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.

Leaf Spots on bodhi tree (Ficus religiosa) caused by Diaporthe tulliensis.

Ficus religiosa L., known as bodhi tree, is an ornamental tree and widely planted as an avenue and roadside tree due to ovate-rounded leaves with narrow, elongated tips. During 2018-2021, circular to oval-shaped leaf spots with pale white centers and brown-black edges surrounded by a chlorotic halo were observed on the leaves of more than 200 bodhi trees all year round in a park in Zhanjiang, Guangdong (N 21°15'22.29''; E110°23'1.03''). The leaf disease incidences were usually 15-80%, in severe cases, up to 100% in autumn and winter every year, and some trees shed all leaves(Fig S1). Repeated annual defoliation may weaken the tree and decrease the aesthetic value in the landscape. Diseased tissues (5 × 5 mm) of five symptomatic infected leaves were surface sterilized in 3% hydrogen peroxide solution for 3 min, rinsed thrice with sterile water, plated on potato dextrose agar (PDA) amended with ampicillin (50mg/L), and incubated at 25-28 ℃ in the dark for 3-7 days. Five strains with similar morphology were obtained by transferring hyphal tips of the colonies to fresh PDA and further isolating by single spore method. Fungal colonies were flat and spreading, with sparse, white aerial mycelium, and black pycnidial conidiomata semi-immersed in PDA after 30-days incubation at 25-28 ℃ in dark. Conidiophores were hyaline and α-conidia were single-celled, oval to fusiform, guttulate, 5.3 × 2.5 µm (n>50), similar to Diaporthe sp. (Crous et al. 2015), but no ß and γ -conidia were observed. The internal transcribed spacer(ITS), large subunit ribosomal RNA gene(LSU), calmodulin (CAL) and ß-tubulin(TUB) gene regions of a representative strain were amplified using specific primers reported by White et al. (1990), Gao et al (2017) and Gomes et al (2013), and submitted to GenBank (ITS: OM200852, LSU: OM228732, CAL: OM244761, TUB: OM244760). NCBI Blastn showed above 99% identity to D. tulliensis (anamorph: Phomopsis heveicola ) isolates of ITS (MT974186, MN393590 and KX457967), LSU (KR936131), CAL (MW759801), and TUB (KR936132 and MN399886), respectively (Crous et al. 2015; Huang et al. 2021; Gong et al. 2020; Bai et al. 2017). Based on the concatenated ITS, CAL, and TUB, a Maximum Likelihood phylogenetic analyses using MEGA 10.1.8 clustered the fungus with D. tulliensis in a clade with a 93% bootstrap support(Fig S2). Therefore, the fungus was identified as D. tulliensis based on morpho- molecular characteristics. Healthy detached leaves were sanitized thrice with 70% alcohol, and rinsed with sterile water. PDA plugs with actively growing 10-days-old mycelium were placed on predetermined sites, put into a sealed box with above 80% relative humidity and incubated at room temperature (25-28℃). Each isolate was inoculated at 25 needle-wounded and unwounded sites, PDA plugs without mycelium served as controls. Symptomatic spots appeared on all wounded leaves by 7 days post-inoculation (dpi) and on all unwounded leaves by 12 dpi. No symptoms appeared on controlled leaves. Pure cultures were recovered from inoculated leaves and showed identical morpho-molecular criteria to the original isolates. More than 70 pathogenic fungi are known to cause diseases on F. religiosa while there is no record of D. tulliensis infecting bodhi according to the U.S. National Fungus Collection (Farr and Rossman 2022). This report could provide basic understanding and alerting role for horticulturist in daily management.