First Report of Leaf Spot of Pineapple (Ananas comosus (L.) Merr Caused by Neoscytalidium dimidiatum in Malaysia

In June 2017, severe leaf spots symptoms were observed by growers on pineapple leaves of Josapine variety in in Alor Pongsu (5°01'60.00" N, 100°34' 59.99" E), Perak, northwest of Peninsular Malaysia. The early infection stage shows that several brown spots could be observed, which then would merge to form large brown to creamy white lesions that cover all the leaf surface. This infection finally caused the plant to die after a while. Disease observations conducted from 2018 - 2023 showed that 10-15% incidences of the disease were observed in several pineapple farms located in Johor, Kedah, and Sarawak. The aim of this study to confirm the causal pathogen of the disease by performing isolation, pathogenicity testing, and identification of the primary causal pathogen from 20 samples of infected leaves collected from Alor Pongsu. The leaf tissues between infected and healthy were cut into small pieces (0.5 cm 0.5 cm), and surface sterilized with 1% sodium hypochlorite for 30 seconds, followed by 70% ethanol for 30 seconds, and rinsed thrice with sterilized water before placing on Potato Dextrose Agar (PDA). The PDA plates were incubated at room temperature (28 ± 2℃) in natural light. After five days of incubation, the potential causal pathogen was purified using a single conidial isolation technique for morphological and molecular characterizations. All 32 isolates displayed similar phenotypes. Based on morphological observation on PDA, the colonies were initially white of aerial mycelia but gradually darkened as the culture aged. Microscopic features of the 14-day-old fungal culture showed that the mycelia were branched with 0- 1 septa, pigmented, and brown. Arthroconidia were ellipsoid to ovoid or round shaped, hyaline, with rounded apex, truncate base, and occurring singly or in chains averaging 9 ± 3 × 5 ± 2 μm (n = 20).  Based on the morphological characteristics, the fungal isolates were tentatively identified as Neoscytalidium species. A representative isolate of Neoscytalidium coded as UiTMPMD2 was further identified through PCR implication of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers and BLAST homology search as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on 100% similarity (575 bp out of 575 bp) to a reference sequence (accession no. KU204558.1). The sequence was deposited in Gen Bank (accession no. OR366479) with reference sequence code of INBio:30A. Pathogenicity tests were performed on 10 whole plants of Josapine pineapple (4 months old) using a leaf inoculating method (Wu et al. 2022) in a glasshouse (25-32°C) and repeated twice. Four mature leaves per each plant were wounded at two points and inoculated with mycelium PDA plugs from 7-days-old cultures of N. dimidiatum. Control plants were wounded in the same manner but inoculated with sterilized PDA plugs. Seven days post inoculation, leaf spot symptoms were observed on treated plants with the pathogen, while the control plants remained symptomless. Pathogen was successfully reisolated from brown leaf spot symptoms in which the cultural and morphological characteristics were identical to those of the originals. Neoscytalidium dimidiatum has a wide range of hosts and it has been reported in Malaysia to cause stem canker on pitahaya (Mohd et al. 2003; Khoo et al. 2023 ) and fruit rot of guava (Ismail et al. 2021). To the best of our knowledge this is the first report of N. dimidiatum causing leaf spots on pineapples in Malaysia. This report establishes a foundation for further study of N. dimidiatum that can effectively address the disease in pineapple.