Papaya (Carica papaya L.) is grown widely in tropical and sub-tropical regions (Ahmed et al. 2008). In Pakistan, papaya production and consumption are increasing due to its medicinal, nutritional, pharmacological properties and a rich source of antioxidant, vitamin B, potassium, and magnesium. In November 2021, 26 to 35% incidence of fruit rot was observed in 15 fields of Lahore, a district of Punjab, Pakistan. Affected fruit developed circular, gray-to-brown lesions (8 to 10 mm in diameter) with white mycelia forming on the surface of lesions. In advanced stages of the disease, the lesions enlarged in size and led to the rot of entire fruit. To isolate the causal agent, small tissue segments (1 to 2 cm) were excised from 15 symptomatic fruit, surface disinfected with 1% NaClO for 30 s, rinsed with sterile distilled water three times, air dried in laminar flow hood, aseptically transferred onto petri dishes containing potato dextrose agar (PDA) and incubated at 25℃ for 5 days with a 12-h photoperiod. Eleven isolates were obtained that produced white mycelia on PDA. Flask-shaped, dark-pigmented pycnidia formed on PDA after 18 days of incubation at 25°C, which produced α-conidia measuring 4.1 to 7.2 × 1.5 to 3.0 μm and β-conidia measuring 16.4 to 25.5 × 1.0 to 1.6 μm (n = 40). α-conidia were hyaline, fusiform, and single-celled, whereas β-conidia were one-celled, hyaline, and filiform. The morphological characteristics of the fungus were compatible with a Diaporthe species (Gomes et al. 2013). The internal transcribed spacer region (ITS) (OM865414 and OM865415), translation elongation factor 1-alpha (tef1) (OM831226 and OM831229), and histone H3 (HIS) (OM831227 and OM831228) of two representative isolates (UO02 and UO03) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and CYLH3F/H3-1b (Chaisiri et al. 2021), respectively. Blast searches showed 99 to 100% nucleotide identity with reference sequences of several Diaporthe amygdali deposited in NCBI GenBank, including the ex-type strain CBS 126679. A pathogenicity test was also performed on harvested fruit of papaya cv. Bombay using isolates UO02 and UO03. Ten mature and healthy papaya fruit were surface disinfected with 1% NaClO solution for 1 min, rinsed with sterile water and dried. Each fruit was wounded twice with a sterile scalpel (4 to 5 mm incision on the peel) and a 5-mm agar disc with mycelia of each isolate was separately placed in each wound. The wounds were wrapped with Parafilm following inoculation. Sterile PDA plugs were used in separate inoculated controls. All wounds were sealed with parafilm. All fruit were maintained in plastic boxes at 25°C with 80% relative humidity. After 6 days of incubation, rot symptoms similar to those appearing on naturally-infected fruit were observed on inoculated fruits while controls remained asymptomatic. The experiment was repeated twice with similar findings. Diaporthe amygdali was re-isolated (100%) from inoculated fruit and the pathogen identification was confirmed by morphological and molecular analysis, thus fulfilling Koch's postulates. Previously, the pathogen has been reported as a causal agent of canker and shoot blight disease in other countries (Ko and Sun, 2003; Beluzan et al. 2021). To our knowledge, this is the first report of D. amygdali on papaya in Punjab Province of Pakistan. Papaya is an emerging fruit crop in Punjab Province and it is important to further investigate the presence of this pathogen in other papaya orchards of the province since D. amygdali may cause rapid disease outbreaks resulting in severe losses.
Keywords: Causal Agent; Crop Type; Fruit; Fungi; Pathogen detection; Subject Areas.