First report of Pseudopestalotiopsis theae causing leaf spot of robusta coffee in the Philippines

Abstract

Robusta coffee (Coffea canephora) is the most produced coffee species in the Philippines, accounting for 70% of the country's total coffee production (Philippine Statistics Authority, 2023). Despite being considered more disease-resistant than C. arabica, a number of diseases have been reported infecting robusta coffee (Cao et al., 2014). In November 2022, necrotic leaf spots (Figure 1) were observed on the leaves of five-year-old robusta coffee trees in New Bantangan, Columbio, Sultan Kudarat, Philippines (6.3427 N; 124.5924 E). The leaf spots initially started as tiny circular or irregular brown spots that gradually enlarged and turned darker. The disease incidence was approximately 85% over an area of 1 hectare with 25% of the leaves on affected plants having disease symptoms. Twenty diseased leaves were collected, one each from 20 plants, and 3 mm discs were cut from the advancing margin of the infection adjoining healthy tissues. The discs were soaked immediately in a 10% solution of NaOCl for one minute, followed by three rinses in sterile distilled water, and blot dried on aseptic, dry tissue paper inside a laminar flow hood. The tissues were then placed equidistantly on potato dextrose agar (PDA) medium and incubated at 28 ±1°C. The active mycelial tip was transferred and incubated for seven days. The colonies produced white aerial mycelium with black conidial masses as they aged (Figure 2). Microscopic examination showed four-celled spindle-shaped conidia (n = 20), measuring 24.42-31.08 × 6.66 μm, and basal appendages 28.86-33.30 μm long (Figure 3). The fungal isolate was deposited in the fungal repository of the Plant Pathology Laboratory of the University of Southern Mindanao Research and Development Center (P3LSS01). To further ascertain its identity, genomic DNA was extracted from a representative seven-day-old isolate (001) grown in PDA broth, using a Zymo Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research, USA). The rDNA ITS region of the representative isolate was amplified and sequenced using universal primers ITS4/ITS5 (White et al., 1990). The sequence was deposited in Genbank (Accession No. OR125548). A BLASTn search revealed that the isolate had 99.61% identity to isolate FAFU03 (MH470257.1). On the basis of the morphological and molecular characters, the fungus was identified as Pseudopestalotiopsis theae. A pathogenicity test was performed thrice on detached healthy leaves of robusta coffee. Ten wounded (pin-pricked) and ten unwounded leaves were sprayed with 30 μl spore suspension (107 spores/ml) taken from a seven-day-old pure culture of the pathogen (isolate P3LSS01). Sterile distilled water was sprayed onto leaves as a control. The leaves were incubated at 26 ±1°C and the development of the infection and symptoms were observed regularly over a seven-day period. The first symptoms, small brown spots, were observed on wounded leaves after three days and these enlarged to 5–6 mm in diameter after seven days. Small brown spots only began to appear in unwounded test samples after seven days. Control leaves did not develop any symptoms (Figure 4). Although Pseudopestalotiopsis theae has originally been associated with tea, it has also been found to infect several important crops in the temperate and tropical areas of the world (Maharachchikumbura et al., 2014). This is the first report of P. theae infection in the Philippines, and the first report in robusta coffee globally. The impact of this pathogen is unknown but could prove to be an economically important disease of coffee. Cross-infection studies with other important crops will also be important as coffee is usually intercropped with other crops in the Philippines. Research on management approaches for this pathogen are required to safeguard coffee production in the Philippines and worldwide. The authors express gratitude towards PhilCafe and World Coffee Research for financial support of this research. Thanks are also due to Dr. Tamie C. Solpot for granting permission to use the Plant Pathology Research Laboratory during pathogen isolation and molecular identification.