First report of Brown blotch disease caused by Diaporthe ueckeri on Hyophorbe lagenicaulis in China.

Bottle palm (Hyophorbe lagenicaulis) is a picturesque evergreen plant in the family Aceraceae, ubiquitously cultivated as an ornamental tree throughout the tropics and subtropics due its attractive shape and small stature. During 2016-2022, brown spots were observed on the leaves of bottle palm on both sides of a campus road in Mazhang, Zhanjiang, Guangdong province (N21°8' 59.9" E110°17' 51.4"). Symptoms appeared as circular, light yellow to brownish red, slightly sunken spots, and brown to black in the center (Fig 1 A-C). The spots expanded to big irregular blotches, which finally led leaves to wither. 0ne hundred percent of 50 plants were infected and 90% of the leaves each plant were covered with brown patches of different sizes. Tissues (5 × 5 mm) from the diseased-healthy junction of the leaf spots were surface disinfected with 75% ethanol for 30 s and 3.5% hydrogen peroxide solution for 5 min, rinsed three times with sterile water and placed on potato dextrose agar (PDA) at 25-28℃ in the dark for 3 days. Fungi with the similar morphology grew from 100% of these inoculated tissues. Hyphal tips from the colony edges were transferred to PDA. Single isolates were obtained by plate dilution method after sporogenesis. Colonies with sparse aerial mycelium were flat, grey. Conidiomata were superficial, black, solitary, scattered. Conidiophores were cylindrical, densely aggregated. Alpha conidia with bi-multiguttulate were hyaline, smooth, ellipsoidal, aseptate, 4.7-7.9×1.6-3.4 µm (Av. 6.4×2.3µm, n>50). Beta conidia were filiform, slightly curved, 15.9-28.3× 0.7-1.1 μm (20.1 × 0.9 μm, n>50) (Fig 1 E-H). Gamma conidia were not observed. The internal transcribed spacer (ITS) regions, large subunit (LSU) rRNA sequence, translation elongation factor (TEF), actin (ACT), calmodulin (CAL), histone H3 (HIS), and beta tubulin (TUB2) genes were amplified using the primers ITS4/ITS1 (White et al. 1990), LR0R/LR5, EF1-728F/EF-2, ACT-512F/ACT-783R, CAL228F/ CAL737R, CYLH3F/ H3-1b, and TUB2Fd /TUB4Rd (Aveskamp et al. 2009; Carbone and Kohn 1999; Crous et al. 2004; O'Donnell et al. 2000), respectively. All seven sequences of the isolate CCAS-JPYZ-4B (ACCC 35493) were submitted to NCBI (OR430112-3, OR451702-6). BLAST search result showed high sequence identity with several Diaporthe ueckeri isolates (Tab1). Phylogenetic analysis of the concatenated data of CAL, HIS, ITS, TEF and TUB genes using Maximum Likelihood method placed CCAS-JPYZ-4B in the D. ueckeri clade. Thus, the isolate was identified as D. ueckeri (Udayanga et al. 2015). Pathogenicity tests were performed on three 5-year-old plants, one healthy new leaf per plant and 10 sites on per leaf were slightly wounded and inoculated with 5-mm mycelial plugs from 10-day-old culture on PDA. The control sites were treated with PDA plugs. Each inoculated leaf was covered with a plastic bag to maintain high humidity and kept at natural temperatures (25-28 ℃). The experiment was repeated once. Symptoms appeared as those described as above 5 to 10 days after inoculation (Fig 1 D). Controls were asymptomatic. The fungus was reisolated from diseased leaves and identified as D. ueckeri. D. ueckeri may infect Arachis hypogaea, Cucumis melo, Camellia sinensis, Glycine max, Mangifera indica and Michelia shiluensis, and this is first report causing brown blotch on bottle palm in China. This disease occurred all year round, which seriously affected plants growth and ornamental value; it is necessary to develop an effective management strategy.