Abstract

Colletotrichum species present a significant threat and posing a serious threat to the economy of Pakistan. This fact is due to suitable environmental conditions for pathogen dispersal. The research was conducted for the accurate identification of Colletotrichum species and studying its pathogenic behaviour responsible for pre-harvest bitter rot disease of apple fruits in Quetta, Pakistan. For this purpose, a survey was conducted in 2016 2017 across ten locations in Quetta to evaluate the disease assessment and sample collection. The findings revealed disease incidence of 39.22% in 2016 and 32.56% in 2017. Symptoms observed sunken brown lesions, measuring 1-4 cm in diameter, on the fruit surface, containing small, pinhead-sized black fruiting structures. A total of 130 fungal isolates were obtained from infected samples and categorized primarily into three groups (A, B, C) based on distinct cultural and morphological characteristics. Pathogenicity test, conducted in triplicate through spore suspension application on healthy apple fruit, confirmed Colletotrichum as the causative pathogen. Furthermore, ten isolates from each group were subjected to molecular analysis to identify the Colletotrichum species using internal transcribed spacer (ITS), beta-tubulin (TUB), and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) gene regions. From sequence analysis, the fungal isolates were identified as three Colletotrichum species mainly Group (A) C. acutatum (B) C. gloeosporioides and (C) C. siamense. Morphological features act as a primary way of identifying Colletotrichum spp. although it is not feasible due to some Colletotrichum genera being morphologically related, consequently molecular method of identification was sufficient for accurate identification and species confirmation. At our knowledge, tion this is the first comprehensive study of Colletotrichum spp. causing bitter rot disease of apple in Pakistan. The findings of our resesearch will pave the way for future disease management experiments, aiming to prevent this disease becoming a serious threat in the region.