Introduction
Crop yield losses of 20-40% of total agriculture productivity can be attributed to pests and diseases (Oerke, 2006, Savary et al., 2012). Of these threats, the pathogen Botrytis cinerea causes annual losses of $10-$100 billion, as it reduces crop yield before harvest or leads to waste and spoilage post-harvest. It is the causative agent of grey mould disease in tomato and many other economically important crops, such as pepper, aubergine, grape, lettuce and raspberry. B. cinerea is a fungal generalist (broad-host range) and considered to be a model necrotrophic pathogen (Williamson et al., 2007). Effective control include the use of conventional crop protectants (e.g. fungicides) and resistant varieties as well as sanitation and environmental control. However, rapid pathogen evolution can result in the loss of efficacy of resistance sources and fungicides (Pappas, 1997, Williamson et al., 2007). In addition, the use of pesticides is strictly limited by European regulations due to human health and environment risk and hazard assessment changes. New alternative strategies are therefore needed. Exploiting the plant’s defence system to provide protection against these threats has emerged as a potential strategy against pathogen infection and disease (Luna, 2016).
Plant endogenous defences is activated by elicitor molecules resulting in induced resistance (IR) (Mauch-Mani et al., 2017), since they are able to mimic pathogen-inducible defence mechanisms (Aranega-Bou et al., 2014). Induced resistance works via two different mechanisms: direct activation of systemic plant defences after signal recognition and; priming, a mechanism that initiates a wide reprogramming of plant processes, considered to be an adaptive component of induced resistance (Mauch-Mani et al., 2017). Priming has been demonstrated to be the most cost-effective mechanism of induced resistance in terms of plant development as there is no direct relocation of plant resources from growth to defence until it is necessary (van Hulten et al., 2006). Studies have already shown that low elicitor doses can enhance resistance to pests without interfering with crop production (Redman et al., 2001). Elicitor-induced priming has been demonstrated to last from a few days (Conrath et al., 2006) to weeks (Worrall et al., 2012) after treatment and even through subsequent generations (Ramírez-Carrasco et al., 2017, Slaughter et al., 2012).
Priming can have multiple effects on plant defences, which vary depending on the type of plant-pathogen interaction. Defence priming enables the plant to fine-tune immunity responses through enhancement of the initial defences. These is achieved through different mechanisms that act at specific defence layers (Mauch-Mani et al., 2017). For instance, cell-wall fortification and effective production of reactive oxygen species (ROS) has been used as a marker for the expression of priming responses. Hexanoic acid (Hx) primes cell-wall defences through callose deposition and redox processes in tomato cultivars againstB.cinerea (Aranega-Bou et al., 2014). In Arabidopsis thaliana , BABA and benzothiadiazole (BTH)-induced priming is also based on an increase in callose deposition (Kohler et al., 2002, Ton et al., 2005). Priming also results in transcriptomic changes. Gene expression analysis of A. thaliana after BABA treatment was used to identify a transient accumulation of SA-dependent transcripts, including that ofNPR1 , which provides resistance against Pseudomonas syringae (Zimmerli et al., 2000). Changes in metabolite accumulation have been shown to mark priming of defence also. For instance, defence hormone profiling has shown that accumulation of JA and JA-derivatives mediates priming of mycorrhizal fungi (Pozo et al., 2015). Moreover, untargeted metabolomic analysis have identified different compounds, including kaempferol (Król et al., 2015), quercetin, and indole 3 carboxylic acid (I3CA) (Gamir et al., 2014), that drive priming responses.
Several elicitors have been described to induce resistance mechanisms in tomato against B. cinerea . For instance, BABA has been demonstrated to provide long-lasting induced resistance against B. cinerea in leaves (Luna et al., 2016) and in fruit (Wilkinson et al., 2018). In addition, the plant defence hormone JA has also been linked to short-term and long-term induced resistance in tomato against B. cinerea (Luna et al., 2016, Worrall et al., 2012). To date, however, few studies have investigated elicitor-induced priming in tomato againstB. cinerea. One of them showed that Hx-induced priming is based on callose deposition, the expression of tomato antimicrobial genes (e.g. protease inhibitor and endochitinase genes), and the fine-tuning of redox processes (Aranega-Bou et al., 2014, Finiti et al., 2014). Therefore, evidence is building in tomato, that induced resistance against B. cinerea can be based on priming also.
In this study we investigated whether the chitin de-acetylated derivative, chitosan, triggers priming of defence in tomato againstB. cinerea . Chitosan as a plant protection product is considered ‘generally recognised as safe’ (Raafat and Sahl, 2009) that is effective in protecting strawberry, tomato and grape against B. cinerea(Muñoz and Moret, 2010, Romanazzi et al., 2013). Different studies have shown that its effect on crop protection results from induction of defence mechanisms (Sathiyabama et al., 2014) and direct antimicrobial activity (Goy et al., 2009). However, treatments with chitosan require infiltration into the leaves to trigger a robust effect (Scalschi et al., 2015) making it an unsuitable method of application in large-scale experiments or studies that take into consideration first barrier defence strategies. Here, we have addressed whether treatment with a water-soluble formulation of chitosan results in induced resistance phenotypes and in priming of cell wall defence and defence hormone accumulation. In addition, whole-scale transcriptome analysis was performed to identify candidate genes that are driving expression of priming. Our findings, together with the outlined characteristics of chitosan, make this substance a suitable candidate for extensive application as a component of Integrated Pests (and disease) Management (IPM) for the protection of crops against fungal pathogens.