First report of postharvest stem end rot disease on MD2 pineapple fruits caused by Neoscytalidium dimidiatum in Malaysia

Repeated sampling conducted from December 2019 to March 2020, and fruit of pineapple (Ananas comosus) var MD2 showing early stem end rot symptoms including brown and rotten fruit skin near the stem end region (Fig.1Aa) or darker skin with black discoloration (Fig.1Ab) indicated a consistent fungal infection. The samples (30 fruits from each location) were collected from store houses in three farmer fields with 60% disease incidence in Serdang, (3.0220oN,101.7055oE), Selangor, West Malaysia. The pulp of infected fruits appeared watery with characteristic spoilage odour. Symptomatic necrotic tissues from stem end region and skin were cut in to pieces (1x1cm), surface sterilized and plated onto potato dextrose agar amended aseptically with 0.5 g L-1 streptomycin sulphate. The plates were incubated at room temperature (28±2oC) in natural light conditions. Five days old cultures were light grey in colour and gradually turned dark brown to black with dense deeply tufted, mycelium as the culture aged (Fig.1B, C). Conidial morphology was observed using compound microscope (Olympus model BX-50F4, Tokyo, Japan) equipped with Dino-Eye. Branched mycelia with 0-1 septate arthospores were evident in 14 days old cultures (Fig.1D). Measured arthroconidia (5 to10x3 to 4.5µm) were ellipsoid to ovoid or round shaped, hyaline with an acutely rounded apex, truncate base, initially aseptate (Fig.1E) and arranged as chain at maturity (Fig.1F). The pathogen was identified through PCR amplification of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers (White et al., 1990) and BLASTn homology search as Neoscytalidium dimidiatum based on 100% similarity to a reference sequence (accession number KJ648577) that was previously deposited (Mohd et al.,2013). The sequence was deposited in Gen Bank ( accession number MW082810). Pathogenicity test was performed using the mycelial plug inoculation method and repeated twice with five replicates. Healthy MD2 pineapple fruits were surface sterilized with 1% NaOCl solution for15 min. followed by washing with sterilized distilled water. One centimeter diameter PDA plug at the margin of actively growing seven days old cultures were inserted in each of two inoculation wounds made on the skin and stem end of each fruit then the wounds were wrapped with moist cotton wool. Non-colonized PDA plugs were used to inoculate the control fruits. Fruits were incubated under 85% RH at room temperature. Five days after inoculation, the fruits showed similar dark necrotic discoloration and confirmed as N.dimidiatum by PCR (Fig.1G). The Koch postulates were fulfilled by inoculation and re-isolation of the fungal pathogen. This pathogen has also been reported previously to cause economic losses on a number of other hosts, such as pitayah fruits in Israel and Malaysia (Erza et al., 2013; Mohd et al., 2013)) and almond in California (Mohomed et al., 2018). To our knowledge this is the first report of N. dimidiatum causing postharvest stem end rot on MD2 pineapple in Malaysia. It may have the possibility to develop postharvest economic losses to pineapple industry, if severely affected fruits with high population of the pathogen left unattended in store houses.