked, and the extent of xylem necrosis was measured. The necrosis was identical to that observed in the field samples. This experiment was conducted twice. For both experiments combined, mean length of xylem lesions was significantly higher (P < 0.001) for N. parvum-inoculated seedlings (P17-79 = 4.0 ± 0.5 cm and P17-80 = 3.7 ± 0.4 cm) versus controls (mean = 0.6 ± 0.1 cm). Fungi reisolated from inoculated tissue were molecularly analyzed using ITS PCR and sequencing, confirming the fungus as N. parvum, thus completing Koch’s postulates. Control plants showed no symptoms. N. parvum is an endophyte or latent pathogen of woody hosts and has been reported in Hawai‘i on Eucalyptus (Sakalidis et al. 2013). To our knowledge, this is the first report of N. parvum on ‘ōhi‘a in Hawai‘i, and causing stem necrosis. We suspect N. parvum is acting as a stress-induced pathogen (Slippers and Wingfield 2007) and not a primary tree killer. Further investigation is needed to determine the interaction of N. parvum with other pathogens of ‘ōhi‘a, especially A. psidii, which is believed to play a significant role in the defoliation and dieback on O‘ahu.