First report of Neovaginatispora fuckelii causing leaf spot on Phoenix canariensis

Phoenix canariensis Chabaud is a vital ornamental and widely planted in the urban landscape of China (Lan et al. 2019). In December 2019, six of seven P. canariensis plants exhibited typical symptoms of leaf spot in Chengdu campus of Sichuan Agricultural University in Sichuan, China, with roughly 80% leaves per plant affected. Symptomatic leaves initially showed dark brown spots with a yellow halo at the periphery. As the disease progressed, spots gradually expanded and eventually formed necrotic spots, with the whole leaf dying when severely infected. 10 diseased leaves from different plants were collected in December 2019 and deposited in Sichuan Agricultural University Herbarium. A single spore isolation was performed following Chomnunti et al. (2014) and transferred to potato dextrose agar (PDA). Five fungal isolates were obtained from five different infected leaves respectively and deposited in Sichuan Agricultural University Culture Collection. The fungal colonies were incipiently greyish-white becoming pale brown at the margins and dark brown in the center. The reverse was the same as the obverse with radial cracking at the center. Morphological characteristics on the host were examined by light microscopy. Ascomata were semi-immersed to immersed, black, pyriform to subglobose, and measured 180-310 × 140-260 μm. The peridium comprised several layers of hyaline cells of textura angularis and measured 9-27 μm wide. Pseudoparaphyses were numerous, septate, hyaline, usually longer than asci. Asci were 8-spored, bitunicate, cylindric-clavate, and measured 50-95 × 8-11.5 μm (n=35). Ascospores were 1-septate, straight or slightly curved, hyaline, usually with globose appendages at both ends, and measured 14-18.5 × 4-6.5 μm (n=50). Conidiogenesis was not observed on PDA incubated four months at 25℃ and 95 % relative humidity on a 12-h fluorescent light/dark incubation. Morphology was consistent with the sexual stage of Neovaginatispora fuckelii (Sacc.) A. Hashim. et al. described by Hashimoto et al. 2018, Tennakoon et al. 2018, and Hyde et al. 2020. The rDNA internal transcribed spacer region (ITS), 28S large subunit (LSU), 18S small subunit (SSU), translation elongation factor 1-alpha (TEF 1-α), and RNA polymerase II subunit 2 (RPB2) gene regions were amplified by using the primers ITS5/ITS4, LR0R/LR5, NS1/NS4, EF1-983F/EF1-2218R, and fRPB2-5F/fRPB2-7cR, respectively. The DNA amplification products of the representative isolate SICAUCC 20-0008 isolated from SICAU 20-0008 were sequenced and accessioned in GenBank, viz MT427731, MT427734, MT427738, MT441923, and MT441920, respectively. The sequences were compared with the GenBank database using nucleotide BLAST, and these five sequences were great identical with the sequences of the fungus N. fuckelii (GenBank accession no. ITS, LC001732, 522/524, 99.62%; LSU, AB619009, 839/840, 99.88%; SSU, AB618690, 976/976, 100%; TEF 1-α, LC001750, 895/912, 98.14%; RPB2, MN482130, 935/935, 100%). Using phylogenetic analysis in which reference sequences collected from GenBank were included, the isolate SICAUCC 20-0008 clustered within N. fuckelii with high bootstrap support (Fig. 1). To conduct Koch's postulates, fifteen healthy P. canariensis 2 to 3-year-old plants were inoculated by placing a mycelium plug from the growing margin of 15-day-old colonies upside down directly onto fresh wounds punctured with a fine needle. Five healthy plants were inoculated with a sterile agar plug as the control. Plants were incubated in a growth chamber at 25 ± 1℃ and 95% relative humidity on a 12-h fluorescent light/dark regimen. After ten days, the leaves of inoculated plants turned brown and gradually expanded and became necrotic spots, similar to symptoms observed in the field. The control plants were symptomless. The pathogen was re-isolated from leaf lesions and identified by morphological characteristics, whereas no fungus was recovered from control treatments. N. fuckelii has been often reported as a saprobe on numerous hosts including species of Acer, Carpinus, Carya, Epilobium, Prunus, Quercus, Rhus, Salix, and Vitis, as well as other genera (Thambugala et al. 2015, Hashimoto et al. 2018, Hyde et al. 2020). To our knowledge, this is the first report of N. fuckelii causing leaf spot on P. canariensis. Some proteaceous plants and other palms are known to be infected by this fungus (Hyde et al. 2000, Taylor et al. 2000), which may result in a great threat to ornamental horticulture. Fungicides treatments should be considered to prevent the damage to plants and spread of this fungus.

Keywords: Causal Agent; Crop Type; Fungi; Leaf spot; Neovaginatispora fuckelii; Trees; ornamentals.