Contents lists available at ScienceDirect Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio Effects of mandarin (Citrus reticulata) peel essential oil as a natural antibiofilm agent against Aspergillus niger in onion bulbs Marwa M. Abdel-Aziz a , Tamer M. Emam b , Elsherbiny A. Elsherbiny c, ⁎ a Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt b Desert Research Center (DRC), Cairo, Egypt c Plant Pathology Department, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt ARTICLE INFO Keywords: Onion black mold Aspergillus niger Fungal biofilm Essential oil GC–MS Electron microscopy ABSTRACT The efficacy of the mandarin peel essential oil (MPEO) was evaluated for antibiofilm activity against A. niger with the effect on the development of black mold in onion bulbs in both curative and preventive applications. The detection of A. niger biofilm in onion bulbs by scanning electron microscopy (SEM), and fluorescence mi- croscopy (FM) was performed for the first time. Twelve constituents were identified in the MPEO by GC–MS analysis, and the predominant compound was limonene by 75.16%. The concentration of 30.72 mL L -1 of MPEO completely inhibited the planktonic growth of A. niger as well as the formation and eradication of A. niger biofilm. The changes in the biofilm morphology treated with MPEO were observed using scanning electron microscopy (SEM), including loss of ability to biofilm formation, absence of extracellular polymeric substances (EPS) and collapse of hyphae. Also, transmission electron microscopy (SEM) observations revealed ultra- structural alterations, including the disintegration of cytoplasmic organelles and folding of the cell membrane at various sites. Also, the fungal cell membrane integrity was affected by MPEO as detected by FM. Furthermore, the MPEO caused a considerable reduction in the development of black mold in onion bulbs inoculated with A. niger in the curative and preventive applications. The MPEO and its active components could be effective natural agent as an alternative to synthetic fungicides against A. niger in both planktonic and biofilm form. 1. Introduction Black mold disease caused by Aspergillus niger is a serious and eco- nomically damaging disease of onion (Allium cepa L.) production worldwide under field and storage conditions. The disease causes sig- nificant economic losses up to 80% of stored bulbs (Özer and Arin, 2014). The infection of onion plants by A. niger is initiated by con- taminated seed or soil and may continue throughout storage (Özer et al., 2009). Furthermore, the fungus is capable of producing myco- toxins such as ochratoxin A and fumonisin B2 and B4 (Nielsen et al., 2009). The use of chemical fungicides and resistant cultivars is an in- sufficient to reduce the disease under favorable environmental condi- tions (Özer, 2011). It is becoming increasingly evident that several plant pathogens are capable of forming biofilms on a number of plant species (Marques et al., 2002; Galiana et al., 2008; Harding et al., 2010; Theodorakopoulos et al., 2011; Li and Wang, 2014; Peiqian et al., 2014). In a biofilm the cells are protected by a matrix of extracellular polymeric substances (EPS), which exhibit an increased survival and resistance to chemical fungicides and host defenses. The matrix consists primarily of exopolysaccharides as the core component, lipids, proteins, extracellular DNA and other adhesive fibers (Balcázar et al., 2015; Villa et al., 2017). In general, the microbial biofilms exhibit up to 1000 times resistant to specific antifungal agents compared with the free-floating cells (Di Bonaventura et al., 2006). Furthermore, the microbial biofilms are a very significant problem in food industry, industrial water sys- tems, medical devices, and human infections (Sadekuzzaman et al., 2015). Hence, it is estimated that biofilms cause more than 80% of human persistent infections, especially in immune-compromised pa- tients (Roy et al., 2018). Currently, the consumption of pesticides is about 1.5 million tons per year worldwide (Liu et al., 2015). As a consequence, there is strong public attention about the safety of chemical pesticides used in the agriculture sector as well as great efforts to the discovery of new eco- sustainable strategies that could be integrated in the IPM practices (Villa et al., 2017). Various natural products such as plant extracts and essential oils have been extensively studied as a promising approach for controlling plant pathogens with prevent the formation and https://doi.org/10.1016/j.postharvbio.2019.110959 Received 14 March 2019; Received in revised form 26 June 2019; Accepted 17 July 2019 ⁎ Corresponding author. E-mail address: sherbiny@mans.edu.eg (E.A. Elsherbiny). Postharvest Biology and Technology 156 (2019) 110959 0925-5214/ © 2019 Elsevier B.V. All rights reserved. T