First Report of Pseudopestalotiopsis ampullacea Causing Leaf Spot on Hevea brasiliensis in Malaysia

Rubber is an economically important crop cultivated extensively in Malaysia for latex production. In March 2024, necrotic spots were observed on mature leaves of rubber plants (clone RRIM2004) during surveys in two rubber plantations, in Sungai Buloh, Selangor state and Kota Tinggi, Johor state, with a disease incidence of 80%. Initial symptoms on leaves appeared as light-yellow, circular, semi-circular to irregular lesions (2–5 mm in diameter) on the adaxial leaf surface, which gradually changed to brown and grey-white spots (Figure 1). Diseased leaves became blighted and the plants defoliated as the disease progressed. To identify the pathogen, fragments (5 × 5 mm) were excised from the margin of the diseased leaf tissues, surface-sterilised with 1% sodium hypochlorite solution for three minutes, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 28°C with a 12 h photoperiod for 7 days. Ten single-spore isolates were obtained from sampled leaves, all isolates exhibited a Pseudopestalotiopsis-like morphology and two representative isolates (PA1 and PA2) were selected for further study. Colonies on PDA were whitish with dense aerial mycelia, forming black gregarious conidiomata and the reverse side was whitish to pale yellow (Figure 2). Conidia were fusoid to ellipsoid, straight to slightly curved, 4-septate, ranging from 21 to 30 ±6.5–9 µm (n = 30) and septa darker than the rest of the cell (Figure 3). The basal cells were conic with a truncate base, hyaline and thin-walled, 2.5–5.0 µm long. Three median cells were doliiform, 13.5 to 19.5 µm long, hyaline, subcylindrical, thin-walled, with 2–3 tubular apical appendages arising from the apical crest, unbranched, filiform, 17–25 µm long. The basal appendages were singular, tubular, unbranched, centric, 3.5–7.0 µm long. On the basis of morphology, both representative isolates were identified as Pseudopestalotiopsis (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF1-α) gene of isolates PA1 and PA2 were amplified using the ITS5/ITS4 and EF1-728F/EF1-986R primer set, respectively (White et al. 1990; Carbone and Kohn 1999). BLASTn analysis of the resulting ITS and TEF1-α sequences indicated 99% identity to ex-holotype Pseudopestalotiopsis ampullacea strain LC6618. The ITS (GenBank Accession Nos. PP779714 and PP779715) and TEF1-α (PP785033 and PP785034) sequences were deposited in the GenBank databases. Phylogenetic analysis using the maximum likelihood analysis based on the concatenated ITS-TEF1-α indicated that the Ps. ampullacea PA1 and PA2 isolates form a strongly supported clade (82 bootstrap value) to the ex-holotype culture of Ps. ampullacea LC6618 and both isolates were most closely related to other Pseudopestalotiopsis species (Figure 4) (Kumar et al. 2024). Five healthy leaves from 6-month-old rubber plants (clone RRIM2004) were inoculated with either isolate PA1 or PA2 according to Liu et al. (2025). Control leaves were mock-inoculated using sterile water. Seven days post-inoculation, necrotic lesions developed on inoculated leaves, closely resembling symptoms observed on naturally infected rubber leaves in the field, whereas the control leaves remained asymptomatic (Figure 5). Pseudopestalotiopsis ampullacea was re-isolated from all symptomatic tissues, verified by molecular identification, confirming Koch's postulates. This is the first report of Ps. ampullacea causing leaf spot symptoms on Hevea brasiliensis in Malaysia. The pathogen is primarily known to infect palm species, particularly oil palm (Ismail et al. 2017). The occurrence of this disease needs to be monitored because it poses a significant threat with the potential to reduce latex production by 28%–46% (Kusdiana and Saputra 2022), adversely affecting overall yield and profitability. Therefore, preventive strategies need to be developed to reduce the incidence of the disease in the field. We thank the Diagnostic Unit, Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, for their support in the plant disease diagnosis. Universiti Putra Malaysia supported this work through the Putra Grant Initiative (GPI), vote project number 9758500.