Three-dimensional epicuticular wax on plant surface reduces attachment and survival rate of Salmonella during storage
Introduction
Salmonella, gram-negative, non-endospore-forming, facultative bacteria, is a common etiologic agent of foodborne illness in the United States, which is accounting for 132 outbreaks and 3047 individual cases of illness in the U.S. in 2016 based on a new surveillance report (Dewey-Mattia et al., 2018). Salmonella is traditionally associated with raw or processed poultry meat products, but has been increasingly linked to fresh produce in recent years (Sivapalasingam et al., 2004). Many common vegetables, including lettuce, have been reported to be contaminated with Salmonella (Horby et al., 2003). Most recently, in 2018 and 2019, Salmonella infection was linked to the outbreaks of papaya (CDC, 2019a), pre-cut melons (CDC, 2019b), and kratoms (CDC, 2019c), all these previously unrecognized food vehicles for Salmonella contamination. In a US and European union-based study, salad and leafy vegetables were reported to be the second most common food vehicle for Salmonella outbreak, and Salmonella was also the leading cause of multistate foodborne outbreaks in the U.S. (Callejón et al., 2015). Although the practice using different sanitizers, including chlorine water and organic acids, in production pipelines has been extensively studied, outbreaks are still reported globally or regionally (Li et al., 2017; Weissinger et al., 2000). Therefore, it is crucial to find out alternative strategies to reduce the incidence of Salmonella outbreak by studying interaction between plant surface and food pathogens.
One source of Salmonella contamination of fresh produce is wash or irrigation water in food preparation environment (Hanning et al., 2009), or animal manure (Franz and van Bruggen, 2008). Salmonella in the contaminated water or manual can adhere to the surface of the produce, or even internalize into the fresh produce from stomata (Kroupitski et al., 2009) or damaged area (Erickson, 2012). In either situation, if the attachment or the colonization of Salmonella can be reduced, the incidence of salmonellosis may be reduced. It has been hypothesized that the special physicochemical characters of plant surface might contribute to less susceptibility to the contamination of food pathogens, which may provide useful strategies to decrease Salmonella recalls and outbreaks.
Epicuticular wax has its unique hydrophobicity and crystalline morphology due to its prolific chemical components with long-chain fatty acids, alcohols, aldehydes, primary-alcohols, alkanes, esters, and lipids (Koch and Ensikat, 2008). Its crystalline morphology occurring as two-dimensional wax films, three-dimensional platelets, rods, and dendritic structures (Ensikat et al., 2006) may be changed by environmental conditions (Jetter and Riederer, 1994) and the epidermal secretion site (Jenks and Ashworth, 1998). Besides, its three-dimensional wax crystal morphology influences the repellent ability of leaf surface water-retaining (Ensikat et al., 2011). Epicuticular wax boundary provides a hydrophobic coating over most above-ground organs and protects plants from various environmental stresses, and was found to interact with the microbiota on lettuce (Lima et al., 2013).
Previous studies have demonstrated that epicuticular wax of the produce affected the microorganism attachment to the surfaces of various produce (Hunter et al., 2015; Kenney and Beuchat, 2002; Ku et al., 2020; Lu et al., 2015). In an early study, Kenney and Beuchat reported that spraying wax onto apples reduced Escherichia coli O157:H7 and Salmonella Muenchen by 1.5 log10 CFU per apple than the control and water treatments (Kenney and Beuchat, 2002). Hunter et al. (2015) reported that the older leaves had higher Salmonella attachment rates than the younger leaves due to the difference of leaf morphology and hydrophobicity caused by the amount of wax between older and younger leaves (Hunter et al., 2015). Lu et al. (2015) reported that cuticular wax levels significantly changed rotavirus attachment among lettuce cultivars (Lu et al., 2015). Our recent study (Ku et al., 2020) also indicates that mature rosette leaves on bolting ‘Two Star’ lettuce with lower wax amount (175 vs 447 mg m−2) showed higher Salmonella attachment than immature leaves (5.72 vs 5.18 log10 CFU g-1) at harvest and after 7 d storage of 4 °C storage (5.16 vs 4.38 log10 CFU g-1). The epicuticular wax morphology of bolting ‘Two Star’ lettuce was found to have three-dimensional wax crystals on mature leaves (Lu et al., 2015). These shreds of evidence suggested a trend that leaf surface that is high in epicuticular three-dimensional wax crystals was associated with fewer numbers of Salmonella attachment. As three-dimensional waxes may contribute to the wettability and anti-adhesion on plant surface (Ren et al., 2007), visible three-dimensional epicuticular wax crystals on the leaf may be the major contributor to the decreased attachment of Salmonella-contaminated water.
While previous research has proposed that epicuticular three-dimensional wax crystals are associated with reduced attachments of food pathogens, this relationship has not yet been verified. The aim of this study is to test the hypothesis that the presence of three-dimensional epicuticular wax crystals decreases the attachment efficiency of Salmonella spp. To further examine this hypothesis, we utilized the leaf of near-isogenic (NIL) broccoli to test the attachment efficiency of Salmonella. The NIL plants imply that only a single DNA fragment, locus, is different between two lines, and therefore, only one trait is different between two lines. The NIL broccoli (Brassica oleracea L. var. italica) developed by (Farnham, 2010) are highly homogenous and phenotypically homogeneous except for the presence of epicuticular wax on the leaf, and thus, they are ideal for this study. To characterize the wax on the leaves, scanning electron microscope (SEM) imaging was employed to visualize the morphology of epicuticular wax associated with the plant materials.
Section snippets
Greenhouse production and phenotype description of near-isogenic broccoli and collard
Seeds of two pairs of NIL broccoli (USVL115-GL (glossy); USVL115-NG (non-glossy; waxy) and USVL188-GL; USVL188-NG, obtained from U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS, U.S. Vegetable Laboratory, Charleston, South Carolina) were used in this study (Fig. 1A and B).
To further apply the experimental results to real production pipelines, different cultivars from the same plant species (bigger genetic difference than NIL lines) Brassica oleracea were also used in
Surface properties and cuticular wax quantification of glossy, wax-removed, and waxy leaves
To investigate the effect of epicuticular wax crystals on pathogen attachment, gum arabic treatment was used to mechanically remove the surface wax crystals. Among various adhesives for this practice, gum arabic was reported to have the best reproducible result on wax removal (Jetter and Schäffer, 2001). On the surface of waxy plants (USVL 188-NG, USVL-115-NG, and ‘Top Bunch’ collard), three-dimensional epicuticular wax crystals were easily visible (Fig. 2). The epicuticular wax crystals on gum
Conclusion
In the present study, the inoculated Salmonella spp. on the waxy leaf surface (with three-dimensional epicuticular waxes) was significantly lower than glossy leaf surface (without three-dimensional epicuticular waxes) after the initial inoculation, and was consistently lower for 14-days in 4 °C storage experiment, which suggested that presence of three-dimensional epicuticular waxes could reduce Salmonella attachment in the initial contamination and in prolonged 4 °C storage. Moreover, the
CRediT authorship contribution statement
Yu-Chun Chiu: Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Cangliang Shen: Conceptualization, Methodology, Resources, Data curation, Writing - original draft, Writing - review & editing, Funding acquisition, Project administration. Mark W. Farnham: Conceptualization, Resources. Kang-Mo Ku: Conceptualization, Methodology, Resources, Supervision, Data curation, Writing - review & editing, Funding acquisition,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This study was supported by WVU-School of Agriculture and Food Research Grant. We ackowledge the use of the WVU Shared Research Facilities for SEM instrumnet.
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Chiu Y.C. and Shen C. contributed equally to this work