First Report of <i>Diaporthe foeniculina</i> Causing Necrotic Leaf Spot of <i>Hoya carnosa</i> in Australia

Hoya spp. (Apocynaceae) comprises over 300 species across the Australasian and Indomalayan regions, and are increasingly cultivated as ornamental and medicinal plants (Baltazar & Buot Jr, 2019). In December 2020, a single leaf from a mature Hoya carnosa from a nursery in Fitzroy, Melbourne, exhibited foliar symptoms, including necrotic lesions along leaf margins and black pycnidia. The symptomatic leaf was surface-sterilised (1% active ingredient sodium hypochlorite for 1 minute) and rinsed three times in sterile water. Tissue from lesion margins was plated on water agar (WA), and emerging hyphae were transferred to potato dextrose agar (PDA) after five days. Pure cultures were obtained by single-spore isolation after three weeks, and isolate UOM 25001 was obtained. Colonies on PDA at 25 °C (12-hour photoperiod) displayed dense white mycelium, irregular filiform margins, and no elevation. The underside showed concentric green and cream-colored mycelium. Conidiomata were dark brown to black with yellow-white cirri. α-conidia were oval, elongated, aseptate, non-guttulate and hyaline (8.65 ± 0.77 × 2.73 ± 0.65 μm, n = 10; range 7.99–10.36 × 2.13–3.93 μm), and β-conidia were filiform, straight, or curved (22.63 ± 1.52 × 1.46 ± 0.18 μm, n = 10; range 20.98–25.81 × 1.24–1.83 μm). Pathogenicity was assessed on detached Hoya carnosa leaves obtained from a single plant (10 wounded with a sterile needle, 10 non-wounded). The leaves were inoculated with PDA plugs of isolate UOM 25001 and incubated at 25˚C (12-hour photoperiod, 60% humidity). Ten control leaves received WA plugs. After 15 days, red discoloration and necrosis developed around the inoculation sites on six of the wounded leaves (60% incidence), from which the pathogen was successfully re-isolated, fulfilling Koch’s postulates. Pycnidia were observed on one leaf at 7 DAI. No pathogen was re-isolated from non-wounded and control leaves. Molecular identification was performed by amplification and sequencing the Internal Transcribed Spacers and the intervening 5.8S region (ITS) using ITS1/ITS4 primers (White et al., 1990), the translation elongation factor one-alpha (TEF1-α) with EF-1/EF-2 primers (O’Donnell et al., 1998), and the beta-tubulin (TUB2) region with T1/Bt2b primers (O'Donnell & Cigelnik, 1997; Glass & Donaldson, 1995). All sequences were deposited in GenBank with accession numbers PX232706 (ITS), PX243416 (TUB), and PX243417 (TEF). BLAST analysis showed 99.82%, 99.68%, and 100% identity to Diaporthe foeniculina type isolate (GenBank: NR_145303, KC343827, KC344069, respectively). Maximum Likelihood and Bayesian phylogenetic analyses, using a three-gene concatenated alignment in RAxML v.8.2.11 and MrBayes v.3.2.7, respectively, placed UOM 25001 with D. foeniculina (CBS 111553) with 100% ML bootstrap support (based on 1000 replicates) and a Bayesian posterior probability of 0.97. Diaporthe foeniculina was previously reported on persimmon in Australia using ITS and TEF1-α (Golzar et al., 2012). This study provides stronger phylogenetic support through multilocus data and represents the first global report of D. foeniculina infecting Hoya carnosa. Given its latent and endophytic behavior (Huang et al., 2015; Udayanga et al., 2014), infections with mild or unapparent symptoms may spread unnoticed within Hoya plants and pose a risk of dissemination to other susceptible hosts before being detected.