First Report of Bitter Rot of Apple Caused by Colletotrichum siamense in Pakistan

Apple (Pyrus malus Linn.) is one the most common and consumed fruit in the world, owing to its high nutritional value. In Pakistan apple is cultivated in the highlands of Baluchistan, Khyber Pakhtunkhwa, and Northern Punjab. Red Delicious (Tur-Kulu) and Golden Delicious (Shin-Kulu) are famous cultivars throughout Pakistan for their attractive color and taste. Bitter rot, caused by Colletotrichum species, is one of the destructive diseases in apple growing countries (Sharma et al. 2013). In September 2017 apple fruits (cv. Golden Delicious) showing symptoms of bitter rot disease were collected from an orchard in Quetta (30.1798° N, 66.9750° E), Baluchistan Province, Pakistan. Symptoms of bitter rot were initially observed as small, circular gray or brown spots surrounded by red halo on lighter skinned apple fruit. Thereafter, the lesions enlarged rapidly, intermingled, and became sunken. Necrotic tissues extended beneath the fruit core in V-shaped pattern. For the isolation of the causal agent, a small piece (1 to 2 mm) of necrotic tissue having the structure of reproduction of the fungus (i.e., acervuli) was cut from diseased fruit, disinfected with 0.5% NaOCl for 2 min followed by rinsing with sterile distilled water, air dried, and plated onto potato dextrose agar medium. Single-spore culture was obtained by incubating plates at 25 ± 2°C for 7 days, with a 12-h light and darkness period. Colonies were initially white and later became light gray on the front and olivaceous gray on reverse side of plates with concentric rings of salmon acervuli. Conidia were hyaline, straight to slightly curved, cylindrical, aseptate, measuring 3.45 to 5.9 × 11.36 to 17.67 µm (average 4.72 × 14.6 µm) (n = 25), with rounded ends. Appressoria were ovate, globose, ranging from 6.37 to 7.55 × 10.11 to 12.3 µm (average 6.96 × 11.2 µm), and dark brown in color. The morphological characteristics of the causal agent resembled species belonging to the Colletotrichum gloeosporioides complex (Prihastuti et al. 2009; Weir et al. 2012). For further confirmation of the fungal isolate, DNA was extracted from single-spore isolates. Internal transcribed spacer (ITS) and translation elongation factor alpha1 (tef1) regions were amplified from rDNA. The sequences were submitted to GenBank with accession numbers MG867589 and MH844549, respectively. The BLAST result of the ITS sequence showed 99% identity with the corresponding sequence (MH700460.1) of the reference strains of C. siamense, and tef1 gene BLAST result showed 100% identity with the sequence (KC790609.1) of C. siamense. Koch’s postulates were accomplished by spraying two drops of the conidial suspension (10⁵ conidia/ml) on 10 healthy and ripe fruits, whereas sterile distilled water drops were applied on control fruits. All fruits were incubated at 25 ± 2°C in a humid chamber for 10 days. The characteristic disease symptoms developed, as described above. The fungus was successfully reisolated from inoculated fruits and identified as C. siamense. To our knowledge, this is the first report of C. siamense causing bitter rot of apple in Pakistan. This pathogenic fungus may pose a serious threat to apple cultivation in Pakistan.