UV-C et laitue

Cette annexe 4 est présentée sous forme d’un article de résultats accepté dans le Journal « Scientia Horticulturae » (Figure 79, version acceptée en 2017). Figure 79 : Article de résultats UV-C/laitue. Pre-harvest hormetic doses of UV-C radiation can decrease susceptibility of lettuce leaves (Lactuca sativa L.) to Botrytis cinerea L. 1. Abstract Post-harvest applications of UV-C radiation have proven very efficient in reducing the development of post-harvest diseases in many species including lettuce (Lactuca sativa L.). Several studies suggest that UV-C radiation is effective not only because of its disinfecting effect but also because it may stimulate plant defenses. Preharvest treatment with UV-C radiation may thus offer an interesting potential for lettuce protection, provided that application doses are effective while excluding any harmful effects on the plants. Here we provide evidence that 0.85 kJ/m2 and 1.70 kJ/m2 represent doses of UV-C radiation that are not deleterious for lettuce plants. We used several criteria to evaluate the effect of UV-C radiation on the plant, including histological observations; the concentration of malondialdehyde, an indicator of membrane integrity, as well as Annexe 4 303 parameters derived from measurements of chlorophyll fluorescence, such as maximal efficiency of photosystem II (Fv/Fm) and the Performance Index of Strasser. We observed that a single dose of 0.85 kJ/m2 slightly increased plant resistance to grey mould (Botrytis cinerea L.) while a single dose of 1.70 kJ/m2 had the opposite effect. When a 0.85 kJ/m2 dose was applied 4 times, at two-day intervals, there was an increase in the total phenol content of leaves, and in PAL, CAT, and MDAHR activities. Leaves inoculated 2 days after the latter UV-C treatment showed significantly decreased sensitivity (- 30%) when compared to the control. Keywords: UV-C radiation, Botrytis cinerea L., Lactuca sativa L., photosystem II, membrane lipid peroxidation, plant resistance 2. Introduction Lettuce (Lactuca sativa L.) is cultivated worldwide and is one of the green leafy vegetables most consumed in raw form for its good taste, low price, and high nutritive value, especially due to its vitamin, fiber, and mineral input to a diet. Several diseases can be particularly damaging for lettuce production. Some cause loss of quality with symptoms on leaves, while infection of young plants often results in plant mortality. Among diseases, grey mould caused by the fungus Botrytis cinerea is of major concern to growers. The most common and effective strategy to control grey mould is the application of fungicides (Barrière et al., 2014). The use of these fungicides, however, can have a negative impact on the environment and human health. In addition, frequent use of fungicides can lead to the development of resistance (Russell, 1995; Komarek et al., 2010). Thus, there are less and less active molecules officially authorized in the EU. There is thus a growing interest for innovative strategies based on the use of abiotic or biotic factors for stimulating plant natural defenses (Conrath, 2009). Light is an important regulator of the plant-pathogen interactions via distinct photoreceptors and signalling pathways (Jenkins, 2009; Magerøy et al., 2010; Demkura and Ballare, 2012). In addition to the visible light, there are other electromagnetic radiations like infra-red light (IR) or ultraviolet light (UV). The UV portion of the electromagnetic spectrum includes longwave UV-A radiations (315 – 400 nm), medium-wave UV-B radiations (280 – 315 nm), Annexe 4 304 and short-wave UV-C radiations (200 – 280 nm) (Barta et al., 2004). UV-B radiation (280- 320 nm) has been reported to increase plant resistance to leaf pathogens (Gunasekera and Paul, 2007; Kunz et al., 2008; Ballare et al., 1996; Kuhlmann and Müller, 2010; Demkura and Ballare, 2012). The resistance may be attributed to changes in plant tissue metabolites induced by UV-B radiation, which include accumulation of protective phenolic compounds and enhancement of jasmonic acid-dependent defense pathway (Rousseaux et al., 1998; Mazza et al., 1999; Izaguirre et al., 2003; Foggo et al., 2007; Izaguirre et al., 2007; Kuhlmann and Müller, 2009; Demkura et al., 2010; Ballare, 2011, Mazid et al., 2011). In addition, UV-B radiation stimulates transcription of genes important for defense, including those encoding for phenylalanine ammonia-lyase and chalcone synthase, two key-enzymes controlling the synthesis of defense-related phenolic compounds, as well as pathogenesis-related proteins such as chitinase and ß-1,3- glucanase (El Ghaouth et al., 2003; Bonomelli et al., 2004; Borie et al., 2004). Conversely, we may expect UV-C radiation to be the most effective (and potentially harmful) due to their high energy level. However, besides the fact that it provokes oxidative stress, little is known about the biological effects of UV-C radiation on vegetative bodies and the mechanisms responsible for the stimulation of plant defenses (Urban et al., 2016). Exposure to UV-C radiation has been reported to reduce post-harvest decay of several species such as Allium cepa (Lu et al., 1987), Ipomea batatas (Stevens et al., 1990), Daucus carota (Mercier et al., 1993a,b) and Solanum lycopersicum (Charles et al., 2008a,b; Scott et al., 2016). Mercier et al. (2001) showed that the control of disease in bell pepper was obtained through induction of disease resistance rather than by fruit surface disinfection. Based on what is known about UV-B radiation and the induction of disease resistance in post-harvest crops by UV-C, it can reasonably be hypothesized that UV-C radiation could also exert an additional positive effect, indirectly by stimulating the resistance of plants against bio-aggressors. A large number of previously published works have shown that UV-C irradiation elicit defense responses in harvested horticultural crops (Charles et al., 2008a-c). The induction of disease resistance in postharvest crops in response to UV-C appears to involve the elicitation and accumulation of phytoalexins (Ben-Yehoshua et al., 1992; Mercier et al., 1993b; Sarig et al., 1997; Charles et al., 2008a), the ultra-structural modification of the epicarp leading to the formation of physical barriers (Charles et al., 2008b), the reinforcement of the cell walls by phenolic compounds, lignin and suberin (Charles et al., 2008c), and the enhancement of constitutive and inducible ß-1,3-glucanases and chitinases (Charles et al., 2009). Annexe 4 305 We have recently shown that lettuce plants treated with a low dose of UV-C after harvest show increased resistance against B. cinerea and Sclerotinia minor (Ouhibi et al., 2015a,b). Surprisingly, observations about the effect of UV-C radiation against pathogens by stimulating natural plant defences during their growth are extremely scarce (Kunz et al., 2008; Darras et al., 2015). These authors observed that treating Arabidopsis or Pelargonium plants with UV-C radiation induces reduction of infection by Hyaloperono sporaparasitica and B. cinerea, respectively. There is still a lack of references on crops destined for human consumption. Moreover the effect of intermittent exposures to UV-C has never been investigated so far.

Production of plant material and UV-C treatment

Romaine lettuce seeds (cv Duende, De Ruiter Seeds) were sown in 1 cm3 rockwool cubes in a glasshouse. One week after sowing, the cubes, each containing one plantlet, were transferred into plastic pots (5 L) containing a commercial growing medium (MetroMix 350, Sun Gro, Canada). They were then grown for 3 or 4 weeks at a temperature of 24/16°C (day/night). Five-week-old seedlings were used to identify non-deleterious doses of UV-C, while 4-week-old seedlings were used for a dose/repetition trial. Three independent experiments were conducted, the first between October and November 2013 (Mean cumulated global energy per m2 and per day is 7.4 MJ/m2 ), the second one between March and April 2014 (Mean cumulated global energy per m2 and per day is 13.7 MJ/m2 ) and the third one between October and November 2015 (Mean cumulated global energy per m2 and per day is 7.5 MJ/m2 ,). Annexe 4 306 To determine non-deleterious doses, the seedlings were exposed to UV-C light (254 nm, Spectroline, Model ZQJ-254, output 300 mW.c/m2, USA) at a distance of 30 cm. The UV-C radiation dose was made to vary by modifying the duration of exposure. Light intensity measurements were performed with a radiometer (Data Logging Radiometer PMA 2100, Glenside, USA) positioned at 30 cm from the ceiling light. The duration of plant UV-C radiation are 1 min (which corresponded to a dose of 0.85 kJ/m2 ), 2 min (1.70 kJ/m2 ), 4 min (3.40 kJ/m2 ), and eventually 8 min (6.80 kJ/m2 ). In the second experiment, the 4-week-old seedlings were exposed for a period of 1 min every two days during one week, which represents a cumulated dose of 3.40 kJ/m2 . In both trials, we used untreated plants as a control batch. To avoid the restorative effect of white light (Mercier et al., 2001), All the UV-C treatments were initiated after sunset. b. Measurements of chlorophyll a fluorescence using the Handy PEA Chlorophyll a fluorescence was measured using a portable non-modulated Handy PEA fluorimeter (Plant Efficiency Analyser, Hansatech Instruments, Kings Lynn, UK). After adaptation of leaves to darkness, a single strong 1s light pulse (3500 μmol photon/ m2 /s1 ) was applied with the help of three light-emitting diodes (650 nm). Recorded fluorescence parameters included: F0which represents the minimal fluorescence when all reaction centers are open, Fv/Fm which represents the maximum quantum yield of photosystem II (where Fm is the maximal fluorescence value and Fv = Fm –F0), Fv/Fm is widely used to assess stress in plants. Fv/F0 estimates the maximum primary yield of photochemistry of photosystem II, Fv/F0 is an indicator of the energy trapping probability (Krause and Weiss, 1991). PI is the so-called performance index of Strasser (Strasser et al., 2000). PI is a multi-parametric expression of three independent steps contributing to photosynthesis, namely RC/ABS, Fv/F0 and (1-Vj)/Vj. RC/ABS is an indicator of the size of the chlorophyll antenna serving each reaction center whereas (1-Vj)/Vj is an indicator of the performance due to the conversion of excitation energy to photosynthetic electron transport. PI is considered as a much more sensitive and discriminating stress indicator than Fv/Fm (Thach et al., 2007). Measurements of chlorophyll fluorescence were performed on 20 leaves per treatment (two leaves per plant).