First report of Lasiodiplodia theobromae causing leaf blight on Coffea canephora in the Philippines

The Soccsksargen (South Cotabato, Cotabato, Sultan Kudarat, Sarangani, and General Santos) region in the southern part of the Philippines is the country's leading area of coffee production. However, a decline in production has been reported in the past few years (Philippine Statistics Authority, 2023). Several factors are important in this decline, including the impact of disease. In November 2022, brown to black necrotic lesions were observed on the leaf margins of 24-year-old robusta coffee trees (Coffea canephora) in a 10-hectare coffee plantation in Ganatan (7.1541°N; 125.1149°E), Arakan, Cotabato (Figure 1). The disease incidence amongst the trees was 100% and 43% of the leaf surface area was diseased. The symptoms were observed to start on younger leaves and eventually progressed to older leaves. Twenty trees were examined, every tenth tree being examined along two intersecting lines in the orchard. One diseased leaf was collected from each infected tree. Fungal isolation was done using a tissue planting technique inside a laminar flow hood. At the advancing margin of the lesion, a 2–3 mm section was cut from each leaf and dipped in 10% sodium hypochlorite for one minute, rinsed in three changes of sterile distilled water, and blot dried on sterilised tissue paper before being planted equidistantly onto potato dextrose agar (PDA) medium and incubated at 28 ±1°C. Subsequent hyphal tip transfers were done to obtain a pure culture of the fungus. The colony was characterised by white aerial mycelium that gradually turned black as it aged with a yellowish-grey colour on the obverse part (Figure 2). Microscopic examination revealed that the mycelia were long, light brown, and smooth-walled; developing conidia (n = 20) were 17.8-21.1 × 8.9-15.8 µm in size, initially hyaline that turned brown, ellipsoidal, and thick-walled (Figure 3). The morpho-cultural features of the isolate closely resembled those of Lasiodiplodia theobromae (Pat.) Griffon & Maubl (Punithalingam, 1976). Genomic DNA of a representative isolate (GSB01) was extracted from a fourteen-day-old culture grown in PDA broth using a Zymo Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research, USA). The rDNA ITS region was amplified using the primer sets ITS4/ITS5 (White et al., 1990). BLASTn analysis of the resulting sequence showed 100% identity to isolate G112-8 (OR453211.1) obtained from guava (Psidium guajava) in China, confirming its identity as L. theobromae. The sequence was deposited in GenBank under Accession No. PP291895. A pathogenicity test was performed thrice on ten wounded (pin-pricked) and ten unwounded leaves of Coffea canephora using 5-mm mycelial plugs taken from a fourteen-day-old pure culture of isolate GSB01. For the control, uninoculated agar plugs were used. Inoculated and control leaves were incubated at 27 ±0.5°C for seven days. The first infection was detected on wounded leaves three days post-inoculation; unwounded test samples developed symptoms two days later. None of the control leaves developed symptoms (Figure 4). The pathogen was re-isolated from the inoculated leaves to satisfy Koch's postulates. Lasiodiplodia theobromae is a significant pathogen of economically important crops including cacao, dragon fruit and jackfruit (Alvindia & Gallema, 2017; Solpot, 2020; Taylaran et al., 2021; Evallo et al., 2022). This is the first report of L. theobromae causing leaf blight of Coffea canephora in the Philippines and globally. Hence, developing management approaches and strategies against this pathogen will be an important countermeasure to mitigate the impact of the disease. Moreover, investigating the susceptibility of other economically important crops would identify the diversity and host range of the pathogen. The authors would like to thank PhilCafe and World Coffee Research for funding and Dr. Tamie C. Solpot for the laboratory assistance.