First Report of Leaf Brown Spot Caused by Diaporthe phoenicicola on Pachira glabra in China.

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Pachira glabra is an increasingly important ornamental landscape tree in southern China. In August 2022, brown spots were observed on P. glabra leaves in Xiangtan City, Hunan Province, China (27.932°N, 113.020°E), affecting up to 40% of the 792 trees surveyed. On each diseased tree, nearly 30% leaves had symptoms, with an average severity of 21.2 ± 5.8% (n=100). The disease initially started as small yellow lesions along leaf margins, which later progressed to pale brown to brown with dark brown borders, eventually coalescing into large necrotic areas. Thirty symptomatic leaf samples (2 × 2 mm) were surfaced-sterilized in 75% ethanol for 10 s, 2% NaOCl for 30 s, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and incubated at 25°C for 5 to 7 days in dark. Eight morphologically similar isolates were obtained from diseased leaf samples through single-spore isolation. On PDA, colonies initially appeared white, turning gray, while the reverse developed a pale yellowish hue. Aerial mycelia were white, cottony, and developed visible black pycnidia with oil droplets at maturity. The α-conidia were unicellular, hyaline, aseptate, oval or fusiform, usually with 1 or 2 guttule(s) and rounded at each end. These conidia were 5.3-8.6 × 1.7-2.5 μm (avg. 6.7 × 2.2 μm, n = 100) and present more frequently than β-conidia.The β-conidia were unicellular, hyaline, aseptate, filiform, straight or hamate, eguttulate, 14.6-23.3 × 0.4-1.3 μm (avg. 18.4 × 0.9 μm, n = 30). Morphologically, the fungi were identified as Diaporthe sp. (Udayanga et al. 2014). For molecular identification, the internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), calmodulin (CAL), tubulin 2 (TUB2), and histone H3 (HIS3) sequences of all isolates were amplified from genomic DNA, using primers ITS4/ITS5 (White et al. 1990), TEF-2/728F and CALD-38F/CALD-752R (Carbone and Kohn 1999), Bt2a/Bt2b and H3-1a/H3-1b (Glass and Donaldson 1995; Crous et al. 2004), respectively. The GenBank accession numbers for a representative isolate gpg2023-1 were OR533573 (ITS), OR570887 (EF1-α), OR570888 (TUB2), OR570890 (CAL), and OR570889 (HIS3). BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences were 99%, 99%, 99%, 99%, and 98% identity, respectively, with those of Diaporthe phoenicicola (GenBank: KC343032.1, KC343758.1, KC344000.1, KC343516.1, and KC343274.1). To confirm the pathogen's identity, phylogenetic analysis using MEGA7.0 based on Maximum Likelihood was constructed. Isolate gpg2023-1 clustered with D. phoenicicola. Based on morphological and molecular data, the fungus was identified as D. phoenicicola. Next, pathogenicity tests were performed three times on one-year-old potted P. glabra plants. For each isolate, twelve healthy leaves on each of three plants were either wounded by a sterile needle or left unwounded, and then sprayed with a conidial suspension (1×106 conidia/ml) for each isolate. Control plants received with sterile water only. Plants were kept in a greenhouse at 25°C, 80% relative humidity, with a 12-h photoperiod. All wounded, inoculated leaves developed brown spot symptoms similar to those observed in the field with six days, while unwounded leaves and control plants remained symptom-free. The fungus was reisolated from all diseased leaves, fulfilling Koch's postulates and proving D. phoenicicola as the causative agent of this brown spot disease on P. glabra. While D. pachirae has been reported to cause leaf spot on P. glabra in Brazil (Milagres et al. 2018), this study marks the first report of D. phoenicicola causing leaf brown spot on P. glabra in China. This finding can help develop control strategies for this disease.