Preharvest high-intensity, pulsed polychromatic light and low-intensity UV-C treatments control Botrytis cinerea on lettuce (Lactuca sativa)

Hormetic treatments using high-intensity, pulsed polychromatic light (HIPPL) and low-intensity UV-C (LIUV) can control Botrytis cinerea on lettuce (Lactuca sativa) in a controlled environment. For the cv. Temira, 48 pulses of HIPPL significantly reduced mean disease progression (26%), while 0.64 kJ/m2 of LIUV gave reductions of 27%. No LIUV or HIPPL treatment significantly reduced disease progression for cv. Amica. A 0.98 kJ/m2 dose of LIUV and 24 pulses of HIPPL, however, reduced mean disease progressions (25% and 15%) when compared to the control. Phytotoxicity was observed at 48 and 72 pulses of HIPPL for Amica and Temira, respectively. LIUV caused phytotoxicity on both cvs. above 0.98 kJ/m2 but at a reduced incidence for Temira. Both technologies delivered a similar level of disease control on cv. Temira and may provide a residue-free alternative to the chemical control of plant pathogens. HIPPL treatments, however, were achieved in 15 s and may prove to be a more commercially feasible alternative to LIUV with a treatment duration of 32 s.

High-intensity, pulsed polychromatic light (HIPPL) emits high-energy, short pulses (1 to 10 4 µs) of electromagnetic radiation from ultraviolet to infrared (180 to 1,100 nm). Interest in HIPPL has increased over the last 15 years as a cost-effective and residue-free decontamination method (Rowan 2019). The majority of previous HIPPL research has been focused on the decontamination of surfaces, including that of fresh produce, with a particular emphasis on human pathogenic microorganisms; neither are the focus of this study. See reviews by de Moraes and Carmen (2018), Schottroff et al. (2018) and Rowan (2019) for further information.
In contrast, this study is focused on hormesis-induced disease resistance rather than the direct germicidal activity of HIPPL. Hormesis is a dose-response phenomenon where non-damaging exposure to a stressor brings about a beneficial response. HIPPL may not only provide a residue-free alternative to the chemical control of plant pathogens but also a faster alternative to other hormetic treatments such low-intensity UV-C (LIUV). Scott et al. (2017) showed that postharvest HIPPL treatments that delay ripening on mature green tomato fruit (Solanum lycopersicum cv. Mecano) can be delivered in a matter of seconds (10 s) when compared to LIUV (370 s). Few investigations into HIPPL hormesis, however, have been performed and early studies were focused on beneficial changes to secondary metabolism and nutritional content rather than disease control (Oms-Oliu et al. 2010;Koyyalamudi et al. 2011;Rodov et al. 2012;Pataro et al. 2015). For instance, postharvest HIPPL treatments of net house-grown figs (Ficus carica) increased phenolic concentration and improved fruit colour with a dose of 18 kJ/m 2 while anthocyanin concentration was increased with a dose of 60 kJ/ m 2 (Rodov et al. 2012).
HIPPL induced disease resistance was observed alongside the delayed ripening of tomato fruit by Scott et al. (2017). A 16 pulse postharvest treatment reduced the disease progression of Botrytis cinerea by 41.7% and 28.5% on mature green and ripe tomato fruit, respectively. HIPPL and LIUV induce disease resistance by changing the plants' defence status through upregulation or priming of defence mechanisms such as pathogenesis related proteins and phytoalexins along with delayed ripening or senescence and physical modifications that inhibit pathogen progression through host tissues (Ben-Yehoshua et al. 1992;D'Hallewin et al. 1999;Charles et al. 2008aCharles et al. , b, 2009Scott et al. 2017Scott et al. , 2018. The changes to gene expression underpinning both HIPPL and LIUV disease resistance in tomato fruit were highly similar and included the upregulation of pathogenesis related proteins CHI9 and GLUB together with the upregulation of biomarkers of, and enzymes involved in, the biosynthesis of phytohormones salicylic acid (P4), jasmonic acid (OPR3) and ethylene (ACO1) along with the downregulation of polygalacturnonase (Scott et al. 2018).
A second study looking into HIPPL disease control showed that a 9 kJ/m 2 treatment reduced the mean lesion area of Fusarium pallidoroseum from 13 mm to 4 mm on melon (Cucumis melo) at 21 d post inoculation (DPI) (Filho et al. 2020). Incidence was reduced from 100-33% when compared to the control. The authors state this was achieved through stimulation of the plant defences; identified by the upregulation of biomarkers pipecolic acid, saponarin and orientin. Further investigation of their experimental protocols, however, indicates that the fungicidal and fungistatic effects cannot be exclusively attributed to induced plant defences as the fruit were inoculated prior to treatment and the inoculum was exposed to the direct germicidal effects of HIPPL.
A previous study on the impact of HIPPL treatments on lettuce (Lactuca sativa) by Fgaier et al. (2019) showed HIPPL can significantly increase flavanol content and also transiently damage or inhibit the photosynthetic machinery, which recovered by 6 days post treatment (DPT). Additionally, a 3 pulse treatment showed an increase in photosynthetic assimilation of 38% at 8 DPT indicating a long-term increase in photosynthetic capacity. As yet, however, no investigations pertaining to HIPPL disease control on lettuce have been reported.
A similar progression was also seen in the development of LIUV's application in food production, with early investigations also being concerned with the decontamination of surfaces including fresh or processed produce. The evidence supporting LIUV hormesis, however, is far more substantial than that for HIPPL with the first report of LIUV hormesis in plants by Lu et al. (1987). The benefits of LIUV treatment are well established and have been shown in numerous plant species. These include improved nutritional content and colour, disease resistance and delayed chlorophyll degradation; see reviews by Shama and Alderson (2005), Ribeiro et al. (2012) and Turtoi (2013).
More recently, postharvest LIUV induced disease control has been shown on lettuce by Ouhibi et al. (2014). A postharvest treatment of 0.85 kJ/m 2 reduced B. cinerea and Sclerotinia minor lesion size by 20 and 33% at 4 DPI, respectively. Moreover, plants showed an increase in reactive oxygen species (H 2 O 2 ) at 4 DPI which may be playing a role in defence, aside from their role in cellular signalling. Furthermore, Vàsquez et al. (2017) showed that a single preharvest LIUV treatment of 0.85 kJ/m 2 on four week old lettuce plants reduced mean B. cinerea lesion area by approx. 20% at 7 DPI. Four successive LIUV treatments, delivered with 48 h between treatments, reduced lesion area by approx. 33% at 7 DPI and increased the concentration of phenols and PAL. The LIUV treatments also led to a transient decrease in chlorophyll fluorescence which recovered by 6 DPI in all but the 6.80 kJ/m 2 treatment; a finding analogous to that observed by Fgaier et al. (2019) with HIPPL.
The objective of this study was to establish whether preharvest, hormetic HIPPL and LIUV treatments can control B. cinerea on lettuce plants grown in a controlled environment. Experimental protocols were as follows, lettuce seeds of the cvs. Amica and Temira (Enza Zaden) were sown individually onto 25 mm rockwool propagation cubes (Grodan). The seeds were placed in a controlled environment with a 16/8 h cycle at 21/12°C and a relative humidity of 70 to 80%. Lighting was provided by 400 W HPS bulbs at approx. 250 µmol/ m/s at canopy level. Propagation cubes were kept moist with water until their roots emerged from the cubes (approx. 14 d).
Propagation cubes were then placed into 7.5 cm Delta Cubes (Grodan) prewetted with 1 g/L Hortimix Standard (HortiFeed) and placed into a nutrient film technique (NFT) irrigation system using a randomised experimental design. The NFT system had a gradient of 1:50, 15 cm spacing between plants, recycled its nutrient solution and had four gutters housing up to a total of 60 plants. The nutrient solution was changed on a weekly basis using 40 L of Hortimix Standard (HortiFeeds) at 1 g/L. Plants were grown to the 6 to 8 true leaf stage and then treated with either HIPPL from 40 cm or LIUV at 2000 µW/cm 2 . Plants were kept in darkness for 8 h posttreatment to prevent photoreversal; whereby exposure to white light immediately following treatment can counteract the hormetic benefits of treatment (Stevens et al. 1998).
HIPPL treatments were performed with a LH-840 16" ozone-free B lamp (XENON) with polychromatic emissions between 240 nm and 1050 nm. The lamp was powered and controlled by RT-847 cabinet and RC-802 controller (XENON), supplied by Lambda Photometrics (Harpenden, Herts). The HIPPL system produced 505 J of energy per pulse, with a pulse width of 360 µs at 3.2 pulses/s. LIUV treatments were performed with a Ushaped amalgam UV source (UVI 12OU2G11 CP15/469) with peak emission at 254 nm and housed within an anodised aluminium parabolic reflector; obtained from Dr Hőnle AG, Gräfelfing, Germany. Lamps were switched on 30 min before use and intensity was measured with a radiometer fitted with a 254 nm sensor (Model UVX, UVP Instruments, Cambridge).
Plants were treated with 0 (control), 12, 24, 36, 48 or 72 pulses of HIPPL or 0 (control), 0.32, 0.64, 0.98, 1.28 or 1.92 kJ/m 2 of LIUV. At 2 DPT plants were visually assessed for signs of phytotoxicity which manifested as vascular browning and dry necrotic lesions. A bioassay based on the methods of Laboh (2009) was then performed to measure the treatments' impact on disease progression. Briefly, a 20 mm leaf disc was taken from the lamina closest to the apex of true leaves 3, 4, 5 and 6 to give four technical repeats per plant. Care was taken to avoid the primary vein when taking discs. Leaf discs were placed into 8 cm square Petri dishes containing 25 ml of 0.8% molecular grade agar (Oxoid) with the lower epidermis positioned towards the agar. Sixteen leaf discs were placed in each Petri-dish using a complete block, randomised design and then inoculated with B. cinerea using a calibrated spore solution (1 × 10 6 ) amended with 50% potato dextrose broth (Sigma Aldrich). A 10 µL aliquot was pipetted into the centre of each leaf disc and Petri-dishes were sealed with parafilm and then stored in the controlled environment under the same conditions as plant growth. Photographs of the leaf discs were taken at 2 and 3 DPI. FIJI image analysis software was used to measure lesion area and area underneath the disease progression curve (AUDPC) was calculated (Eq. 1) (Schindelin et al. 2012).
Area underneath the disease progression curve (AUDPC) formula where n = total number of observations per inoculation point, i = observation, y = lesion area and t = time (Jeger and Viljanen-Rollinson 2001).
Two independent experimental replicates were performed giving a total of 10 biological and 40 technical repeats per treatment group. Technical repeats were averaged prior to any additional data processing or analysis. Due to inter-experimental variability in the virulence of B. cinerea, factor correction was performed following the methods of Ruijter et al. (2006) and as previously used in Scott et al. (2019). Data were analysed in R by one-way ANOVA followed by Tukey's post-hoc test (R Core Team 2019). Statistical significance is defined as p < 0.05 for all experiments.  Fig. 1 The extent of phytotoxicity at two days post-treatment following (a) high-intensity, pulsed polychromatic light (HIPPL) and (b) low-intensity UV-C (LIUV) treatments at the 6 to 8 true leaf stage Data were visualised in R using the ggplot2 package (Wickham 2016).
Visual inspection of plants identified a potential difference in sensitivity to HIPPL between the two cultivars with 20% of Amica showing signs of phytotoxicity at 48 pulses and 100% at 72 pulses (Fig. 1a). Only 60% of Temira plants, however, were damaged by 72 pulses. A slight difference in LIUV sensitivity was seen with 100% and 70% of Amica and Temira showing signs of phytotoxicity at 1.28 kJ/m 2 , respectively (Fig. 1b). This is in contrast to 60 and 70% of Amica and Temira being damaged at 0.98 kJ/m 2 .
HIPPL treatments of 48 and 72 pulses significantly reduced the disease progression of B. cinerea on Temira at 26 and 28%, respectively (Fig. 1a). The 72 pulse treatment, however, led to phytotoxicity. The most effective LIUV treatments for Temira were 0.64 and 1.92 kJ/ m 2 , reducing disease progression by 27 and 45% respectively (Fig. 1b). As with HIPPL, the most efficacious LIUV treatment (1.92 kJ/m 2 ) caused damage to the crop. No HIPPL or LIUV treatments significantly reduced disease progression on Amica, 24 pulses of HIPPL and a 0.98 kJ/m 2 dose of LIUV, however, reduced disease progression by 15 and 25%, respectively ( Fig. 1c and d). Labelling indicates statistical significance at p < 0.05. Groups sharing the same letter are not significantly different from each other. Error bars are ± 1 S.E.M and grey markers show the data for each biological repeat; n = 10 In this study, preharvest HIPPL and LIUV treatments that control the disease progression of B. cinerea on lettuce have been identified. This is the first report of preharvest HIPPL induced disease control and HIPPL disease control on lettuce. Furthermore, in comparison to LIUV, disease control was achieved in 15 s, a 53% reduction in treatment time. HIPPL may, therefore, prove to have increased feasibility for commercial production systems as a residue-free alternative to chemical control. The mechanisms underpinning disease control on lettuce have not been explored, but the direct germicidal effects of LIUV and HIPPL can be discounted as artificial inoculations were performed after exposure to HIPPL or LIUV. It is likely, therefore, that disease control is achieved through the activation or priming of plant defences (Fig. 2).
Additionally, the observation that preharvest LIUV treatments of 0.64 kJ/m 2 successfully controlled B. cinerea on Temira support the findings by Vàsquez et al. (2017)  Future investigations should be focused on identifying the treatment dose and application frequency for successful HIPPL and LIUV disease control in the glasshouse and for different cultivars. During a year of preliminary glasshouse trials, reductions in the disease progression of B. cinerea, Sclerotinia sclerotiourm and Rhizoctonia solani were variable (2 to 54%) and optimal treatments fluctuated accross the season (data not shown). This variability could be expected as physiological adaptation to environmental changes may alter the plants' sensitivity to HIPPL and LIUV and the dose required to induce disease resistance or cause damage. A situation in which LIUV or HIPPL treatments may fail to control disease without causing phytotoxicity could, therefore, be hypothesised. Understanding why this variability occurs and how treatment doses should be altered across the season may be key to the successful implementation of HIPPL and LIUV treatments in the commercial glasshouse.
Finally, consumer-focused characteristics such as colour, flavour and nutritional content and producerfocused interests such as plant mass and compactness should be monitored to gain a greater understanding of the impacts of using HIPPL and LIUV to control disease. If the treatments are found to be commercially feasible then investigations into the mechanisms underpinning disease control should be performed.