Papaya (Carica papaya) is a fruit cultivated in tropical and subtropical regions but appreciated worldwide. It is known for its high nutritional and economic potential (Parven et al., 2020). Papaya fruit is rich in vitamins A and C, and in gallic acid, alkaloids, flavonoids, other phenolic compounds, and papain, an enzyme with extensive uses in the pharmaceutical, medical, and food industries (Brishti et al., 2013; Jarisarapurin et al., 2019). However, being a climacteric fruit, it is subject to intense metabolic activity, fast maturation, high susceptibility to fungal diseases, and short shelf life (Batista et al., 2020).

The most common fungal disease of papaya fruit is anthracnose, which is caused by Colletotrichum spp. and can result in 30–50% postharvest losses (Gunathilake et al., 2018). Several technologies have been used to extend the postharvest shelf life of fruit, including fungicides, low-temperature storage, thermal processing, diverse packaging conditions, and preserving compounds obtained from natural sources that are “generally recognized as safe” (Droby and Wisniewski, 2018). Exports of papaya fruit have been projected to grow at 1.7% per year over the medium term by the United Nations Food and Agriculture Organization, to potentially reach 3,18,000 t of fruit by 2028 (Food and Agricultural Organization of the United Nations, 2020). This, thus, indicates the opportunity for significant trade growth.

Chitosan (CS) is a natural biocompatible polysaccharide that is known to be an effective eco-friendly alternative to synthetic fungicides (Mutjaba et al., 2019; Rajestary et al., 2021). In recent years, CS has been used as a natural fungicide and plant defense booster based on its antimicrobial, film-forming, and eliciting defense activities (Feliziani et al., 2015; Romanazzi et al., 2018; Duan et al., 2019). Because of its film-forming properties, it can be used as a coating for many fruits and vegetables, to create a modified atmosphere around the product that prolongs the shelf life and retains the physicochemical and sensory properties (Valencia-Chamorro et al., 2011; Romanazzi et al., 2018). CS can elicit defense mechanisms of papaya (Ali et al., 2012), peach (Ma et al., 2013), banana (Hernández-Ibáñez et al., 2013), strawberry (Landi et al., 2014, 2017), orange (Coqueiro et al., 2015), avocado (Obianom et al., 2019), and grapes (Zhang Z. et al., 2020).

The physicochemical properties of CS relate to its hydrophilic nature, and these can be reinforced with the introduction of hydrophobic compounds, such as some essential oils (EOs). EOs such as those obtained from Cymbopogon citratus, Origanum vulgare, and Thymus capitatus, among others, have shown encouraging benefits when used as postharvest strategies for food preservation (Pisoschi et al., 2018; Sivakumar and Romanazzi, 2019). However, EOs are highly volatile, and thus their persistence on products when applied can be low (Alonso-Gato et al., 2021). This inconvenience could be reduced by encapsulating such EOs in polymers such as CS to potentially use them as alternatives to traditional fungicides (Rodríguez et al., 2016). In recent years, the use of CS-based composite coatings that incorporate EOs has been proposed as postharvest treatments for fruit (Yuan et al., 2016; Grande-Tovar et al., 2018). In most cases, these studies have confirmed the conservation of fruit physicochemical properties, inhibition of pathogenic microorganisms, and the extension of shelf life of fruit (Munhuweyi et al., 2017; Lima Oliveira et al., 2018). More recently, it was reported that an emulsion formed from 2% CS combined with different concentrations of Ruta graveolens (rue) EO (REO) has efficacy as a coating of postharvest fruit, with extended shelf life seen for guava (Grande Tovar et al., 2019), gooseberry (González-Locarno et al., 2020), tomato (Peralta-Ruiz et al., 2020b), pear (Peralta-Ruiz et al., 2021), and papaya (Peralta-Ruiz et al., 2020a). However, to our knowledge, the mechanisms associated with the protective effect induced by REO and CS-REO treatments in fruit are not well understood. It is well known that plant immune regulation is a defensive strategy of plants for protection against pathogen invasion, in addition, some substances can induce plant autoimmunity regulation mechanisms. In this context as mentioned above, CS is well known as an elicitor of plant defense responses, while the defense mechanism induced by REO and CS-REO combination was not investigated. Thus, the objectives of this study were to determine if the treatments of the papaya fruit with 0.5% CS, 0.5% REO, and 0.5% CS-REO combination were able to induce resistance and/or the activation of plant defense mechanisms, testing the early gene expression of key genes involved in plant response against biotic and abiotic stress. Furthermore, the effectiveness of postharvest control of anthracnose following treatments was analyzed.

In this study, we evaluated the effectiveness against postharvest anthracnose in papaya fruit of commercial formulations of CS and REO, both individually and combined. Furthermore, we also investigated the effects of these treatments on the activation of the transcription of key genes involved in plant defense mechanisms. In these fruits under postharvest storage at room temperature, and compared to the CS and REO treatments, the CS-REO combination indeed showed the greatest control of fungal decay and severity, as also for the McKinney’s Index. Similar results were observed in previous studies on papaya artificially inoculated with C. gloeosporioides, where the fruit treated with CS- 0.5% REO showed an incidence decay of 60% and a lower severity lesion (Peralta-Ruiz et al., 2020a). According to the AI, CS-REO showed an additive effect from CS and REO alone, as also indicated by Peralta-Ruiz et al. (2020a). Our findings agree with several previous studies that have reported on synergistic or additive effects of CS and EOs in combination for fruit preservation (Yuan et al., 2016; Lima Oliveira et al., 2018; de Oliveira et al., 2020; Elshamy et al., 2021).

The antifungal properties of CS are usually related to the interactions of its positive amino groups with the fungal membrane, which can induce changes to the permeability of the plasma membrane (Grande-Tovar et al., 2018). Also, the barrier effect of CS inhibits the germination of fungal spores and reduces the fungal decay on fruit (Romanazzi et al., 2018; Rajestary et al., 2021). This can preserve the physicochemical properties of the fruit for longer, thus also prolonging the shelf life (Nair et al., 2020).

In the present study, the effects of CS were reinforced by the addition of REO, which has been observed for other EOs (Sivakumar and Bautista-Baños, 2014; Huang et al., 2021), through their antimicrobial, antifungal, and antioxidant activities (Grande Tovar et al., 2019; González-Locarno et al., 2020; Peralta-Ruiz et al., 2020a; Zhang D. et al., 2020). It has been reported that the mode of action of such EOs is based on the cytotoxic effects including the induction of cell death by activation of apoptosis and/or necrosis processes (Nazzaro et al., 2017; Sharifi-Rad et al., 2017). Indeed, recent studies have reported that the fungal membrane of C. gloeosporioides was compromised after 1 h with 1% REO (Peralta-Ruiz et al., 2020a) and that under similar treatment, Candida yeast showed irreversible cell membrane damage with increased intracellular leakage of macromolecules (Donadu et al., 2021). In addition to the antimicrobial actions, several studies have shown activities as resistance inducers and/or activators of plant defense mechanisms for both CS (Ali et al., 2012; Landi et al., 2014, 2017; Coqueiro et al., 2015; Obianom et al., 2019) and EOs (Banani et al., 2018; Hou et al., 2020).

In the present study, we investigated for the first time, the expression of a range of plant defense genes induced by CS and REO and their combination in the papaya fruit. We selected genes that are involved in key metabolic pathways of plant defense responses to provide basic information on the main mechanisms involved in these actions of such CS–EOs combinations.

We first validated a useful protocol for this gene expression study in papaya fruit using RT-qPCR. This technique has been widely used to evaluate gene expression because of its speed and sensitivity. However, reliable quantification of gene expression mainly depends on accurate normalization. For this reason, the selection and validation of the reference genes were among the most crucial steps in the setting up of this RT-qPCR (Vandesompele et al., 2002; Bustin et al., 2009; Köhsler et al., 2020). This indicated two new suitable genes for gene expression studies in papaya fruit: H1 and tub. In this papaya fruit investigation, and independent of the treatments used in this study, these genes showed greater stability than 18S rRNA, as suggested by Zhu et al. (2012), and the tuf gene.

Based on this transcript analysis, this study showed that both the 0.5% CS and 0.5% REO treatments affected gene expression in the papaya fruits. However, according to the transcription of the individual genes analyzed in this study, some differences were seen. The jasmonic acid (JA)-responsive genes of JMT and LOX2 (Nakamura et al., 2011) were not affected in these papaya fruits by CS treatment, while they were involved in REO and CS-REO treatments. On the other hand, both CS and REO modulated the expression of genes linked to the SA pathway (i.e., SABP2, PR-1, and SNC1) and ET pathway (i.e., ET-2 and RAP2-13). Also, the PR-5 gene, which is linked to the SA pathway (Fu et al., 2012; Ali et al., 2018), was upregulated by REO treatment.

Many studies have indicated that the SA-mediated defense signaling pathway is important for activation of pathogen-associated molecular patterns that trigger immunity and for effector-triggered immunity, as well as for systemic acquired resistance (Jones and Dangl, 2006; Walters and Fountaine, 2009; Spoel and Dong, 2012). As well as the SA-mediated defense signaling pathways are indicated as linked to biotrophic pathogen infection, the jasmonate/ET pathways are indicated as involved with regulators of stress responses against necrotrophic fungi (Wasternack and Hause, 2013), and induction of volatile compounds in response to insect herbivores (Rodriguez-Saona et al., 2013) and during abiotic stress (Wang et al., 2020). However, investigations carried out in recent years have shown the complexity of the plant regulatory network against stress. Indeed, cross talk between the SA-dependent, JA-dependent, and ET-dependent signaling pathways is believed to be involved in the fine-tuning of the defense reaction, to lead to the activation of an optimal mix of defense responses to resist any particular pathogen (Pieterse et al., 2009; Jia et al., 2018). In support of this, a previous study showed that oregano EO can trigger the plant’s innate immune system that involves SA, JA, and ET synthesis and signaling, with the activation of PR proteins and phytoalexin synthesis (Rienth et al., 2019).

However, overall, our study showed that both CS and REO, and their combination CS-REO, can trigger signaling defense mechanisms to induce genes involved in phenylpropanoid biosynthesis and in cell wall metabolism, which demonstrates key roles for both secondary metabolite and cell wall genes in postharvest defense pathways (Landi et al., 2014, 2017; Xoca-Orozco et al., 2019; Zhang et al., 2020).

The present study underlines the involvement of genes linked to heat stress tolerance and cellular apoptotic change, in terms of NAC and HSP70 strongly upregulated in the early phase after REO treatment, while mainly for NAC gene, downregulation and /or unaffected gene expression was observed according to the other treatments. Part of the core of the data presented here is the difference detected between the CS and REO treatments according to activation times. The CS treatment affected gene expression mainly after 24 and 48 hpt, while the REO treatment strongly upregulated the gene transcripts earlier, at 0.5 hpt, then generally the gene expression drastically decreased at 6 hpt, then increased again mainly at 24 hpt, but to a lesser extent. In both cases, changes in gene expression over time are not surprising, given that gene expression is a complex stochastic process that represents the combination of numerous enzymatic reactions with unknown cell variables (Dal Co et al., 2017; Park et al., 2018). These changes in gene expression have been proposed to occur as the result of an optimization process, due to a trade-off between speed and cost (to the cell) of transcript production (Zaslaver et al., 2004). Also, the changes in gene expression might be linked to adaptation to the changes in stress (Koch and Guillaume, 2020). The present study suggests that these differences in gene expression over time can be correlated to the different natures of these two compounds, CS and REO. EOs are volatile, thermolabile, and unstable, which results in natural fluctuations in their components and compositions. They are highly reactive substances, and their antimicrobial activities might be impaired by changes in pH or temperature (Turek and Stintzing, 2013). On the other hand, CS has excellent film-forming properties, which will provide a mechanical barrier for the control of the respiration rate and decrease the loss of volatiles. The highly reactive volatile EOs can thus be stabilized and incorporated into the biodegradable, nontoxic CS, to produce transparent elastic films that can improve the effectiveness of their postharvest actions. This additive effect was evident in the gene expression patterns. Indeed, the genes analyzed after the CS-REO treatment generally showed greatly increased expression levels starting at 6 hpt, rather than at 0.5 hpt, as observed after the REO treatment. This might be due to the encapsulation of REO within the CS emulsion. Then, later on after the CS-REO treatment (i.e., beyond 6 hpt), for most of the genes, the expression levels were maintained relatively high for longer. This was seen for genes linked to the phenylpropanoid pathway, as PAL, FLS, and UFGT, as well as for genes involved in the signaling pathways that regulate plant defense, as SABP2, SNC1, M-JA, LOX2, ETR-2, and RAP2-13, but not for genes more closely with abiotic stresses such as NAC and HSP70, which suggests greater control of the cell stress by CS. This shows that the increase in the effectiveness of the disease control was associated with a broader and constant physiological change in the levels of the gene transcripts with roles in the induction of postharvest defense responses.

This study initially confirms that the incorporation of REO into the edible CS coating improves the control of postharvest decay of papaya fruit compared to the use of these treatments individually. For the first time, the main molecular mechanism in the triggering of defense pathways linked to the CS-REO combination is also indicated, as compared to their application. Indeed, CS largely showed effects on genes involved in the regulation of plant defense at 6 hpt, while REO showed strong induction of overexpression in the early phase, at 0.5 hpt. The CS-REO treatment also demonstrated additive actions on gene expression in these papaya fruits. This was supported by the delay in gene upregulation for CS-REO compared with REO, from 0.5 to 6 hpt, and kept longer over time. Indeed, this effect might be associated with CS such that it incorporates the volatile substances of the rue oil, and then releases them more slowly, to improve the regulation of cell stress.

This study thus represents an important first step in our better understanding of the molecular mechanisms involved in the combined effects of CS and EOs for postharvest control of fruit diseases. Similar studies are important for the control of postharvest decay, to suggest new strategies for induction of defense reactions in plants, and their possible use for the production of new active biological preparations.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

LL and YP-R: methodology, statistical analysis, software, and writing—review and editing. LL, YP-R, CC-L, and GR: conceptualization, design of the study, and review and editing of the manuscript. CC-L and GR: supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.