Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions

Semakaleng Mpai; Dharini Sivakumar

doi:10.3390/agronomy10111654

Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions

Mpai, Semakaleng;Sivakumar, Dharini 2020-10-27 00:00:00 agronomy Article Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions Semakaleng Mpai and Dharini Sivakumar * Department of Crop Sciences, Phytochemical Food Network, Tshwane University of Technology, Pretoria 0001, South Africa; semkalengmpai@gmail.com * Correspondence: SivakumarD@tut.ac.za Received: 19 August 2020; Accepted: 19 October 2020; Published: 27 October 2020 Abstract: The ability of light-emitting diode (LED) light treatment to reduce the anthracnose decay via its eliciting eects and thus induce resistance in the avocado (Persea americana), was investigated in this study to replace the current postharvest fungicide treatment. In experiment 1, the eect of blue or red LED lights (6 h per day) on the incidence of anthracnose in artiﬁcially inoculated (Colletotrichum gloesposorioides) and naturally infected avocados (cv. Fuerte and Hass) at 12–14 C (simulated market shelf) for 4, 8, 14, and 16 days was investigated. In experiment 2, the eect of blue or red LED lights on the induced defence mechanism, fruit metabolites, antioxidant activity, and percentage of fruit reaching ready-to-eat stage was determined. Exposure to red LED light signiﬁcantly reduced the anthracnose decay incidence in naturally infected cv. Fuerte on day 12 and in cv. Hass on day 16 compared to the prochloraz fungicide treatment by upregulating the PAL genes and maintaining the epicatechin content. Blue LED light accelerated the ripening in both cultivars, probably due to reduced D-mannoheptulose content. Red LED light exposure for 6 h per day and 12 days storage showed potential to replace the prochloraz treatment with improved ascorbic acid content and antioxidant activity. Keywords: Persea americana; phenolic compounds; ripening; postharvest decay; D-mannoheptulose; phenylalanine ammonia lyase 1. Introduction Avocado (Persea americana) is a popular subtropical fruit amongst consumers for its health beneﬁts. Consumption of half an avocado (68 g) reportedly provides dietary ﬁbre (4.6 g), potassium (345 mg), vitamin C (6.0 mg), vitamin E (1.3 mg), folate (60 mg), choline (10 mg), lutein/zeaxanthin (185 g), phytosterols (57 mg), and a high level of monounsaturated fatty acids (6.7 g) [1]. Among the avocado cultivars, consumers mainly prefer the dark-skinned cv. Hass because of its creamy and smooth texture and nutty taste, whilst the green-skinned cv. Fuerte is popular due to its good ﬂavour and oily texture [2]. Consumer preference is high for “ready-to-eat stage” avocados [3], but due to postharvest decay anthracnose (Colletotrichum gloeosporioides Penz.), the quality of fruit can be negatively aected, and postharvest losses during marketing are high [4]. Avocado is a climacteric fruit, which reaches ready to ripe stage after harvest at the climacteric peak with ethylene emission, resulting in structural and biochemical changes and reduction of antifungal compounds to alleviate the dormancy of C. gloeosporioides, the latent pathogen [4]. The symptoms are expressed in the fruit at trigger-ripe stage, especially during marketing on the supermarket cold shelf at 12–14 C [5]. Agronomy 2020, 10, 1654; doi:10.3390/agronomy10111654 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1654 2 of 25 In most avocado-exporting countries, Prochloraz, a chemically synthesised fungicide, is applied as a spray or dip in the packing line to provide residual protection against the anthracnose-causing organism during marketing [4]. However, Prochloraz has hazardous health eects, and the maximum residue limits (MRLs) for the South African avocado limits its access to the quality-stringent export markets [6]. Consequently, numerous alternative green technologies have actively undergone research during the past 5 years to replace the Prochloraz fungicide treatment [7]. The use of a light-emitting diode (LED), as a nonchemical treatment for maintaining quality attributes of horticultural produce, is becoming popular [8]. Light-emitting diode (LED) lights are becoming increasingly used in horticulture because of their energy eciency, durability, longer lifetimes, low thermal energy, and cost eectiveness [9,10]. Due to the aforementioned desirable eects of LED lights, their use is highly recommended during storage (cold rooms) or transportation (refrigerated trucks) as an alternative green technology to minimise postharvest losses while maintaining product quality [8,10]. Moreover, the speciﬁc monochromatic spectrum of red LED lights was found to improve bioactive compounds, total phenols, ascorbic acid content, and antioxidant activity in yellow and green fresh-cut sweet peppers for 7 and 11 days, respectively [10]. Blue LED light ameliorated the accumulation of the aforementioned bioactive compounds in fresh-cut red sweet pepper up to 11 days at 7 C and 85% RH, indicating dierent responses or interactions in the accumulation of bioactive compounds with regard to dierent genotypes and the type of LED light [10]. Additionally, blue LED light pretreatment delayed ripening and fruit softening and extended the shelf life of tomatoes (climacteric crop) [11]. Conversely, blue LED light treatment (emission peak of 465 nm and ﬂuency of 2 1 8 molm s ) accelerated ripening in bananas (climacteric fruit) and signiﬁcantly reduced the blue mould (Penicillium italicum) infection in Satsuma Mandarin (citrus) fruit [12]. However, the inﬂuence of monochromatic LED light color and exposure time during storage on avocado fruit quality and postharvest decay (anthracnose) are unknown in the eort to replace the commercially used Prochloraz application. Therefore, the study comprises four objectives to evaluate the inﬂuence of red or blue LED light treatments and storage periods compared to the commercial control, i.e., Prochloraz treatment, on (i) anthracnose incidence, phenylalanine ammonia lyase (PAL) activity, PAL gene expression, and skin epicatechin content; (ii) percentage of ready-to-eat stage fruit; (iii) changes in targeted phenolic compounds and C7 sugar (D-mannoheptulose); and (iv) phytonutritional changes to replace the currently commercially used Prochloraz application. 2. Materials and Methods 2.1. Chemicals and Reagents Chemical standards; D-mannoheptulose; pyrogallol; epicatechin; catechin; and gallic, vanillic, protocatechuic, syringic, chlorogenic, 2,5-dihydroxybezoic, p-coumaric, ellagic acids at > 95–98% purity and other chemicals were purchased from Sigma-Aldrich (Johannesburg, South Africa). For gene expressions, the LunaScript RT SuperMix, Luna Universal qPCR Master Mix, and ZR RNA MiniPrep kits were purchased from Epigenetics Company (Johannesburg, South Africa), whilst primer sequences for actin, lipoxygenase (LOX), phenylalanine ammonia lyase (PAL), chitinase (CHI), and -1,3-glucanase (GLU) genes were obtained from Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa. 2.2. Anthracnose Incidence in Inoculated Avocados Avocado fruit cvs. Fuerte and Hass, harvested at 67 and 70% moisture content, were sorted according to the quality standards at the Bassan Packers in Tzaneen, South Africa. Each fruit weighed between 249 and 289 g, and a set of 200 fruit per each cultivar were brought to the laboratory, ripened at 25 C and 85% RH within two days to reach the trigger-ripe stage at ﬁnger feel ﬁrmness stage 2 (ﬁrmness 1.5 kg; 1 = hard, 2 = slightly soft, just starting to ripen, 3 = very soft). Prior to the artiﬁcial inoculation, fruit were surface sterilised with 0.1 mL L NaOCl for 5 min, and the fruit surfaces of both avocado cultivars were wounded (2 mm deep and 6 mm wide) with a sterilised Agronomy 2020, 10, 1654 3 of 25 cork-borer and inoculated with 20 L of a spore suspension of C. gloeosporioides spore suspension 6 1 (10 spores mL ), as previously described by Obianom et al. [13]. After incubating the fruit for 16 h after inoculation, the fruit was packed in polystyrene trays and wrapped with macro perforated (atmosphere gas composition) biaxially oriented polypropylene (BOPP) ﬁlm to reduce moisture loss. Ten replicate tray packs, each containing four fruit, were placed in a random position, directly exposing the inoculated site, and subsequently exposed to the following postharvest treatments: (i) red LED 2 1 (660 nm, 150 mol m s ) [10] for 6 h per day (as per preliminary experiments); (ii) blue LED (450 nm, 2 1 100 mol m s ) [10] for 6 h per day (as per preliminary experiments); (iii) commercial control Prochloraz (450 g L ; imidazole) (Adama SA (Pty) Ltd., Cape Town, South Africa) at 12 C for 4, 8, 12, or 16 days to simulate market shelf conditions. The shelves were ﬁtted with LED lights and the distance between the fruit and the LED lights was 100 mm. Thereafter, the fruit were held at 25 C for 2 days for the development of anthracnose incidence, and the results were expressed in percentages. 2.3. Gene Expression in Avocado Cultivars Gene expression was performed using fruit skin (10 mm around the inoculated area) diced into pieces, snap-frozen in liquid nitrogen, and stored at 80 C. The gene expression determined was according to the method of Obianom et al. [13] with RT-qPCR using a SYBR green dye system without any modiﬁcations. To extract the total RNA in avocado pulp, using a MiniPrep kit, involved using a volume of 500 mg of fruit skin. In total, 1 L of RNA was used for cDNA synthesis with reverse transcription PCR, using LunaScript RT SuperMix cDNA synthesis kits, according to the manufacturer ’s instructions. The selected genes included those coded for the endogenous control gene (actin), pathogenesis-related protein (chitinase and -1,3-glucanase), and PAL and LOX sequence of Persea americana deposited in NCBI GenBank. The actin was used as a house-keeping gene in this study. 2.4. Naturally Infected Fruit and Incidence of Anthracnose As reported in Section 2.2, 10 replicate punnets exposed to three dierent postharvest treatments were subsequently stored at 12 C and 78% RH for 4, 8, 12, or 16 day to simulate the commercial market shelf conditions. After being removed from the shelf at designated intervals, fruit were kept for an additional 5 days to determine the (i) number of days taken to reach ready-to-eat stage, (ii) anthracnose incidence, and (iii) biochemical characteristics. A set of 10 fruit per treatment (i.e., one fruit from each punnet) was taken for biochemical analysis. After cutting the fruit into pieces, it was mixed together and frozen at 80 C. A set of six samples from the cumulative sample were used for biochemical analysis to reduce the variation. Thereafter, fruit were frozen and freeze-dried using a Virtis SP Scientiﬁc with Sentry 2.0 controller (SP Scientiﬁc, New York, NY, USA) equipped with condenser at 59.6 C and vacuum at 62 mT before being ground to powder for analysis of phenolic metabolites. 2.5. Percentage of Ripe and Ready-to-Eat Stage Fruit under Dierent Postharvest Treatments and Storage Periods The percentage of fruit that reached ready-to-eat ripe stage (ﬁrmness of less than 1.0 kg) was determined for both avocado cultivars. Fruit ﬁrmness was determined at two points of the equatorial area by the puncture method using a penetrometer, with a puncture (or penetrometer) test using a 6.35 mm diameter ﬂat-head stainless-steel probe with a conical tip (Chatillon DFG 50, John Chatillon & Sons, Inc., New York, NY, USA) driven 8 mm into the fruit (skin intact) at a speed of 180 mm min . The advantage of using a conical probe is that fruit ﬁrmness is measurable without needing to remove the skin [14]. Agronomy 2020, 10, 1654 4 of 25 2.6. Biochemical Analysis 2.6.1. D-Mannoheptulose (C7 Sugar) Content D-mannoheptulose content was determined according to Glowacz et al. [15] and Mapi and Sivakumar [16], without any modiﬁcations, using avocado pulp powder (100 mg) dissolved in 1.4 mL of methanol and 50 L of an internal standard (ribitol 2 g L (w/v) in water). D-mannoheptulose content was quantiﬁed using GC-MS system 7890A gas chromatograph equipped with an MS (5975C VL0) detector (Agilent Technologies, Johannesburg, South Africa). The GC conditions and run parameters were set up according to Glowacz et al. [15]. 2.6.2. Extraction and Quantiﬁcation of Fruit Metabolites Extraction of untargeted phenolic metabolites was as per the following method described by Mpai and Sivakumar [17] without any modiﬁcations. Two grams of fruit pulp and 2 mL acidiﬁed methanol containing 80% methanol, 19.5% distilled water, and 0.5% HCl were mixed in a thermostatic shaking water bath at 70 C for 30 min. Subsequently, the mixture was centrifuged at 10,000 rpm for 15 min at 4 C, and the supernatant was ﬁltered through Whatman No. 1 ﬁlter paper. The pooled ﬁltrates were dried under N gas ﬂow at 35 C and then resuspended with 1.5 mL of extraction solution prior to targeted metabolite analysis. Metabolite proﬁling of avocado cultivars was carried out adopting a method similar to that described by Mpai and Sivakumar [16]. Waters Acquity Ultra Performance Liquid Chromatographic (UPLC) system attached to a PDA detector (Waters, Milford, MA, USA) equipped with an Acquity UPLC HSS C18 column (150 2.1 mm, 1.8 m particle size, Waters) was used for the detection and quantiﬁcation of the phenolic metabolites. Mobile phases A and B consisted of water with 0.1% formic acid and acetonitrile, respectively. Optimum separation was achieved using gradient elution executed as follows: 0 min, 95% A and 5% B; 1 min, 85% A and 15% B; 15 min, 5% A and 95% B; coming back to the initial condition and being calibrated. The ﬂow rate used was 0.3 mL min at 30 C and injection volume was 2.0 L (full-loop injection). Chromatographic software Masslynx 4.1 processed and obtained all the chromatographic data. Since it was dicult to purchase authentic standards, catechin (Y= 24.4855x + 246.089, r = 0.99) was to quantify the ﬂavonoids, such as feruloyl-quinic acid, coumaric glucose, and tryptophan. All chromatography operations were performed at ambient temperature, in triplicate, and coumaric acid hexoside (325.091 m/z; 11.1 RT), 4-feruloyl quinic acid (367.1025 m/z; 14.44 RT), and tryptophan (203.0825 m/z; 8.46 RT) were expressed in milligrams per kilogram on a dry fresh weight basis. 2.6.3. Fatty Acid Composition Fatty acid composition was determined according to the method described by Glowacz et al. [15], using avocado pulp (1 g) and homogenising with 30 mL of hexane for 30 s. Oil extraction was performed following the method used by Meyer and Terry [17], without any modiﬁcations. After dissolving the derived avocado oil extract in 2 mL of hexane and mixing with 0.2 mL of 0.2 mol L potassium hydroxide in methanol, the mixture underwent vigorous shaking for 30 s. The upper hexane layer containing methyl esters was decanted and diluted 1:100 (v/v) with n-hexane directly prior to injection into the GC-MS system (7890A gas chromatograph equipped with an MS (5975C VL0) detector (Agilent Technologies, Johannesburg, South Africa)). The conditions for the GC-MS system were similar to those of Glowacz et al. [15], with the fatty acids expressed in milligrams per gram. 2.6.4. Ascorbic Acid Content Ascorbic acid content was determined using the 2,6-dichlorophenolindophenol dye titration method described for the dierent samples. These results were expressed as grams per kilogram on a fresh weight basis [18]. Agronomy 2020, 10, 1654 5 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 5 of 25 2.6.5. The 2,2,1-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay 2.6.5. The 2,2,1-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay The DPPH assay was determined according to the method described by Tinyane et al. [19], and The DPPH assay was determined according to the method described by Tinyane et al. [19], and the the results were expressed as the concentration of antioxidants required to decrease the initial DPPH results were expressed as the concentration of antioxidants required to decrease the initial DPPH absorbance by 50% (IC50) in milligrams of gallic acid equivalent per gram of fruit. absorbance by 50% (IC ) in milligrams of gallic acid equivalent per gram of fruit. 2.7. Statistical Analysis 2.7. Statistical Analysis Assessments of the studied quality attributes and biochemical parameters were conducted Assessments of the studied quality attributes and biochemical parameters were conducted following a two-factorial type, which consisted of three postharvest treatments (red LED, blue LED, following a two-factorial type, which consisted of three postharvest treatments (red LED, blue LED, and commercial condition (prochloraz + darkness)) and four storage periods (days 4, 8, 12, and 16) of and commercial condition (prochloraz + darkness)) and four storage periods (days 4, 8, 12, and 16) of storage separately for cvs. Fuerte and Hass displayed in a completely randomised design. Data were storage separately for cvs. Fuerte and Hass displayed in a completely randomised design. Data were subjected to a two-way analysis of variance (light treatment × days of exposure) (ANOVA) at Fisher’s subjected to a two-way analysis of variance (light treatment days of exposure) (ANOVA) at Fisher ’s protected least significant difference at p < 0.05 level, using Genstat (for Windows 08, version 64-bit protected least signiﬁcant dierence at p < 0.05 level, using Genstat (for Windows 08, version 64-bit Release 18:2, International Ltd., England, UK) package to obtain the mean values for the interaction Release 18:2, International Ltd., England, UK) package to obtain the mean values for the interaction of “light treatment and days of exposure”. There was no significant difference on single factor “light of “light treatment and days of exposure”. There was no signiﬁcant dierence on single factor treatment” or “days of exposure”. During the growing season, the experiment was repeated three “light treatment” or “days of exposure”. During the growing season, the experiment was repeated times. three times. 3. Results 3. Results 3.1. Effect of LED Light Treatments on Incidence of Anthracnose in Inoculated Avocado Cultivars 3.1. Eect of LED Light Treatments on Incidence of Anthracnose in Inoculated Avocado Cultivars Inoculated cv Inoculated cv.. F Fuerte uerte expo exposed sed to r to re ed d L LED ED light show light showed ed 32% 32% anthr anthracnose acnose inc incidence idence on d on day ay 4 4 in in storage, whereas fruit exposed to blue LED light or treated with commercial fungicide showed 42 storage, whereas fruit exposed to blue LED light or treated with commercial fungicide showed 42 and and 40% 4 anthracnose 0% anthracnose incidence, incidence, re respectively specti.vely. Conversely Convers , inoculated ely, inoculat fre uit d fr exposed uit expoto sed t red o red LED LE light D li and ght and stored for 8 and 12 days showed significantly lower anthracnose incidence (25%) compared to stored for 8 and 12 days showed signiﬁcantly lower anthracnose incidence (25%) compared to the fruit the f exposed ruit exposed to bl to blue LED light ue LED l or trieated ght or trea with ted wi commer th co cial mmercia fungicide l fu (Figur ngicid ee ( 1A). FigOn ure 1A day). On d 16, fruit ay exposed 16, fruit exposed to red LED light showed 40% anthracnose incidence, whereas the inoculated fruit exposed to red LED light showed 40% anthracnose incidence, whereas the inoculated fruit exposed to blue LED to bl lightue or LED treated light or trea with commer ted wi cial th comm fungicide ercia showed l fungthe icide showe highest anthracnose d the highest incidences anthracnose of 60 inc and idence 56%, s of 60 and 56%, respectively (Figure 1A). respectively (Figure 1A). (A) Figure 1. Cont. Agronomy 2020, 10, 1654 6 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 6 of 25 (B) Figure 1. (A) Inﬂuence of light-emitting diode (LED) light exposure on inoculated avocado cultivar Figure 1. (A) Influence of light-emitting diode (LED) light exposure on inoculated avocado cultivar Fuerte. (B) Inﬂuence of LED light exposure on inoculated avocado cultivar Hass. The exposure Fuerte. (B) Influence of LED light exposure on inoculated avocado cultivar Hass. The exposure of the of the fruit to blue or red LED light was for 6 h per day during storage. Ten replicate tray packs, fruit to blue or red LED light was for 6 h per day during storage. Ten replicate tray packs, each each containing four fruit, underwent dierent postharvest treatments. Mean values of bars marked by containing four fruit, underwent different postharvest treatments. Mean values of bars marked by dierent letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, different letters were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. blue LED light; Red, red LED light; Com.T, commercial treatment. Inoculated cv. Hass, exposed to red LED light or treated with commercial fungicide showed more Inoculated cv. Hass, exposed to red LED light or treated with commercial fungicide showed or less similar anthracnose incidence (30 to 32%) on day 4 in storage, whilst the fruit exposed to blue more or less similar anthracnose incidence (30 to 32%) on day 4 in storage, whilst the fruit exposed LED light showed higher (42%) anthracnose incidence (Figure 1B). At the same time, signiﬁcantly lower to blue LED light showed higher (42%) anthracnose incidence (Figure 1B). At the same time, anthracnose incidence was observed in inoculated fruit exposed to red LED on days 8, 12, and 16 in significantly lower anthracnose incidence was observed in inoculated fruit exposed to red LED on storage compared to fruit exposed to blue LED light or treated with commercial fungicide and stored days 8, 12, and 16 in storage compared to fruit exposed to blue LED light or treated with commercial for the respective periods. The highest anthracnose incidence (54%) appeared in inoculated fruits fungicide and stored for the respective periods. The highest anthracnose incidence (54%) appeared treated with commercial fungicide and held in storage for 16 days (Figure 1B). in inoculated fruits treated with commercial fungicide and held in storage for 16 days (Figure 1B). 3.2. Eect of LED Light Treatments on Incidence of Anthracnose in Naturally Infected Avocado Cultivars 3.2. Effect of LED Light Treatments on Incidence of Anthracnose in Naturally Infected Avocado Cultivars The lowest anthracnose incidence (25%) was observed in naturally infected cv. Fuerte exposed The lowest anthracnose incidence (≤25%) was observed in naturally infected cv. Fuerte exposed to red LED light and stored up to 4, 8, and 12 days when compared to blue LED light or commercial to red LED fungicide treatment light and (Figur storee d up to 2A). Highest 4, 8, and 12 d anthracnose ays wincidence hen compwas aredon to b day lue16 LE in Dfr liuit ghtexposed or comm to er blue cial fungicide treatment (Figure 2A). Highest anthracnose incidence was on day 16 in fruit exposed to LED light (50% incidence) or treated with commercial fungicide treatment (54% incidence) (Figure 2A). blue LED In naturally light (5 infected 0% incidence) cv. Hass, or trea red ted wi LED light th co exposur mmercia e l f signiﬁcantly ungicide trreat educed ment (5 the 4% inc anthracnose idence) (Figure 2A). incidence to 12% in fruit stored up to 8, 12, and 16 days when compared with the blue LED and commer In natur cial fungicide ally infected cv. H treated frauit ss, re (Figur d LED e 2 light B). Cultivar exposure si Hassgstor nific ed antl up y redu to 4 ced the a days showed nthracnose ~20% incidence to 12% in fruit stored up to 8, 12, and 16 days when compared with the blue LED and anthracnose incidence, whilst fruit exposed to blue LED light or treated with commercial fungicide commercia showed 36 l f and ungic 30%ide t anthracnose reated fru incidence, it (Figure r2B) espectively . Cultiv.ar Sign Haiﬁcantly ss stored up highest to 4 anthracnose days showed incidence ~20% anthracnose incidence, whilst fruit exposed to blue LED light or treated with commercial fungicide (47%) was observed on day 16 in fruit exposed to blue LED light (Figure 2B). The key ﬁnding is showed 36 that employing and 30% anthr red LED a light cnose inc exposur idene ce, respecti for 6 h per vely. day Sign and ific storage antly hifor ghest 8 or ant 12 hracnose days can inci reduce dence (47%) was observed on day 16 in fruit exposed to blue LED light (Figure 2B). The key finding is that the anthracnose incidence in green thin-skinned cultivar Fuerte for marketing. Similar postharvest employin treatmentg re cand LED light be recommended exposure to reduce for 6 h per the anthracnose day and stor incidence age for 8 or in dark-skinned 12 days c cultivar an red Hass uce the for anthracnose incidence in green thin-skinned cultivar Fuerte for marketing. Similar postharvest fruit stored after treatment for 8, 12, and 16 days. treatment can be recommended to reduce the anthracnose incidence in dark-skinned cultivar Hass for fruit stored after treatment for 8, 12, and 16 days. Agronomy 2020, 10, 1654 7 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 7 of 25 (A) (B) Figure 2. (A) Inﬂuence of LED light exposure on naturally infected avocado cultivar Fuerte. (B) Inﬂuence Figure 2. (A) Influence of LED light exposure on naturally infected avocado cultivar Fuerte. (B) of LED light exposure on naturally infected avocado cultivar Hass. The exposure of fruit to blue Influence of LED light exposure on naturally infected avocado cultivar Hass. The exposure of fruit to or red LED light was for 6 h per day during storage. Ten replicate tray packs, each containing four blue or red LED light was for 6 h per day during storage. Ten replicate tray packs, each containing fruit, underwent dierent postharvest treatments. Mean values of bars marked by dierent letters four fruit, underwent different postharvest treatments. Mean values of bars marked by different were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; letters were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED Red, red LED light; Com.T, commercial treatment. light; Red, red LED light; Com.T, commercial treatment. 3.3. Eect of LED Light Treatments on PAL and LOX Gene Expression and Exocarp Epicatechin Content 3.3. Effect of LED Light Treatments on PAL and LOX Gene Expression and Exocarp Epicatechin Content Signiﬁcantly highest upregulation of PAL genes was observed in cv. Fuerte exposed to red Significantly highest upregulation of PAL genes was observed in cv. Fuerte exposed to red LED LED light and stored up to 12 days compared to the fruit stored for 4, 8, and 16 days (Figure 3A). light and stored up to 12 days compared to the fruit stored for 4, 8, and 16 days (Figure 3A). Simultaneously, cv. Fuerte exposed to red LED light showed the signiﬁcantly highest upregulation Simultaneously, cv. Fuerte exposed to red LED light showed the significantly highest upregulation of PAL genes compared to the fruit exposed to blue LED light or commercial fungicide treatment in of PAL genes compared to the fruit exposed to blue LED light or commercial fungicide treatment in storage for 4, 8, 12, and 16 days (Figure 3A). storage for 4, 8, 12, and 16 days (Figure 3A). Similarly, the highest upregulation of PAL genes was noted in cv. Hass fruit exposed to red Similarly, the highest upregulation of PAL genes was noted in cv. Hass fruit exposed to red LED LED light and stored up to 12 days, but the level of upregulation of PAL genes was higher in cv. light and stored up to 12 days, but the level of upregulation of PAL genes was higher in cv. Hass Hass when compared to cv. Fuerte (Figure 3B). Furthermore, red LED light exposure also showed a when compared to cv. Fuerte (Figure 3B). Furthermore, red LED light exposure also showed a higher upregulation of PAL genes in fruit stored up to 8 and 16 days than in fruit exposed to blue LED light or treated with commercial fungicide and stored for similar storage periods (Figure 3B). Agronomy 2020, 10, 1654 8 of 25 higher upregulation of PAL genes in fruit stored up to 8 and 16 days than in fruit exposed to blue LED Agronomy 2020, 10, x FOR PEER REVIEW 8 of 25 light or treated with commercial fungicide and stored for similar storage periods (Figure 3B). (A) (B) Figure Figure 3. 3. ( (AA ) ) I Inﬂuence nfluencof e o LED f Llight ED li exposur ght expo e on suPreAL o(phenylalanine n PAL (phenylalanine ammonia lyase gene ammonia lyase gene expression) in avocado cultivar Fuerte. (B) Inﬂuence of LED light exposure on PAL (phenylalanine ammonia lyase expression) in avocado cultivar Fuerte. (B) Influence of LED light exposure on PAL gene expression) in avocado cultivar Hass. The exposure of the fruit to blue or red LED light was for (phenylalanine ammonia lyase gene expression) in avocado cultivar Hass. The exposure of the fruit 6 h per day during storage. Number of replicates per treatment n = 5. Mean values of bars marked by to blue or red LED light was for 6 h per day during storage. Number of replicates per treatment n = 5. dierent letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, Mean values of bars marked by different letters were significantly different at p < 0.05, according to blue LED light; Red, red LED light; Com. T, commercial treatment. Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. Signiﬁcantly higher epicatechin content in the pericarp was detected in cv. Fuerte exposed to Significantly higher epicatechin content in the pericarp was detected in cv. Fuerte exposed to red LED light and stored for 8 and 12 days when compared to the fruit stored for 4 and 16 days red LED light and stored for 8 and 12 days when compared to the fruit stored for 4 and 16 days (Figure 4A). It is interesting to note that cv. Fuerte exposed to red LED light and stored for 8 and (Figure 4A). It is interesting to note that cv. Fuerte exposed to red LED light and stored for 8 and 12 days showed higher epicatechin content than the fruit exposed to blue LED light or treated with commercial fungicide and stored for 4, 8, 12, and 16 days (Figure 4A). Cultivar Hass exposed to red LED light significantly retained the highest epicatechin content in the pericarp at 12 days of storage, followed by the fruit kept on the shelf for 8 days, when compared to the other two treatments at similar storage periods (Figure 4B). Agronomy 2020, 10, 1654 9 of 25 12 days showed higher epicatechin content than the fruit exposed to blue LED light or treated with commercial fungicide and stored for 4, 8, 12, and 16 days (Figure 4A). Cultivar Hass exposed to red LED light signiﬁcantly retained the highest epicatechin content in the pericarp at 12 days of storage, followed by the fruit kept on the shelf for 8 days, when compared to Agronomy 2020, 10, x FOR PEER REVIEW 9 of 25 the other two treatments at similar storage periods (Figure 4B). (A) (B) Figure Figure 4. 4. ((A A)).. IInﬂuence nfluence o of f L LED ED llight ight e exposur xposure e o on n s skin kin e epicatechin picatechin content in av content in avocado ocado cul cultivar tivar Fuerte. Fuerte. ( (B B) ).. Inﬂuence Influenceof of L LED ED light ligexposur ht exposu e on re on sk skin epicatechin in epicatechin content content in in avocado avo cultivar cado cultivar Hass. TheHass. The exposure of fruit to blue or red LED light was for 6 h per day during storage. Number of replicates per treatment exposure of fruit to blue or red LED light was for 6 h per day during storage. Number of replicates n = 5. Mean values of bars marked by dierent letters were signiﬁcantly dierent at p < 0.05, according per treatment n = 5. Mean values of bars marked by different letters were significantly different at p < to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. Overall, cv. Fuerte exposed to LED light and stored for 4, 8, 12, and 16 days showed signiﬁcantly lower upregulation of LOX gene expression when compared to the other two postharvest treatments Overall, cv. Fuerte exposed to LED light and stored for 4, 8, 12, and 16 days showed significantly and similar storage periods, as shown in Figure 5A. The highest upregulation of LOX gene expression lower upregulation of LOX gene expression when compared to the other two postharvest treatments was noted in fruit treated with commercial fungicide and stored for 16 days. In general, all the fruit and similar storage periods, as shown in Figure 5A. The highest upregulation of LOX gene expression treated with commercial fungicide showed signiﬁcantly higher gene expression compared to the fruit was noted in fruit treated with commercial fungicide and stored for 16 days. In general, all the fruit exposed to the blue LED light and stored at the respective storage periods. treated with commercial fungicide showed significantly higher gene expression compared to the fruit exposed to the blue LED light and stored at the respective storage periods. Likewise, in cv. Hass, exposure to red LED light significantly reduced the upregulation of LOX when compared with the fruit exposed to blue LED light or treated with commercial fungicide and stored at 4, 8, 12, and 16 days (Figure 5B). Agronomy 2020, 10, 1654 10 of 25 Likewise, in cv. Hass, exposure to red LED light signiﬁcantly reduced the upregulation of LOX when compared with the fruit exposed to blue LED light or treated with commercial fungicide and Agronomy 2020, 10, x FOR PEER REVIEW 10 of 25 stored at 4, 8, 12, and 16 days (Figure 5B). (A) (B) Figure 5. (A). Influence of LED light exposure on LOX (lipoxygenase) gene expression in avocado Figure 5. (A). Inﬂuence of LED light exposure on LOX (lipoxygenase) gene expression in avocado cultivar Fuerte. (B). Influence of LED light exposure on LOX (lipoxygenase) gene expression in cultivar Fuerte. (B). Inﬂuence of LED light exposure on LOX (lipoxygenase) gene expression in avocado avocado cultivar Hass. The exposure of fruit to blue or red LED light was for 6 h per day during cultivar Hass. The exposure of fruit to blue or red LED light was for 6 h per day during storage. Number storage. Number of replicates per treatment n = 5. Mean values of bars marked by different letters of replicates per treatment n = 5. Mean values of bars marked by dierent letters were signiﬁcantly were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; Red, red LED light; Com. T, commercial treatment. Com. T, commercial treatment. 3.4. Eect of LED Light Treatments on Coumaric Acid Hexoside, 4-Feruloyl Quinic Acid, 3.4. Effect of LED Light Treatments on Coumaric Acid Hexoside, 4-Feruloyl Quinic Acid, and Tryptophan and Tryptophan Content Content UPLC-QTOF/MS analysis helped to detect the coumaric acid hexoside, 4-feruloyl quinic acid, UPLC-QTOF/MS analysis helped to detect the coumaric acid hexoside, 4-feruloyl quinic acid, and tryptophan in avocado cvs. Fuerte and Hass after storage. Overall, cv. Fuerte treated with and tryptophan in avocado cvs. Fuerte and Hass after storage. Overall, cv. Fuerte treated with commercial fungicide and stored for 12 to 16 days or exposed to blue LED treatments and stored commercial fungicide and stored for 12 to 16 days or exposed to blue LED treatments and stored for for 8 to 12 days showed higher concentrations of coumaric acid hexoside (Figure 6A). The highest 8 to 12 days showed higher concentrations of coumaric acid hexoside (Figure 6A). The highest concentration of coumaric acid hexoside was detected in fruit that underwent commercial fungicide concentration of coumaric acid hexoside was detected in fruit that underwent commercial fungicide treatment and was stored up to 12 days (Figure 6A). Conversely, fruit exposed to red LED light treatment and was stored up to 12 days (Figure 6A). Conversely, fruit exposed to red LED light retained lower concentrations of coumaric acid hexoside throughout the storage time, and the lowest concentration was detected on day 12 (Figure 6A). Similar reduced concentration of coumaric acid hexoside was found in cv. Hass exposed to red LED light in different storage periods (4, 8, 12, and 16 days) compared to the other two postharvest treatments. The significantly lowest concentrations of coumaric acid hexoside were found in red LED light exposed cv. Hass stored for 12 and 16 days (Figure 6B). Agronomy 2020, 10, 1654 11 of 25 retained lower concentrations of coumaric acid hexoside throughout the storage time, and the lowest concentration was detected on day 12 (Figure 6A). Similar reduced concentration of coumaric acid hexoside was found in cv. Hass exposed to red LED light in dierent storage periods (4, 8, 12, and 16 days) compared to the other two postharvest treatments. The signiﬁcantly lowest concentrations of coumaric acid hexoside were found in red LED light exposed cv. Hass stored for 12 and 16 days (Figure 6B). Conversely, the signiﬁcantly lowest concentrations of 3-feruloyl quinic acid were in cv. Fuerte fruit exposed to red LED light treatment and stored for 12 and 16 days (Figure 6C) when compared to the fruit treated with commercial fungicide or exposed to blue LED light and stored for similar extended storage periods. (Figure 6B). Red light exposure showed a similar trend in cv. Hass, but signiﬁcantly lowest concentrations of 3-feruloyl quinic acid were found in fruit exposed to red LED light and stored for 8 and 12 days when compared to all other postharvest treatments adopted in this study (Figure 6D). Tryptophan amino acid accumulation was highest in cultivar Fuerte on day 12 in fruit exposed to red LED light compared to the fruit exposed to blue LED light or treated with commercial fungicide (Figure 6E). In cv. Hass, the highest accumulation of tryptophan was in fruit exposed to red LED light and stored for 8 and 12 days when compared to the fruit that underwent the other two postharvest treatments and was stored at dierent storage periods (Figure 6F). Agronomy 2020, 10, x FOR PEER REVIEW 12 of 25 Agronomy 2020, 10, 1654 12 of 25 Figure 6. Cont. Agronomy 2020, 10, 1654 13 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 13 of 25 Figure 6. Cont. Agronomy 2020, 10, 1654 14 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 14 of 25 Figure 6. Inﬂuence of LED light treatments on (A) coumaric acid hexoside in cv. Fuerte and (B) coumaric acid hexoside in cv. Hass. Inﬂuence of LED light treatments on Figure 6. Influence of LED light treatments on (A) coumaric acid hexoside in cv. Fuerte and (B) coumaric acid hexoside in cv. Hass. Influence of LED light treatments on (C) 3-feruloyl quinic acid in cv. Fuerte and (D) 3-feruloyl quinic acid in cv. Hass. Inﬂuence of LED light treatments on (E) tryptophan in cv. Fuerte and (F) tryptophan in (C) 3-feruloyl quinic acid in cv. Fuerte and (D) 3-feruloyl quinic acid in cv. Hass. Influence of LED light treatments on (E) tryptophan in cv. Fuerte and (F) tryptophan in cv. Hass. Mean standard deviation values for each bar were calculated based on ﬁve fruits. Number of replicates per treatment n = 5. Mean values of bars marked by cv. Hass. Mean ± standard deviation values for each bar were calculated based on five fruits. Number of replicates per treatment n = 5. Mean values of bars marked by dierent letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. different letters were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Agronomy 2020, 10, 1654 15 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 15 of 25 3.5. Eect of LED Light Treatments on Percentage of Fruit Reaching Ready-to-Eat Stage 3.5. Effect of LED Light Treatments on Percentage of Fruit Reaching Ready-to-Eat Stage Cultivar Fuerte fruit exposed to red LED light and stored for 12 days were signiﬁcantly ﬁrm when Cultivar Fuerte fruit exposed to red LED light and stored for 12 days were significantly firm when compared to the fruit exposed to blue LED light or commercial fungicide treatment and stored for compared to the fruit exposed to blue LED light or commercial fungicide treatment and stored for a similar a similar storage period (Figure 7A). Overall, the percentages of ﬁrm fruit obtained from red LED storage period (Figure 7A). Overall, the percentages of firm fruit obtained from red LED light treatment and light treatment and storage periods of 8, 12, and 16 days were signiﬁcantly higher when compared to storage periods of 8, 12, and 16 days were significantly higher when compared to those of the other two those of the other two postharvest treatments. Approximately 70 to 80% ﬁrm fruit were obtained from postharvest treatments. Approximately 70 to 80% firm fruit were obtained from red LED light treatment and 8 red LED light treatment and 8 to 12 days cold shelf storage (Figure 7A). Exposure to blue LED light to 12 days cold shelf storage (Figure 7A). Exposure to blue LED light showed 11, 20, and 40% overripe fruit after showed 11, 20, and 40% overripe fruit after 8, 12, and 16 days of cold storage, respectively. Moreover, 8, 12, and 16 days of cold storage, respectively. Moreover, 30% of cv. Fuerte fruit became overripe after 30% of cv. Fuerte fruit became overripe after commercial fungicide treatment and 16 days of cold commercial fungicide treatment and 16 days of cold shelf storage (Figure 7A). The significantly highest shelf storage (Figure 7A). The signiﬁcantly highest percentages (65 to 70%) of ready-to-eat ripe stage percentages (65 to 70%) of ready-to-eat ripe stage were obtained from blue LED light treatment (8 and 12 days were obtained from blue LED light treatment (8 and 12 days storage periods), red LED light treatment (4 days storage) and commercial fungicide treatment (16 days storage). storage periods), red LED light treatment (4 days storage) and commercial fungicide treatment (16 days storage). A similar ripening trend was observed in cv. Hass as well, with the fruit exposed to red LED light A similar ripening trend was observed in cv. Hass as well, with the fruit exposed to red LED light and and stored for 8, 12, and 16 days being ﬁrmer than fruit that underwent other postharvest treatments stored for 8, 12, and 16 days being firmer than fruit that underwent other postharvest treatments and were and were stored at dierent storage periods (Figure 7B). Approximately 70 to 75% of cv. Hass fruit stored at different storage periods (Figure 7B). Approximately 70 to 75% of cv. Hass fruit exposed to red LED exposed to red LED and stored for 8 to 16 days were ﬁrmer when compared to the fruit from blue LED and stored for 8 to 16 days were firmer when compared to the fruit from blue LED light or chemical fungicide light or chemical fungicide treatment (Figure 7B). Conversely, commercial fungicide treated fruit stored treatment (Figure 7B). Conversely, commercial fungicide treated fruit stored for 12 days or fruit exposed blue for 12 days or fruit exposed blue LED light and stored up to 8 days showed the highest percentages of LED light and stored up to 8 days showed the highest percentages of ready-to-eat stage ripe fruit. ready-to-eat stage ripe fruit. Furthermore, cv. Hass fruit exposed to blue LED light and stored for 12 Furthermore, cv. Hass fruit exposed to blue LED light and stored for 12 and 16 days showed around 22 to 28% and 16 days showed around 22 to 28% overripe fruits, and commercial fungicide treated fruit stored overripe fruits, and commercial fungicide treated fruit stored for 16 days also showed 30% overripe fruit. for 16 days also showed 30% overripe fruit. (A) Figure 7. Cont. Agronomy 2020, 10, 1654 16 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 16 of 25 (B) Figure 7. (A) Inﬂuence of LED light treatment on ripening in avocado cultivar Fuerte. (B) Inﬂuence Figure 7. (A) Influence of LED light treatment on ripening in avocado cultivar Fuerte. (B) Influence of LED of LED light treatment on ripening in avocado cultivar Hass. Fruit were exposed to blue or red light treatment on ripening in avocado cultivar Hass. Fruit were exposed to blue or red LED light for 6 h per LED light for 6 h per day during storage and left at 25 C for 2 days after storage. Mean values for day during storage and left at 25 °C for 2 days after storage. Mean values for each bar were calculated based each bar were calculated based on 100 fruits for each treatment. Bars with similar letters were not on 100 fruits for each treatment. Bars with similar letters were not significantly different at p < 0.05 according signiﬁcantly dierent at p < 0.05 according to LSD. Blue, blue LED light; Red, red LED light; Com. T, to LSD. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. commercial treatment. 3.6. Eect of LED Light Treatments on D-Mannoheptulose Content 3.6. Effect of LED Light Treatments on D-Mannoheptulose Content The D-mannoheptulose content was signiﬁcantly highest in cv. Fuerte exposed to red LED light The D-mannoheptulose content was significantly highest in cv. Fuerte exposed to red LED light and and stored for 12 days when compared to the other two postharvest treatments and at similar storage stored for 12 days when compared to the other two postharvest treatments and at similar storage periods periods (Figure 8A). Fruit exposed to red LED light and stored for 8 and 16 days and commercial (Figure 8A). Fruit exposed to red LED light and stored for 8 and 16 days and commercial fungicide treated fungicide treated fruit stored for 4 days showed higher concentrations of D-mannoheptulose sugar fruit stored for 4 days showed higher concentrations of D-mannoheptulose sugar than the other counterpart than the other counterpart treatments and storage periods. It is interesting to note that the fruit exposed treatments and storage periods. It is interesting to note that the fruit exposed to blue LED light for 12 and to blue LED light for 12 and 16 days or commercial fungicide treated fruit stored for 12 and 16 days 16 days or commercial fungicide treated fruit stored for 12 and 16 days showed significantly lower showed signiﬁcantly lower concentrations of D-mannoheptulose sugar (Figure 8A). concentrations of D-mannoheptulose sugar (Figure 8A). Cultivar Hass exposed to red LED light and stored for 8, 12, and 16 days showed signiﬁcantly Cultivar Hass exposed to red LED light and stored for 8, 12, and 16 days showed significantly higher higher and similar D-mannoheptulose content compared to the commercial fungicide treated fruit stored 4 days on the cold shelf (Figure 8B). However, cv. Hass exposed to blue LED light and stored and similar D-mannoheptulose content compared to the commercial fungicide treated fruit stored 4 days for 16 days showed the highest D-mannoheptulose content compared to the other two postharvest on the cold shelf (Figure 8B). However, cv. Hass exposed to blue LED light and stored for 16 days showed treatments and storage periods (Figure 8B). the highest D-mannoheptulose content compared to the other two postharvest treatments and storage periods (Figure 8B). Agronomy 2020, 10, 1654 17 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 17 of 25 (A) (B) Figure Figure 8. (A 8. ) I(n A fluence of ) Inﬂuence LED of LED lights on lights D-mannoheptulose and on D-mannoheptulose o and leic ac oleic id cont acidecontent nt in the mes in the oc mesocarp arp of avo of cado avocado cultivar Fuerte. (B) Inﬂuence of LED lights on D-mannoheptulose and oleic acid content in cultivar Fuerte. (B) Influence of LED lights on D-mannoheptulose and oleic acid content in the mesocarp of the mesocarp of avocado cultivar Hass. Mean standard deviation values for each bar were calculated avocado cultivar Hass. Mean ± standard deviation values for each bar were calculated based on five fruits. based on ﬁve fruits. Number of replicates per treatment n = 5. Mean values of bars marked by dierent Number of replicates per treatment n = 5. Mean values of bars marked by different letters were significantly letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, light; Red, red LED light; Com.T, commercial treatment. commercial treatment. 3.7. Eect of LED Light Treatments on Oleic Acid Content in the Mesocarp 3.7. Effect of LED Light Treatments on Oleic Acid Content in the Mesocarp Oleic acid content was determined in ready-to-eat stage ripe fruit. Overall, all blue or red LED Oleic acid content was determined in ready-to-eat stage ripe fruit. Overall, all blue or red LED light light treatments signiﬁcantly increased the oleic acid (18:1) content in cv. Fuerte compared to the fruit treatments significantly increased the oleic acid (18:1) content in cv. Fuerte compared to the fruit treated treated with commercial fungicide, irrespective of the storage period. However, fruit exposed to red with commercial fungicide, irrespective of the storage period. However, fruit exposed to red LED light and LED light and stored for 8 days showed lower concentrations than the fruit exposed to blue LED light stored for 8 days showed lower concentrations than the fruit exposed to blue LED light and stored for and stored for similar storage periods (Figure 9A). A similar increased trend in accumulation of oleic similar storage periods (Figure 9A). A similar increased trend in accumulation of oleic acid (18:1) content in acid (18:1) content in cv. Hass fruit exposed to blue LED or red LED light compared to the commercial cv. Hass fruit exposed to blue LED or red LED light compared to the commercial fungicide treated fruit was fungicide treated fruit was observed, irrespective of the storage period (Figure 9B). observed, irrespective of the storage period (Figure 9B). Agronomy 2020, 10, 1654 18 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 18 of 25 (A) (B) Figu Figure re 9. (A 9.) I (A n)flInﬂuence uence of LED lights on ole of LED lights on oleic ic acid content acid content in the in themesoca mesocarp rp of av of avocado ocado cultiva cultivar r Fue Fuerte. rte. (B) Infl(uence o B) Inﬂuence f LED lights on olei of LED lights on c acid content in the oleic acid content in mthe esocarp of avocado cu mesocarp of avocado ltivars Hass and F cultivars Hass and uerte. Mean Fuerte. ± standa Mean rd devia standar tion val d u deviation es for each ba values r we for re ceach alculated bar wer based e calculated on five fruits. N based umbe on ﬁve r of replic fruits. ates pe Number r treat of ment n = 5 r.eplicates Mean valper ues o tr featment bars marn ke= d b 5. y d Mean ifferen values t letters of wbars ere simarked gnificantby ly d di iff eer reent nt at letters p < 0.05 wer , according to Fis e signiﬁcantly her’s dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Com.T, commercial treatment. 3.8. Effect of LED Light Treatments on Ascorbic Acid Content in the Mesocarp 3.8. Eect of LED Light Treatments on Ascorbic Acid Content in the Mesocarp Overall, the ascorbic acid content was significantly lower in commercial fungicide treated cv. Fuerte Overall, the ascorbic acid content was signiﬁcantly lower in commercial fungicide treated cv. fruit compared to the fruit exposed to blue LED light and stored for 6, 12, and 16 days or red LED light and Fuerte fruit compared to the fruit exposed to blue LED light and stored for 6, 12, and 16 days or red stored for 4, 8, 12, and 16 days in storage (Figure 10A). Likewise, there were lower concentrations of ascorbic LED light and stored for 4, 8, 12, and 16 days in storage (Figure 10A). Likewise, there were lower acid content detected in commercial fungicide treated fruit when compared to the blue or red LED light concentrations of ascorbic acid content detected in commercial fungicide treated fruit when compared treated fruit stored for 4 to 16 days onwards. In other words, exposure to blue or red LED light improved to the blue or red LED light treated fruit stored for 4 to 16 days onwards. In other words, exposure to the biosynthesis of ascorbic acid in cv. Hass (Figure 10B). blue or red LED light improved the biosynthesis of ascorbic acid in cv. Hass (Figure 10B). Agronomy 2020, 10, 1654 19 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 19 of 25 (A) (B) Figure 10. (A) Influence of LED lights on ascorbic acid content in the mesocarp of avocado cultivar Fuerte. (B) Figure 10. (A) Inﬂuence of LED lights on ascorbic acid content in the mesocarp of avocado cultivar Influ Fuerte. ence of LE (B)D lights o Inﬂuencen asc of LED orbic acid co lights on ntent i ascorbic n the m acid esocarp of avocad content in the mesocarp o cultivar Hass of avocado . Mean ± st cultivar andard deviatio Hass. n valu Mean es for each b standardar deviation were calcvalues ulated based on five for each bar wer frui e ts. Num calculated ber of re basedplicates on ﬁveper t fruits. reaNumber tment n = 5. Mean v of replicates alues of bars per tr ma eatment rked byn diffe = 5. re Mean nt lette values rs weof re si bars gnific marked antly diff by ere din er t ent at pletters < 0.05, acc were ording t signiﬁcantly o Fisher’s dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Com.T, commercial treatment. 3.9. Effect of LED Light Treatments on Antioxidant Activity 3.9. Eect of LED Light Treatments on Antioxidant Activity Antioxidant activity was significantly lowest in commercial fungicide treated cv. Fuertes from 4 days Antioxidant activity was signiﬁcantly lowest in commercial fungicide treated cv. Fuertes from onwards up to 16 days compared to the fruit that underwent the other two postharvest treatments and was 4 days onwards up to 16 days compared to the fruit that underwent the other two postharvest stored at similar storage periods. However, the significantly highest antioxidant activity was observed in treatments and was stored at similar storage periods. However, the signiﬁcantly highest antioxidant fruit exposed to red LED light and stored for 12 days. In general, red LED light exposed fruit stored for 4 to activity was observed in fruit exposed to red LED light and stored for 12 days. In general, red LED 16 days and fruit exposed to blue LED light and stored for 12 days showed higher antioxidant activity light exposed fruit stored for 4 to 16 days and fruit exposed to blue LED light and stored for 12 days (Figure 11A). showed higher antioxidant activity (Figure 11A). Agronomy 2020, 10, 1654 20 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 20 of 25 Notably, cv. Hass had similar significantly increased antioxidant activity in fruit exposed to red LED Notably, cv. Hass had similar signiﬁcantly increased antioxidant activity in fruit exposed to red light and stored from 4 days to 16 days compared to the fruits fruit from the other two postharvest LED light and stored from 4 days to 16 days compared to the fruits fruit from the other two postharvest treatments. Likewise, commercial fungicide treated cv. Hass showed significantly lowest antioxidant treatments. Likewise, commercial fungicide treated cv. Hass showed signiﬁcantly lowest antioxidant activity than the fruit from the other two postharvest treatments (Figure 11B). Ascorbic acid showed a strong activity than the fruit from the other two postharvest treatments (Figure 11B). Ascorbic acid showed a positive correlation with antioxidant scavenging activity of cultivars Hass (r = 0. 77, p < 0.05) and Fuerte (r strong positive correlation with antioxidant scavenging activity of cultivars Hass (r = 0. 77, p < 0.05) = 0.72, p < 0.05). and Fuerte (r = 0.72, p < 0.05). (A) (B) Figu Figure re 11. (A 11. ) Antio (A) Antioxidant xidant metabolit metabolite e conten contents ts in avocado cultivar Fue in avocado cultivar rte t F ruerte eated post treated harve postharvest st with LED lights. with (B) Antio LED lights. xidant m (B)eAntioxidant tabolite conte metabolite nts in avocado cu contents ltivar in avocado Hass treated cultivar Hass postharvest with L treated postharvest ED lights. Mean ± with LED lights. Mean standard deviation values for each bar were calculated based on ﬁve fruits. Number standard deviation values for each bar were calculated based on five fruits. Number of replicates per treatment of replicates per treatment n = 5. Mean values of bars marked by dierent letters were signiﬁcantly n = 5. Mean values of bars marked by different letters were significantly different at p < 0.05, according to Fisher’s dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Com.T, commercial treatment. Agronomy 2020, 10, x FOR PEER REVIEW 21 of 25 Agronomy 2020, 10, 1654 21 of 25 The performing of PCA analysis was to summarise the interrelated biochemical changes with different LED light treatments and storage periods for both avocado cultivars Hass and Fuerte. The obtaining of the The performing of PCA analysis was to summarise the interrelated biochemical changes with two principal components with their factor loading was with respect to Eigen values > 1. The principal dierent LED light treatments and storage periods for both avocado cultivars Hass and Fuerte. components explained 71.4% (PC1 38.2% and 33.2%) (Figure 12). In PC1, the components coefficients or The obtaining of the two principal components with their factor loading was with respect to Eigen correlation coefficients (r) for the following parameters (variables): - oleic acid, ascorbic acid, and values > 1. The principal components explained 71.4% (PC1 38.2% and 33.2%) (Figure 12). In PC1, antioxidant activity, were positively loaded, and tryptophan was negatively loaded in PC1. In PC2: - 3- the components coecients or correlation coecients (r) for the following parameters (variables): feruloyl quinic acid, coumaric acid hexoside, ready-to-eat ripeness were loaded positively and D- - oleic acid, ascorbic acid, and antioxidant activity, were positively loaded, and tryptophan was manoheptulose, firm fruit were loaded negatively. The PCA analysis confirmed the increase of 3-feruloyl negatively loaded in PC1. In PC2: - 3-feruloyl quinic acid, coumaric acid hexoside, ready-to-eat quinic acid, coumaric acid hexoside in ready to ripe stage Hass and Fuerte avocado fruit exposed under ripeness were loaded positively and D- manoheptulose, ﬁrm fruit were loaded negatively. The PCA blue LED light or treated with commercial fungicide and stored for 12 and 16 days. Both cultivars, stored analysis conﬁrmed the increase of 3-feruloyl quinic acid, coumaric acid hexoside in ready to ripe stage for 8 and 12 days under red light, confirmed increased antioxidant activity, D- manoheptulose, tryptophan Hass and Fuerte avocado fruit exposed under blue LED light or treated with commercial fungicide and and skin epicatechin concentration and firm fruit. stored for 12 and 16 days. Both cultivars, stored for 8 and 12 days under red light, conﬁrmed increased antioxidant activity, D- manoheptulose, tryptophan and skin epicatechin concentration and ﬁrm fruit. Figure 12. Principal component analysis (PCA) showing correlation loadings of fruit phenolic compounds; epicatechin in the pericarp; tryptophan, D-mannoheptulose sugar, ascorbic acid, oleic Figure 12. Principal component analysis (PCA) showing correlation loadings of fruit phenolic compounds; acid, and antioxidant activity in the mesocarp; and fruit ripeness related to the LED light treatments in epicatechin in the pericarp; tryptophan, D-mannoheptulose sugar, ascorbic acid, oleic acid, and antioxidant comparison to the commercial treatment. F-QA, 3-feruloyl quinic acid; C-AH, coumaric acid hexoside; activity in the mesocarp; and fruit ripeness related to the LED light treatments in comparison to the D, days; H, cv. Hass; F, Cv. Fuerte. commercial treatment. F-QA, 3-feruloyl quinic acid; C-AH, coumaric acid hexoside; D, days; H, cv. Hass; F, Cv. 4. Discussion Fuerte. In the fruit industry, there is a growing interest in protecting the fruit against postharvest decay using elicitors or resistance inducers to stimulate the natural compounds responsible for resistance against the postharvest pathogens, as this will promote sustainable plant protection against pathogens Agronomy 2020, 10, 1654 22 of 25 and reduce the loss of fruit during marketing. Use of LEDs is becoming a popular and handy tool for sustainable agricultural practices [20]. Antimicrobial properties of light are gaining more attention in fungi, as they are developing resistance against the conventionally used fungicides. Blue LEDs elicited resistance against green mould (Penicillium digitatum) decay in citrus (nonclimacteric fruit) by altering the phenolic proﬁle, especially the scoparone content [21]. However, in our study, red LED light caused signiﬁcant reduction in anthracnose incidence in both cultivars Fuerte and Hass, although the impact was higher in cv. Hass, primarily by eliciting resistance by upregulating the PAL genes and increasing the epicatechin concentration (Figure 2A). A similar increase in PAL activity and PAL gene expression induced by postharvest treatments (e.g., thyme oil vapours) demonstrated the control of anthracnose decay in avocados [22]. Antifungal diene compound (AFD; 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12, 15-diene) is responsible for the resistance of unripe avocado fruit against the anthracnose pathogen [23]. The AFD compound declines as the fruit ripens due to the activity of lipoxygenase enzyme and facilitates the entry of the pathogen into the fruit (host). Alternatively, emission of ethylene further facilitates the infection process of C. gloeosporioides. The concentration of epicatechin, a product of the phenylpropanoid pathway, regulates the activity of lipoxygenase and the levels of AFD compound [24–26]. Furthermore, upregulation of PAL gene expression was found to stimulate the biosynthesis of epicatechin content in the skin of avocado [22,23]. Results presented herein indicate that both cultivars (Fuerte and Hass) exposed to red LED for 12 day, via activation of the phenylpropanoid pathway, revealed PAL gene expression and epicatechin content in the skin (Figure 3A,B and Figure 4A,B). This research reported this by demonstrating the low regulation of LOX genes in fruit exposed to LED light, the higher concentration of epicatechin in the fruit skin, and the observed reduction in anthracnose-infected fruits (Figure 2B). Accumulation of tryptophan and the biosynthetic pathway guide the production of dierent secondary metabolites [27]. Fundamentally, tryptophan biosynthesis and the enzymes are induced by the stress reaction caused by biotic elicitors [27]. There is reportedly an interdependence of the shikimate pathway and the key phenylalanine-derived phenolics and phytoalexin via phenylalanine ammonia lyase in dierent fruits and vegetables [28,29]; similarly, red LED lights stimulated the accumulation of tryptophan amino acid in this study (Figure 6E,F). Sensitivity or response of the fruit or plant and the pathogen regarding the control of anthracnose during storage on the market shelf towards a speciﬁc monochromatic light is more likely due to the sensitivity of their light receptors than the energy or the wavelength of the speciﬁc light [30]. This could be the reason for the observed dierences in response towards the dierent LED light treatments. Blue LED light was found to accelerate the ripening in bananas (climacteric fruit), while red LED light delayed the ripening [31]. A similar trend was observed in both avocado cultivars (Fuerte and Hass) in this study (Figure 3); however, blue LED light showed a remarkable impact in ripening cvs. Fuerte and Hass on days 8 and 16 respectively. Huang et al. [31] investigated the impact of dierent LED lights and wavelengths on climacteric pattern and reported the blue light was the most eective in inducing the onset of the climacteric peak, but the red LED light slowed the ripening. The accelerated ripening in fruit exposed to blue LED light and reduction of epicatechin content in the avocado fruit skin could have aggravated the anthracnose of decay and the entry of C. gloesoporioides into the fruit (Figure 3A,B). The C7 D-mannoheptulos correlated with time of ripening [16] and avocado (cv. Hass) grown under the red nets demonstrated higher concentration of D-mannoheptulos and the fruits took time to reach ready stage in our previous study [32]. Detected in all avocado cultivars, except cv. Bacon, during ripening were p-coumaric and ferulic acids (hydroxycinnamic acids) [33]. Fruit ripening of climacteric fruits such as tomato and kiwi showed an intense modiﬁcation of nonvolatile secondary metabolites in the phenolic proﬁle [34]. Some derivatives of phenolic compounds, such as coumaric and ferulic acid, reportedly increased linearly during development, and accumulation continued in the fully ripe fruit [34]. Likewise, our results showed an increase in coumaric acid hexoside and 3-feruloyl quinic acid related to the number of fruit reaching ready-to-eat stage ripeness exposed to blue LED Agronomy 2020, 10, 1654 23 of 25 light or commercial fungicide treatment (Figure 3A,B). Variability in avocado metabolites occurs due to dierent photoreceptors that sense each light, since the blue light is absorbed by the phototropin and cryptochrome and red light is absorbed by the phytochrome. Phytochromes mediate the red/FAR light related responses in plants and biosynthesis of phenolic compounds (phytochemicals). In our previous research, red net grown cv. Hass avocados showed higher PAL activity and accumulation of epicatechin and phenolic acid components [19]. The ascorbic acid content (vitamin C) was stimulated in cabbage stored under blue LED lights, which improved the ascorbic acid accumulation. However, based on the results presented here, in avocado cultivars Fuerte and Hass, the increase of ascorbic acid is favoured by the red LED light (Figure 10A,B). Furthermore, blue LED light was found to promote the synthesis of all free amino acids, except glutamic and aspartic acids, during postharvest ripening of tomatoes from green to yellow to red [11]. Likewise, in this study, blue and red LED light inﬂuenced the oleic acid biosynthesis in both avocado cultivars (Figure 9A,B). It is noteworthy that the production of antioxidants, ascorbic acid, and phenolic compounds and amino acid biosynthesis occur under more stressful conditions [35]. 5. Conclusions Application of the LED light technology to avocado at postharvest stage can be a useful tool to address the demand for fruits that have reached the ready-to-eat stage without compromising the fruit quality. Applying the blue LED light in cold storage can accelerate fruit ripening, whereas employing the red LED light can induce fruit resistance against the anthracnose postharvest disease in both avocado cvs. Fuerte and Hass for up to 12 days on the market shelf at 10–12 C. Further studies on the application of red and blue LED lights as a combined spectrum are imperative. Author Contributions: S.M. performed the experiment, was responsible for the data collection, conducted formal analysis and the analysis of phenolic compounds, conducted statistical analysis, and wrote the ﬁrst draft. D.S. conceptualised the research idea; supervised the ﬁrst author; participated in discussion, review, editing, and data validation; was the grant holder and project administrator; provided resources; and revised the ﬁnal draft. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Research Foundation South Africa: [98352]. Acknowledgments: The ﬁnancial support from the National Research Foundation, South Africa, and Grant number 98352 for Phytochemical Food Network to Improve Nutritional Quality for Consumers is greatly acknowledged. Conﬂicts of Interest: Authors have no conﬂict of interest to declare. References 1. Dreher, M.L.; Davenport, A.J. Hass avocado composition and potential health eects. Crit. Rev. Food Sci. Nutr. 2013, 53, 738–750. [CrossRef] [PubMed] 2. South African Avocado Grower ’s Association 2020. Available online: https://www.avocado.co.za/varieties/ (accessed on 15 May 2020). 3. 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[CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agronomy Multidisciplinary Digital Publishing Institute http://www.deepdyve.com/lp/multidisciplinary-digital-publishing-institute/stimulation-of-light-emitting-diode-treatment-on-defence-system-and-osax9ZnFhl

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DOI: 10.3390/agronomy10111654
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Abstract

agronomy Article Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions Semakaleng Mpai and Dharini Sivakumar * Department of Crop Sciences, Phytochemical Food Network, Tshwane University of Technology, Pretoria 0001, South Africa; semkalengmpai@gmail.com * Correspondence: SivakumarD@tut.ac.za Received: 19 August 2020; Accepted: 19 October 2020; Published: 27 October 2020 Abstract: The ability of light-emitting diode (LED) light treatment to reduce the anthracnose decay via its eliciting eects and thus induce resistance in the avocado (Persea americana), was investigated in this study to replace the current postharvest fungicide treatment. In experiment 1, the eect of blue or red LED lights (6 h per day) on the incidence of anthracnose in artiﬁcially inoculated (Colletotrichum gloesposorioides) and naturally infected avocados (cv. Fuerte and Hass) at 12–14 C (simulated market shelf) for 4, 8, 14, and 16 days was investigated. In experiment 2, the eect of blue or red LED lights on the induced defence mechanism, fruit metabolites, antioxidant activity, and percentage of fruit reaching ready-to-eat stage was determined. Exposure to red LED light signiﬁcantly reduced the anthracnose decay incidence in naturally infected cv. Fuerte on day 12 and in cv. Hass on day 16 compared to the prochloraz fungicide treatment by upregulating the PAL genes and maintaining the epicatechin content. Blue LED light accelerated the ripening in both cultivars, probably due to reduced D-mannoheptulose content. Red LED light exposure for 6 h per day and 12 days storage showed potential to replace the prochloraz treatment with improved ascorbic acid content and antioxidant activity. Keywords: Persea americana; phenolic compounds; ripening; postharvest decay; D-mannoheptulose; phenylalanine ammonia lyase 1. Introduction Avocado (Persea americana) is a popular subtropical fruit amongst consumers for its health beneﬁts. Consumption of half an avocado (68 g) reportedly provides dietary ﬁbre (4.6 g), potassium (345 mg), vitamin C (6.0 mg), vitamin E (1.3 mg), folate (60 mg), choline (10 mg), lutein/zeaxanthin (185 g), phytosterols (57 mg), and a high level of monounsaturated fatty acids (6.7 g) [1]. Among the avocado cultivars, consumers mainly prefer the dark-skinned cv. Hass because of its creamy and smooth texture and nutty taste, whilst the green-skinned cv. Fuerte is popular due to its good ﬂavour and oily texture [2]. Consumer preference is high for “ready-to-eat stage” avocados [3], but due to postharvest decay anthracnose (Colletotrichum gloeosporioides Penz.), the quality of fruit can be negatively aected, and postharvest losses during marketing are high [4]. Avocado is a climacteric fruit, which reaches ready to ripe stage after harvest at the climacteric peak with ethylene emission, resulting in structural and biochemical changes and reduction of antifungal compounds to alleviate the dormancy of C. gloeosporioides, the latent pathogen [4]. The symptoms are expressed in the fruit at trigger-ripe stage, especially during marketing on the supermarket cold shelf at 12–14 C [5]. Agronomy 2020, 10, 1654; doi:10.3390/agronomy10111654 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1654 2 of 25 In most avocado-exporting countries, Prochloraz, a chemically synthesised fungicide, is applied as a spray or dip in the packing line to provide residual protection against the anthracnose-causing organism during marketing [4]. However, Prochloraz has hazardous health eects, and the maximum residue limits (MRLs) for the South African avocado limits its access to the quality-stringent export markets [6]. Consequently, numerous alternative green technologies have actively undergone research during the past 5 years to replace the Prochloraz fungicide treatment [7]. The use of a light-emitting diode (LED), as a nonchemical treatment for maintaining quality attributes of horticultural produce, is becoming popular [8]. Light-emitting diode (LED) lights are becoming increasingly used in horticulture because of their energy eciency, durability, longer lifetimes, low thermal energy, and cost eectiveness [9,10]. Due to the aforementioned desirable eects of LED lights, their use is highly recommended during storage (cold rooms) or transportation (refrigerated trucks) as an alternative green technology to minimise postharvest losses while maintaining product quality [8,10]. Moreover, the speciﬁc monochromatic spectrum of red LED lights was found to improve bioactive compounds, total phenols, ascorbic acid content, and antioxidant activity in yellow and green fresh-cut sweet peppers for 7 and 11 days, respectively [10]. Blue LED light ameliorated the accumulation of the aforementioned bioactive compounds in fresh-cut red sweet pepper up to 11 days at 7 C and 85% RH, indicating dierent responses or interactions in the accumulation of bioactive compounds with regard to dierent genotypes and the type of LED light [10]. Additionally, blue LED light pretreatment delayed ripening and fruit softening and extended the shelf life of tomatoes (climacteric crop) [11]. Conversely, blue LED light treatment (emission peak of 465 nm and ﬂuency of 2 1 8 molm s ) accelerated ripening in bananas (climacteric fruit) and signiﬁcantly reduced the blue mould (Penicillium italicum) infection in Satsuma Mandarin (citrus) fruit [12]. However, the inﬂuence of monochromatic LED light color and exposure time during storage on avocado fruit quality and postharvest decay (anthracnose) are unknown in the eort to replace the commercially used Prochloraz application. Therefore, the study comprises four objectives to evaluate the inﬂuence of red or blue LED light treatments and storage periods compared to the commercial control, i.e., Prochloraz treatment, on (i) anthracnose incidence, phenylalanine ammonia lyase (PAL) activity, PAL gene expression, and skin epicatechin content; (ii) percentage of ready-to-eat stage fruit; (iii) changes in targeted phenolic compounds and C7 sugar (D-mannoheptulose); and (iv) phytonutritional changes to replace the currently commercially used Prochloraz application. 2. Materials and Methods 2.1. Chemicals and Reagents Chemical standards; D-mannoheptulose; pyrogallol; epicatechin; catechin; and gallic, vanillic, protocatechuic, syringic, chlorogenic, 2,5-dihydroxybezoic, p-coumaric, ellagic acids at > 95–98% purity and other chemicals were purchased from Sigma-Aldrich (Johannesburg, South Africa). For gene expressions, the LunaScript RT SuperMix, Luna Universal qPCR Master Mix, and ZR RNA MiniPrep kits were purchased from Epigenetics Company (Johannesburg, South Africa), whilst primer sequences for actin, lipoxygenase (LOX), phenylalanine ammonia lyase (PAL), chitinase (CHI), and -1,3-glucanase (GLU) genes were obtained from Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa. 2.2. Anthracnose Incidence in Inoculated Avocados Avocado fruit cvs. Fuerte and Hass, harvested at 67 and 70% moisture content, were sorted according to the quality standards at the Bassan Packers in Tzaneen, South Africa. Each fruit weighed between 249 and 289 g, and a set of 200 fruit per each cultivar were brought to the laboratory, ripened at 25 C and 85% RH within two days to reach the trigger-ripe stage at ﬁnger feel ﬁrmness stage 2 (ﬁrmness 1.5 kg; 1 = hard, 2 = slightly soft, just starting to ripen, 3 = very soft). Prior to the artiﬁcial inoculation, fruit were surface sterilised with 0.1 mL L NaOCl for 5 min, and the fruit surfaces of both avocado cultivars were wounded (2 mm deep and 6 mm wide) with a sterilised Agronomy 2020, 10, 1654 3 of 25 cork-borer and inoculated with 20 L of a spore suspension of C. gloeosporioides spore suspension 6 1 (10 spores mL ), as previously described by Obianom et al. [13]. After incubating the fruit for 16 h after inoculation, the fruit was packed in polystyrene trays and wrapped with macro perforated (atmosphere gas composition) biaxially oriented polypropylene (BOPP) ﬁlm to reduce moisture loss. Ten replicate tray packs, each containing four fruit, were placed in a random position, directly exposing the inoculated site, and subsequently exposed to the following postharvest treatments: (i) red LED 2 1 (660 nm, 150 mol m s ) [10] for 6 h per day (as per preliminary experiments); (ii) blue LED (450 nm, 2 1 100 mol m s ) [10] for 6 h per day (as per preliminary experiments); (iii) commercial control Prochloraz (450 g L ; imidazole) (Adama SA (Pty) Ltd., Cape Town, South Africa) at 12 C for 4, 8, 12, or 16 days to simulate market shelf conditions. The shelves were ﬁtted with LED lights and the distance between the fruit and the LED lights was 100 mm. Thereafter, the fruit were held at 25 C for 2 days for the development of anthracnose incidence, and the results were expressed in percentages. 2.3. Gene Expression in Avocado Cultivars Gene expression was performed using fruit skin (10 mm around the inoculated area) diced into pieces, snap-frozen in liquid nitrogen, and stored at 80 C. The gene expression determined was according to the method of Obianom et al. [13] with RT-qPCR using a SYBR green dye system without any modiﬁcations. To extract the total RNA in avocado pulp, using a MiniPrep kit, involved using a volume of 500 mg of fruit skin. In total, 1 L of RNA was used for cDNA synthesis with reverse transcription PCR, using LunaScript RT SuperMix cDNA synthesis kits, according to the manufacturer ’s instructions. The selected genes included those coded for the endogenous control gene (actin), pathogenesis-related protein (chitinase and -1,3-glucanase), and PAL and LOX sequence of Persea americana deposited in NCBI GenBank. The actin was used as a house-keeping gene in this study. 2.4. Naturally Infected Fruit and Incidence of Anthracnose As reported in Section 2.2, 10 replicate punnets exposed to three dierent postharvest treatments were subsequently stored at 12 C and 78% RH for 4, 8, 12, or 16 day to simulate the commercial market shelf conditions. After being removed from the shelf at designated intervals, fruit were kept for an additional 5 days to determine the (i) number of days taken to reach ready-to-eat stage, (ii) anthracnose incidence, and (iii) biochemical characteristics. A set of 10 fruit per treatment (i.e., one fruit from each punnet) was taken for biochemical analysis. After cutting the fruit into pieces, it was mixed together and frozen at 80 C. A set of six samples from the cumulative sample were used for biochemical analysis to reduce the variation. Thereafter, fruit were frozen and freeze-dried using a Virtis SP Scientiﬁc with Sentry 2.0 controller (SP Scientiﬁc, New York, NY, USA) equipped with condenser at 59.6 C and vacuum at 62 mT before being ground to powder for analysis of phenolic metabolites. 2.5. Percentage of Ripe and Ready-to-Eat Stage Fruit under Dierent Postharvest Treatments and Storage Periods The percentage of fruit that reached ready-to-eat ripe stage (ﬁrmness of less than 1.0 kg) was determined for both avocado cultivars. Fruit ﬁrmness was determined at two points of the equatorial area by the puncture method using a penetrometer, with a puncture (or penetrometer) test using a 6.35 mm diameter ﬂat-head stainless-steel probe with a conical tip (Chatillon DFG 50, John Chatillon & Sons, Inc., New York, NY, USA) driven 8 mm into the fruit (skin intact) at a speed of 180 mm min . The advantage of using a conical probe is that fruit ﬁrmness is measurable without needing to remove the skin [14]. Agronomy 2020, 10, 1654 4 of 25 2.6. Biochemical Analysis 2.6.1. D-Mannoheptulose (C7 Sugar) Content D-mannoheptulose content was determined according to Glowacz et al. [15] and Mapi and Sivakumar [16], without any modiﬁcations, using avocado pulp powder (100 mg) dissolved in 1.4 mL of methanol and 50 L of an internal standard (ribitol 2 g L (w/v) in water). D-mannoheptulose content was quantiﬁed using GC-MS system 7890A gas chromatograph equipped with an MS (5975C VL0) detector (Agilent Technologies, Johannesburg, South Africa). The GC conditions and run parameters were set up according to Glowacz et al. [15]. 2.6.2. Extraction and Quantiﬁcation of Fruit Metabolites Extraction of untargeted phenolic metabolites was as per the following method described by Mpai and Sivakumar [17] without any modiﬁcations. Two grams of fruit pulp and 2 mL acidiﬁed methanol containing 80% methanol, 19.5% distilled water, and 0.5% HCl were mixed in a thermostatic shaking water bath at 70 C for 30 min. Subsequently, the mixture was centrifuged at 10,000 rpm for 15 min at 4 C, and the supernatant was ﬁltered through Whatman No. 1 ﬁlter paper. The pooled ﬁltrates were dried under N gas ﬂow at 35 C and then resuspended with 1.5 mL of extraction solution prior to targeted metabolite analysis. Metabolite proﬁling of avocado cultivars was carried out adopting a method similar to that described by Mpai and Sivakumar [16]. Waters Acquity Ultra Performance Liquid Chromatographic (UPLC) system attached to a PDA detector (Waters, Milford, MA, USA) equipped with an Acquity UPLC HSS C18 column (150 2.1 mm, 1.8 m particle size, Waters) was used for the detection and quantiﬁcation of the phenolic metabolites. Mobile phases A and B consisted of water with 0.1% formic acid and acetonitrile, respectively. Optimum separation was achieved using gradient elution executed as follows: 0 min, 95% A and 5% B; 1 min, 85% A and 15% B; 15 min, 5% A and 95% B; coming back to the initial condition and being calibrated. The ﬂow rate used was 0.3 mL min at 30 C and injection volume was 2.0 L (full-loop injection). Chromatographic software Masslynx 4.1 processed and obtained all the chromatographic data. Since it was dicult to purchase authentic standards, catechin (Y= 24.4855x + 246.089, r = 0.99) was to quantify the ﬂavonoids, such as feruloyl-quinic acid, coumaric glucose, and tryptophan. All chromatography operations were performed at ambient temperature, in triplicate, and coumaric acid hexoside (325.091 m/z; 11.1 RT), 4-feruloyl quinic acid (367.1025 m/z; 14.44 RT), and tryptophan (203.0825 m/z; 8.46 RT) were expressed in milligrams per kilogram on a dry fresh weight basis. 2.6.3. Fatty Acid Composition Fatty acid composition was determined according to the method described by Glowacz et al. [15], using avocado pulp (1 g) and homogenising with 30 mL of hexane for 30 s. Oil extraction was performed following the method used by Meyer and Terry [17], without any modiﬁcations. After dissolving the derived avocado oil extract in 2 mL of hexane and mixing with 0.2 mL of 0.2 mol L potassium hydroxide in methanol, the mixture underwent vigorous shaking for 30 s. The upper hexane layer containing methyl esters was decanted and diluted 1:100 (v/v) with n-hexane directly prior to injection into the GC-MS system (7890A gas chromatograph equipped with an MS (5975C VL0) detector (Agilent Technologies, Johannesburg, South Africa)). The conditions for the GC-MS system were similar to those of Glowacz et al. [15], with the fatty acids expressed in milligrams per gram. 2.6.4. Ascorbic Acid Content Ascorbic acid content was determined using the 2,6-dichlorophenolindophenol dye titration method described for the dierent samples. These results were expressed as grams per kilogram on a fresh weight basis [18]. Agronomy 2020, 10, 1654 5 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 5 of 25 2.6.5. The 2,2,1-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay 2.6.5. The 2,2,1-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay The DPPH assay was determined according to the method described by Tinyane et al. [19], and The DPPH assay was determined according to the method described by Tinyane et al. [19], and the the results were expressed as the concentration of antioxidants required to decrease the initial DPPH results were expressed as the concentration of antioxidants required to decrease the initial DPPH absorbance by 50% (IC50) in milligrams of gallic acid equivalent per gram of fruit. absorbance by 50% (IC ) in milligrams of gallic acid equivalent per gram of fruit. 2.7. Statistical Analysis 2.7. Statistical Analysis Assessments of the studied quality attributes and biochemical parameters were conducted Assessments of the studied quality attributes and biochemical parameters were conducted following a two-factorial type, which consisted of three postharvest treatments (red LED, blue LED, following a two-factorial type, which consisted of three postharvest treatments (red LED, blue LED, and commercial condition (prochloraz + darkness)) and four storage periods (days 4, 8, 12, and 16) of and commercial condition (prochloraz + darkness)) and four storage periods (days 4, 8, 12, and 16) of storage separately for cvs. Fuerte and Hass displayed in a completely randomised design. Data were storage separately for cvs. Fuerte and Hass displayed in a completely randomised design. Data were subjected to a two-way analysis of variance (light treatment × days of exposure) (ANOVA) at Fisher’s subjected to a two-way analysis of variance (light treatment days of exposure) (ANOVA) at Fisher ’s protected least significant difference at p < 0.05 level, using Genstat (for Windows 08, version 64-bit protected least signiﬁcant dierence at p < 0.05 level, using Genstat (for Windows 08, version 64-bit Release 18:2, International Ltd., England, UK) package to obtain the mean values for the interaction Release 18:2, International Ltd., England, UK) package to obtain the mean values for the interaction of “light treatment and days of exposure”. There was no significant difference on single factor “light of “light treatment and days of exposure”. There was no signiﬁcant dierence on single factor treatment” or “days of exposure”. During the growing season, the experiment was repeated three “light treatment” or “days of exposure”. During the growing season, the experiment was repeated times. three times. 3. Results 3. Results 3.1. Effect of LED Light Treatments on Incidence of Anthracnose in Inoculated Avocado Cultivars 3.1. Eect of LED Light Treatments on Incidence of Anthracnose in Inoculated Avocado Cultivars Inoculated cv Inoculated cv.. F Fuerte uerte expo exposed sed to r to re ed d L LED ED light show light showed ed 32% 32% anthr anthracnose acnose inc incidence idence on d on day ay 4 4 in in storage, whereas fruit exposed to blue LED light or treated with commercial fungicide showed 42 storage, whereas fruit exposed to blue LED light or treated with commercial fungicide showed 42 and and 40% 4 anthracnose 0% anthracnose incidence, incidence, re respectively specti.vely. Conversely Convers , inoculated ely, inoculat fre uit d fr exposed uit expoto sed t red o red LED LE light D li and ght and stored for 8 and 12 days showed significantly lower anthracnose incidence (25%) compared to stored for 8 and 12 days showed signiﬁcantly lower anthracnose incidence (25%) compared to the fruit the f exposed ruit exposed to bl to blue LED light ue LED l or trieated ght or trea with ted wi commer th co cial mmercia fungicide l fu (Figur ngicid ee ( 1A). FigOn ure 1A day). On d 16, fruit ay exposed 16, fruit exposed to red LED light showed 40% anthracnose incidence, whereas the inoculated fruit exposed to red LED light showed 40% anthracnose incidence, whereas the inoculated fruit exposed to blue LED to bl lightue or LED treated light or trea with commer ted wi cial th comm fungicide ercia showed l fungthe icide showe highest anthracnose d the highest incidences anthracnose of 60 inc and idence 56%, s of 60 and 56%, respectively (Figure 1A). respectively (Figure 1A). (A) Figure 1. Cont. Agronomy 2020, 10, 1654 6 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 6 of 25 (B) Figure 1. (A) Inﬂuence of light-emitting diode (LED) light exposure on inoculated avocado cultivar Figure 1. (A) Influence of light-emitting diode (LED) light exposure on inoculated avocado cultivar Fuerte. (B) Inﬂuence of LED light exposure on inoculated avocado cultivar Hass. The exposure Fuerte. (B) Influence of LED light exposure on inoculated avocado cultivar Hass. The exposure of the of the fruit to blue or red LED light was for 6 h per day during storage. Ten replicate tray packs, fruit to blue or red LED light was for 6 h per day during storage. Ten replicate tray packs, each each containing four fruit, underwent dierent postharvest treatments. Mean values of bars marked by containing four fruit, underwent different postharvest treatments. Mean values of bars marked by dierent letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, different letters were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. blue LED light; Red, red LED light; Com.T, commercial treatment. Inoculated cv. Hass, exposed to red LED light or treated with commercial fungicide showed more Inoculated cv. Hass, exposed to red LED light or treated with commercial fungicide showed or less similar anthracnose incidence (30 to 32%) on day 4 in storage, whilst the fruit exposed to blue more or less similar anthracnose incidence (30 to 32%) on day 4 in storage, whilst the fruit exposed LED light showed higher (42%) anthracnose incidence (Figure 1B). At the same time, signiﬁcantly lower to blue LED light showed higher (42%) anthracnose incidence (Figure 1B). At the same time, anthracnose incidence was observed in inoculated fruit exposed to red LED on days 8, 12, and 16 in significantly lower anthracnose incidence was observed in inoculated fruit exposed to red LED on storage compared to fruit exposed to blue LED light or treated with commercial fungicide and stored days 8, 12, and 16 in storage compared to fruit exposed to blue LED light or treated with commercial for the respective periods. The highest anthracnose incidence (54%) appeared in inoculated fruits fungicide and stored for the respective periods. The highest anthracnose incidence (54%) appeared treated with commercial fungicide and held in storage for 16 days (Figure 1B). in inoculated fruits treated with commercial fungicide and held in storage for 16 days (Figure 1B). 3.2. Eect of LED Light Treatments on Incidence of Anthracnose in Naturally Infected Avocado Cultivars 3.2. Effect of LED Light Treatments on Incidence of Anthracnose in Naturally Infected Avocado Cultivars The lowest anthracnose incidence (25%) was observed in naturally infected cv. Fuerte exposed The lowest anthracnose incidence (≤25%) was observed in naturally infected cv. Fuerte exposed to red LED light and stored up to 4, 8, and 12 days when compared to blue LED light or commercial to red LED fungicide treatment light and (Figur storee d up to 2A). Highest 4, 8, and 12 d anthracnose ays wincidence hen compwas aredon to b day lue16 LE in Dfr liuit ghtexposed or comm to er blue cial fungicide treatment (Figure 2A). Highest anthracnose incidence was on day 16 in fruit exposed to LED light (50% incidence) or treated with commercial fungicide treatment (54% incidence) (Figure 2A). blue LED In naturally light (5 infected 0% incidence) cv. Hass, or trea red ted wi LED light th co exposur mmercia e l f signiﬁcantly ungicide trreat educed ment (5 the 4% inc anthracnose idence) (Figure 2A). incidence to 12% in fruit stored up to 8, 12, and 16 days when compared with the blue LED and commer In natur cial fungicide ally infected cv. H treated frauit ss, re (Figur d LED e 2 light B). Cultivar exposure si Hassgstor nific ed antl up y redu to 4 ced the a days showed nthracnose ~20% incidence to 12% in fruit stored up to 8, 12, and 16 days when compared with the blue LED and anthracnose incidence, whilst fruit exposed to blue LED light or treated with commercial fungicide commercia showed 36 l f and ungic 30%ide t anthracnose reated fru incidence, it (Figure r2B) espectively . Cultiv.ar Sign Haiﬁcantly ss stored up highest to 4 anthracnose days showed incidence ~20% anthracnose incidence, whilst fruit exposed to blue LED light or treated with commercial fungicide (47%) was observed on day 16 in fruit exposed to blue LED light (Figure 2B). The key ﬁnding is showed 36 that employing and 30% anthr red LED a light cnose inc exposur idene ce, respecti for 6 h per vely. day Sign and ific storage antly hifor ghest 8 or ant 12 hracnose days can inci reduce dence (47%) was observed on day 16 in fruit exposed to blue LED light (Figure 2B). The key finding is that the anthracnose incidence in green thin-skinned cultivar Fuerte for marketing. Similar postharvest employin treatmentg re cand LED light be recommended exposure to reduce for 6 h per the anthracnose day and stor incidence age for 8 or in dark-skinned 12 days c cultivar an red Hass uce the for anthracnose incidence in green thin-skinned cultivar Fuerte for marketing. Similar postharvest fruit stored after treatment for 8, 12, and 16 days. treatment can be recommended to reduce the anthracnose incidence in dark-skinned cultivar Hass for fruit stored after treatment for 8, 12, and 16 days. Agronomy 2020, 10, 1654 7 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 7 of 25 (A) (B) Figure 2. (A) Inﬂuence of LED light exposure on naturally infected avocado cultivar Fuerte. (B) Inﬂuence Figure 2. (A) Influence of LED light exposure on naturally infected avocado cultivar Fuerte. (B) of LED light exposure on naturally infected avocado cultivar Hass. The exposure of fruit to blue Influence of LED light exposure on naturally infected avocado cultivar Hass. The exposure of fruit to or red LED light was for 6 h per day during storage. Ten replicate tray packs, each containing four blue or red LED light was for 6 h per day during storage. Ten replicate tray packs, each containing fruit, underwent dierent postharvest treatments. Mean values of bars marked by dierent letters four fruit, underwent different postharvest treatments. Mean values of bars marked by different were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; letters were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED Red, red LED light; Com.T, commercial treatment. light; Red, red LED light; Com.T, commercial treatment. 3.3. Eect of LED Light Treatments on PAL and LOX Gene Expression and Exocarp Epicatechin Content 3.3. Effect of LED Light Treatments on PAL and LOX Gene Expression and Exocarp Epicatechin Content Signiﬁcantly highest upregulation of PAL genes was observed in cv. Fuerte exposed to red Significantly highest upregulation of PAL genes was observed in cv. Fuerte exposed to red LED LED light and stored up to 12 days compared to the fruit stored for 4, 8, and 16 days (Figure 3A). light and stored up to 12 days compared to the fruit stored for 4, 8, and 16 days (Figure 3A). Simultaneously, cv. Fuerte exposed to red LED light showed the signiﬁcantly highest upregulation Simultaneously, cv. Fuerte exposed to red LED light showed the significantly highest upregulation of PAL genes compared to the fruit exposed to blue LED light or commercial fungicide treatment in of PAL genes compared to the fruit exposed to blue LED light or commercial fungicide treatment in storage for 4, 8, 12, and 16 days (Figure 3A). storage for 4, 8, 12, and 16 days (Figure 3A). Similarly, the highest upregulation of PAL genes was noted in cv. Hass fruit exposed to red Similarly, the highest upregulation of PAL genes was noted in cv. Hass fruit exposed to red LED LED light and stored up to 12 days, but the level of upregulation of PAL genes was higher in cv. light and stored up to 12 days, but the level of upregulation of PAL genes was higher in cv. Hass Hass when compared to cv. Fuerte (Figure 3B). Furthermore, red LED light exposure also showed a when compared to cv. Fuerte (Figure 3B). Furthermore, red LED light exposure also showed a higher upregulation of PAL genes in fruit stored up to 8 and 16 days than in fruit exposed to blue LED light or treated with commercial fungicide and stored for similar storage periods (Figure 3B). Agronomy 2020, 10, 1654 8 of 25 higher upregulation of PAL genes in fruit stored up to 8 and 16 days than in fruit exposed to blue LED Agronomy 2020, 10, x FOR PEER REVIEW 8 of 25 light or treated with commercial fungicide and stored for similar storage periods (Figure 3B). (A) (B) Figure Figure 3. 3. ( (AA ) ) I Inﬂuence nfluencof e o LED f Llight ED li exposur ght expo e on suPreAL o(phenylalanine n PAL (phenylalanine ammonia lyase gene ammonia lyase gene expression) in avocado cultivar Fuerte. (B) Inﬂuence of LED light exposure on PAL (phenylalanine ammonia lyase expression) in avocado cultivar Fuerte. (B) Influence of LED light exposure on PAL gene expression) in avocado cultivar Hass. The exposure of the fruit to blue or red LED light was for (phenylalanine ammonia lyase gene expression) in avocado cultivar Hass. The exposure of the fruit 6 h per day during storage. Number of replicates per treatment n = 5. Mean values of bars marked by to blue or red LED light was for 6 h per day during storage. Number of replicates per treatment n = 5. dierent letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, Mean values of bars marked by different letters were significantly different at p < 0.05, according to blue LED light; Red, red LED light; Com. T, commercial treatment. Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. Signiﬁcantly higher epicatechin content in the pericarp was detected in cv. Fuerte exposed to Significantly higher epicatechin content in the pericarp was detected in cv. Fuerte exposed to red LED light and stored for 8 and 12 days when compared to the fruit stored for 4 and 16 days red LED light and stored for 8 and 12 days when compared to the fruit stored for 4 and 16 days (Figure 4A). It is interesting to note that cv. Fuerte exposed to red LED light and stored for 8 and (Figure 4A). It is interesting to note that cv. Fuerte exposed to red LED light and stored for 8 and 12 days showed higher epicatechin content than the fruit exposed to blue LED light or treated with commercial fungicide and stored for 4, 8, 12, and 16 days (Figure 4A). Cultivar Hass exposed to red LED light significantly retained the highest epicatechin content in the pericarp at 12 days of storage, followed by the fruit kept on the shelf for 8 days, when compared to the other two treatments at similar storage periods (Figure 4B). Agronomy 2020, 10, 1654 9 of 25 12 days showed higher epicatechin content than the fruit exposed to blue LED light or treated with commercial fungicide and stored for 4, 8, 12, and 16 days (Figure 4A). Cultivar Hass exposed to red LED light signiﬁcantly retained the highest epicatechin content in the pericarp at 12 days of storage, followed by the fruit kept on the shelf for 8 days, when compared to Agronomy 2020, 10, x FOR PEER REVIEW 9 of 25 the other two treatments at similar storage periods (Figure 4B). (A) (B) Figure Figure 4. 4. ((A A)).. IInﬂuence nfluence o of f L LED ED llight ight e exposur xposure e o on n s skin kin e epicatechin picatechin content in av content in avocado ocado cul cultivar tivar Fuerte. Fuerte. ( (B B) ).. Inﬂuence Influenceof of L LED ED light ligexposur ht exposu e on re on sk skin epicatechin in epicatechin content content in in avocado avo cultivar cado cultivar Hass. TheHass. The exposure of fruit to blue or red LED light was for 6 h per day during storage. Number of replicates per treatment exposure of fruit to blue or red LED light was for 6 h per day during storage. Number of replicates n = 5. Mean values of bars marked by dierent letters were signiﬁcantly dierent at p < 0.05, according per treatment n = 5. Mean values of bars marked by different letters were significantly different at p < to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. Overall, cv. Fuerte exposed to LED light and stored for 4, 8, 12, and 16 days showed signiﬁcantly lower upregulation of LOX gene expression when compared to the other two postharvest treatments Overall, cv. Fuerte exposed to LED light and stored for 4, 8, 12, and 16 days showed significantly and similar storage periods, as shown in Figure 5A. The highest upregulation of LOX gene expression lower upregulation of LOX gene expression when compared to the other two postharvest treatments was noted in fruit treated with commercial fungicide and stored for 16 days. In general, all the fruit and similar storage periods, as shown in Figure 5A. The highest upregulation of LOX gene expression treated with commercial fungicide showed signiﬁcantly higher gene expression compared to the fruit was noted in fruit treated with commercial fungicide and stored for 16 days. In general, all the fruit exposed to the blue LED light and stored at the respective storage periods. treated with commercial fungicide showed significantly higher gene expression compared to the fruit exposed to the blue LED light and stored at the respective storage periods. Likewise, in cv. Hass, exposure to red LED light significantly reduced the upregulation of LOX when compared with the fruit exposed to blue LED light or treated with commercial fungicide and stored at 4, 8, 12, and 16 days (Figure 5B). Agronomy 2020, 10, 1654 10 of 25 Likewise, in cv. Hass, exposure to red LED light signiﬁcantly reduced the upregulation of LOX when compared with the fruit exposed to blue LED light or treated with commercial fungicide and Agronomy 2020, 10, x FOR PEER REVIEW 10 of 25 stored at 4, 8, 12, and 16 days (Figure 5B). (A) (B) Figure 5. (A). Influence of LED light exposure on LOX (lipoxygenase) gene expression in avocado Figure 5. (A). Inﬂuence of LED light exposure on LOX (lipoxygenase) gene expression in avocado cultivar Fuerte. (B). Influence of LED light exposure on LOX (lipoxygenase) gene expression in cultivar Fuerte. (B). Inﬂuence of LED light exposure on LOX (lipoxygenase) gene expression in avocado avocado cultivar Hass. The exposure of fruit to blue or red LED light was for 6 h per day during cultivar Hass. The exposure of fruit to blue or red LED light was for 6 h per day during storage. Number storage. Number of replicates per treatment n = 5. Mean values of bars marked by different letters of replicates per treatment n = 5. Mean values of bars marked by dierent letters were signiﬁcantly were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; Red, red LED light; Com. T, commercial treatment. Com. T, commercial treatment. 3.4. Eect of LED Light Treatments on Coumaric Acid Hexoside, 4-Feruloyl Quinic Acid, 3.4. Effect of LED Light Treatments on Coumaric Acid Hexoside, 4-Feruloyl Quinic Acid, and Tryptophan and Tryptophan Content Content UPLC-QTOF/MS analysis helped to detect the coumaric acid hexoside, 4-feruloyl quinic acid, UPLC-QTOF/MS analysis helped to detect the coumaric acid hexoside, 4-feruloyl quinic acid, and tryptophan in avocado cvs. Fuerte and Hass after storage. Overall, cv. Fuerte treated with and tryptophan in avocado cvs. Fuerte and Hass after storage. Overall, cv. Fuerte treated with commercial fungicide and stored for 12 to 16 days or exposed to blue LED treatments and stored commercial fungicide and stored for 12 to 16 days or exposed to blue LED treatments and stored for for 8 to 12 days showed higher concentrations of coumaric acid hexoside (Figure 6A). The highest 8 to 12 days showed higher concentrations of coumaric acid hexoside (Figure 6A). The highest concentration of coumaric acid hexoside was detected in fruit that underwent commercial fungicide concentration of coumaric acid hexoside was detected in fruit that underwent commercial fungicide treatment and was stored up to 12 days (Figure 6A). Conversely, fruit exposed to red LED light treatment and was stored up to 12 days (Figure 6A). Conversely, fruit exposed to red LED light retained lower concentrations of coumaric acid hexoside throughout the storage time, and the lowest concentration was detected on day 12 (Figure 6A). Similar reduced concentration of coumaric acid hexoside was found in cv. Hass exposed to red LED light in different storage periods (4, 8, 12, and 16 days) compared to the other two postharvest treatments. The significantly lowest concentrations of coumaric acid hexoside were found in red LED light exposed cv. Hass stored for 12 and 16 days (Figure 6B). Agronomy 2020, 10, 1654 11 of 25 retained lower concentrations of coumaric acid hexoside throughout the storage time, and the lowest concentration was detected on day 12 (Figure 6A). Similar reduced concentration of coumaric acid hexoside was found in cv. Hass exposed to red LED light in dierent storage periods (4, 8, 12, and 16 days) compared to the other two postharvest treatments. The signiﬁcantly lowest concentrations of coumaric acid hexoside were found in red LED light exposed cv. Hass stored for 12 and 16 days (Figure 6B). Conversely, the signiﬁcantly lowest concentrations of 3-feruloyl quinic acid were in cv. Fuerte fruit exposed to red LED light treatment and stored for 12 and 16 days (Figure 6C) when compared to the fruit treated with commercial fungicide or exposed to blue LED light and stored for similar extended storage periods. (Figure 6B). Red light exposure showed a similar trend in cv. Hass, but signiﬁcantly lowest concentrations of 3-feruloyl quinic acid were found in fruit exposed to red LED light and stored for 8 and 12 days when compared to all other postharvest treatments adopted in this study (Figure 6D). Tryptophan amino acid accumulation was highest in cultivar Fuerte on day 12 in fruit exposed to red LED light compared to the fruit exposed to blue LED light or treated with commercial fungicide (Figure 6E). In cv. Hass, the highest accumulation of tryptophan was in fruit exposed to red LED light and stored for 8 and 12 days when compared to the fruit that underwent the other two postharvest treatments and was stored at dierent storage periods (Figure 6F). Agronomy 2020, 10, x FOR PEER REVIEW 12 of 25 Agronomy 2020, 10, 1654 12 of 25 Figure 6. Cont. Agronomy 2020, 10, 1654 13 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 13 of 25 Figure 6. Cont. Agronomy 2020, 10, 1654 14 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 14 of 25 Figure 6. Inﬂuence of LED light treatments on (A) coumaric acid hexoside in cv. Fuerte and (B) coumaric acid hexoside in cv. Hass. Inﬂuence of LED light treatments on Figure 6. Influence of LED light treatments on (A) coumaric acid hexoside in cv. Fuerte and (B) coumaric acid hexoside in cv. Hass. Influence of LED light treatments on (C) 3-feruloyl quinic acid in cv. Fuerte and (D) 3-feruloyl quinic acid in cv. Hass. Inﬂuence of LED light treatments on (E) tryptophan in cv. Fuerte and (F) tryptophan in (C) 3-feruloyl quinic acid in cv. Fuerte and (D) 3-feruloyl quinic acid in cv. Hass. Influence of LED light treatments on (E) tryptophan in cv. Fuerte and (F) tryptophan in cv. Hass. Mean standard deviation values for each bar were calculated based on ﬁve fruits. Number of replicates per treatment n = 5. Mean values of bars marked by cv. Hass. Mean ± standard deviation values for each bar were calculated based on five fruits. Number of replicates per treatment n = 5. Mean values of bars marked by dierent letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. different letters were significantly different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Agronomy 2020, 10, 1654 15 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 15 of 25 3.5. Eect of LED Light Treatments on Percentage of Fruit Reaching Ready-to-Eat Stage 3.5. Effect of LED Light Treatments on Percentage of Fruit Reaching Ready-to-Eat Stage Cultivar Fuerte fruit exposed to red LED light and stored for 12 days were signiﬁcantly ﬁrm when Cultivar Fuerte fruit exposed to red LED light and stored for 12 days were significantly firm when compared to the fruit exposed to blue LED light or commercial fungicide treatment and stored for compared to the fruit exposed to blue LED light or commercial fungicide treatment and stored for a similar a similar storage period (Figure 7A). Overall, the percentages of ﬁrm fruit obtained from red LED storage period (Figure 7A). Overall, the percentages of firm fruit obtained from red LED light treatment and light treatment and storage periods of 8, 12, and 16 days were signiﬁcantly higher when compared to storage periods of 8, 12, and 16 days were significantly higher when compared to those of the other two those of the other two postharvest treatments. Approximately 70 to 80% ﬁrm fruit were obtained from postharvest treatments. Approximately 70 to 80% firm fruit were obtained from red LED light treatment and 8 red LED light treatment and 8 to 12 days cold shelf storage (Figure 7A). Exposure to blue LED light to 12 days cold shelf storage (Figure 7A). Exposure to blue LED light showed 11, 20, and 40% overripe fruit after showed 11, 20, and 40% overripe fruit after 8, 12, and 16 days of cold storage, respectively. Moreover, 8, 12, and 16 days of cold storage, respectively. Moreover, 30% of cv. Fuerte fruit became overripe after 30% of cv. Fuerte fruit became overripe after commercial fungicide treatment and 16 days of cold commercial fungicide treatment and 16 days of cold shelf storage (Figure 7A). The significantly highest shelf storage (Figure 7A). The signiﬁcantly highest percentages (65 to 70%) of ready-to-eat ripe stage percentages (65 to 70%) of ready-to-eat ripe stage were obtained from blue LED light treatment (8 and 12 days were obtained from blue LED light treatment (8 and 12 days storage periods), red LED light treatment (4 days storage) and commercial fungicide treatment (16 days storage). storage periods), red LED light treatment (4 days storage) and commercial fungicide treatment (16 days storage). A similar ripening trend was observed in cv. Hass as well, with the fruit exposed to red LED light A similar ripening trend was observed in cv. Hass as well, with the fruit exposed to red LED light and and stored for 8, 12, and 16 days being ﬁrmer than fruit that underwent other postharvest treatments stored for 8, 12, and 16 days being firmer than fruit that underwent other postharvest treatments and were and were stored at dierent storage periods (Figure 7B). Approximately 70 to 75% of cv. Hass fruit stored at different storage periods (Figure 7B). Approximately 70 to 75% of cv. Hass fruit exposed to red LED exposed to red LED and stored for 8 to 16 days were ﬁrmer when compared to the fruit from blue LED and stored for 8 to 16 days were firmer when compared to the fruit from blue LED light or chemical fungicide light or chemical fungicide treatment (Figure 7B). Conversely, commercial fungicide treated fruit stored treatment (Figure 7B). Conversely, commercial fungicide treated fruit stored for 12 days or fruit exposed blue for 12 days or fruit exposed blue LED light and stored up to 8 days showed the highest percentages of LED light and stored up to 8 days showed the highest percentages of ready-to-eat stage ripe fruit. ready-to-eat stage ripe fruit. Furthermore, cv. Hass fruit exposed to blue LED light and stored for 12 Furthermore, cv. Hass fruit exposed to blue LED light and stored for 12 and 16 days showed around 22 to 28% and 16 days showed around 22 to 28% overripe fruits, and commercial fungicide treated fruit stored overripe fruits, and commercial fungicide treated fruit stored for 16 days also showed 30% overripe fruit. for 16 days also showed 30% overripe fruit. (A) Figure 7. Cont. Agronomy 2020, 10, 1654 16 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 16 of 25 (B) Figure 7. (A) Inﬂuence of LED light treatment on ripening in avocado cultivar Fuerte. (B) Inﬂuence Figure 7. (A) Influence of LED light treatment on ripening in avocado cultivar Fuerte. (B) Influence of LED of LED light treatment on ripening in avocado cultivar Hass. Fruit were exposed to blue or red light treatment on ripening in avocado cultivar Hass. Fruit were exposed to blue or red LED light for 6 h per LED light for 6 h per day during storage and left at 25 C for 2 days after storage. Mean values for day during storage and left at 25 °C for 2 days after storage. Mean values for each bar were calculated based each bar were calculated based on 100 fruits for each treatment. Bars with similar letters were not on 100 fruits for each treatment. Bars with similar letters were not significantly different at p < 0.05 according signiﬁcantly dierent at p < 0.05 according to LSD. Blue, blue LED light; Red, red LED light; Com. T, to LSD. Blue, blue LED light; Red, red LED light; Com. T, commercial treatment. commercial treatment. 3.6. Eect of LED Light Treatments on D-Mannoheptulose Content 3.6. Effect of LED Light Treatments on D-Mannoheptulose Content The D-mannoheptulose content was signiﬁcantly highest in cv. Fuerte exposed to red LED light The D-mannoheptulose content was significantly highest in cv. Fuerte exposed to red LED light and and stored for 12 days when compared to the other two postharvest treatments and at similar storage stored for 12 days when compared to the other two postharvest treatments and at similar storage periods periods (Figure 8A). Fruit exposed to red LED light and stored for 8 and 16 days and commercial (Figure 8A). Fruit exposed to red LED light and stored for 8 and 16 days and commercial fungicide treated fungicide treated fruit stored for 4 days showed higher concentrations of D-mannoheptulose sugar fruit stored for 4 days showed higher concentrations of D-mannoheptulose sugar than the other counterpart than the other counterpart treatments and storage periods. It is interesting to note that the fruit exposed treatments and storage periods. It is interesting to note that the fruit exposed to blue LED light for 12 and to blue LED light for 12 and 16 days or commercial fungicide treated fruit stored for 12 and 16 days 16 days or commercial fungicide treated fruit stored for 12 and 16 days showed significantly lower showed signiﬁcantly lower concentrations of D-mannoheptulose sugar (Figure 8A). concentrations of D-mannoheptulose sugar (Figure 8A). Cultivar Hass exposed to red LED light and stored for 8, 12, and 16 days showed signiﬁcantly Cultivar Hass exposed to red LED light and stored for 8, 12, and 16 days showed significantly higher higher and similar D-mannoheptulose content compared to the commercial fungicide treated fruit stored 4 days on the cold shelf (Figure 8B). However, cv. Hass exposed to blue LED light and stored and similar D-mannoheptulose content compared to the commercial fungicide treated fruit stored 4 days for 16 days showed the highest D-mannoheptulose content compared to the other two postharvest on the cold shelf (Figure 8B). However, cv. Hass exposed to blue LED light and stored for 16 days showed treatments and storage periods (Figure 8B). the highest D-mannoheptulose content compared to the other two postharvest treatments and storage periods (Figure 8B). Agronomy 2020, 10, 1654 17 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 17 of 25 (A) (B) Figure Figure 8. (A 8. ) I(n A fluence of ) Inﬂuence LED of LED lights on lights D-mannoheptulose and on D-mannoheptulose o and leic ac oleic id cont acidecontent nt in the mes in the oc mesocarp arp of avo of cado avocado cultivar Fuerte. (B) Inﬂuence of LED lights on D-mannoheptulose and oleic acid content in cultivar Fuerte. (B) Influence of LED lights on D-mannoheptulose and oleic acid content in the mesocarp of the mesocarp of avocado cultivar Hass. Mean standard deviation values for each bar were calculated avocado cultivar Hass. Mean ± standard deviation values for each bar were calculated based on five fruits. based on ﬁve fruits. Number of replicates per treatment n = 5. Mean values of bars marked by dierent Number of replicates per treatment n = 5. Mean values of bars marked by different letters were significantly letters were signiﬁcantly dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED different at p < 0.05, according to Fisher’s protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, light; Red, red LED light; Com.T, commercial treatment. commercial treatment. 3.7. Eect of LED Light Treatments on Oleic Acid Content in the Mesocarp 3.7. Effect of LED Light Treatments on Oleic Acid Content in the Mesocarp Oleic acid content was determined in ready-to-eat stage ripe fruit. Overall, all blue or red LED Oleic acid content was determined in ready-to-eat stage ripe fruit. Overall, all blue or red LED light light treatments signiﬁcantly increased the oleic acid (18:1) content in cv. Fuerte compared to the fruit treatments significantly increased the oleic acid (18:1) content in cv. Fuerte compared to the fruit treated treated with commercial fungicide, irrespective of the storage period. However, fruit exposed to red with commercial fungicide, irrespective of the storage period. However, fruit exposed to red LED light and LED light and stored for 8 days showed lower concentrations than the fruit exposed to blue LED light stored for 8 days showed lower concentrations than the fruit exposed to blue LED light and stored for and stored for similar storage periods (Figure 9A). A similar increased trend in accumulation of oleic similar storage periods (Figure 9A). A similar increased trend in accumulation of oleic acid (18:1) content in acid (18:1) content in cv. Hass fruit exposed to blue LED or red LED light compared to the commercial cv. Hass fruit exposed to blue LED or red LED light compared to the commercial fungicide treated fruit was fungicide treated fruit was observed, irrespective of the storage period (Figure 9B). observed, irrespective of the storage period (Figure 9B). Agronomy 2020, 10, 1654 18 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 18 of 25 (A) (B) Figu Figure re 9. (A 9.) I (A n)flInﬂuence uence of LED lights on ole of LED lights on oleic ic acid content acid content in the in themesoca mesocarp rp of av of avocado ocado cultiva cultivar r Fue Fuerte. rte. (B) Infl(uence o B) Inﬂuence f LED lights on olei of LED lights on c acid content in the oleic acid content in mthe esocarp of avocado cu mesocarp of avocado ltivars Hass and F cultivars Hass and uerte. Mean Fuerte. ± standa Mean rd devia standar tion val d u deviation es for each ba values r we for re ceach alculated bar wer based e calculated on five fruits. N based umbe on ﬁve r of replic fruits. ates pe Number r treat of ment n = 5 r.eplicates Mean valper ues o tr featment bars marn ke= d b 5. y d Mean ifferen values t letters of wbars ere simarked gnificantby ly d di iff eer reent nt at letters p < 0.05 wer , according to Fis e signiﬁcantly her’s dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Com.T, commercial treatment. 3.8. Effect of LED Light Treatments on Ascorbic Acid Content in the Mesocarp 3.8. Eect of LED Light Treatments on Ascorbic Acid Content in the Mesocarp Overall, the ascorbic acid content was significantly lower in commercial fungicide treated cv. Fuerte Overall, the ascorbic acid content was signiﬁcantly lower in commercial fungicide treated cv. fruit compared to the fruit exposed to blue LED light and stored for 6, 12, and 16 days or red LED light and Fuerte fruit compared to the fruit exposed to blue LED light and stored for 6, 12, and 16 days or red stored for 4, 8, 12, and 16 days in storage (Figure 10A). Likewise, there were lower concentrations of ascorbic LED light and stored for 4, 8, 12, and 16 days in storage (Figure 10A). Likewise, there were lower acid content detected in commercial fungicide treated fruit when compared to the blue or red LED light concentrations of ascorbic acid content detected in commercial fungicide treated fruit when compared treated fruit stored for 4 to 16 days onwards. In other words, exposure to blue or red LED light improved to the blue or red LED light treated fruit stored for 4 to 16 days onwards. In other words, exposure to the biosynthesis of ascorbic acid in cv. Hass (Figure 10B). blue or red LED light improved the biosynthesis of ascorbic acid in cv. Hass (Figure 10B). Agronomy 2020, 10, 1654 19 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 19 of 25 (A) (B) Figure 10. (A) Influence of LED lights on ascorbic acid content in the mesocarp of avocado cultivar Fuerte. (B) Figure 10. (A) Inﬂuence of LED lights on ascorbic acid content in the mesocarp of avocado cultivar Influ Fuerte. ence of LE (B)D lights o Inﬂuencen asc of LED orbic acid co lights on ntent i ascorbic n the m acid esocarp of avocad content in the mesocarp o cultivar Hass of avocado . Mean ± st cultivar andard deviatio Hass. n valu Mean es for each b standardar deviation were calcvalues ulated based on five for each bar wer frui e ts. Num calculated ber of re basedplicates on ﬁveper t fruits. reaNumber tment n = 5. Mean v of replicates alues of bars per tr ma eatment rked byn diffe = 5. re Mean nt lette values rs weof re si bars gnific marked antly diff by ere din er t ent at pletters < 0.05, acc were ording t signiﬁcantly o Fisher’s dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Com.T, commercial treatment. 3.9. Effect of LED Light Treatments on Antioxidant Activity 3.9. Eect of LED Light Treatments on Antioxidant Activity Antioxidant activity was significantly lowest in commercial fungicide treated cv. Fuertes from 4 days Antioxidant activity was signiﬁcantly lowest in commercial fungicide treated cv. Fuertes from onwards up to 16 days compared to the fruit that underwent the other two postharvest treatments and was 4 days onwards up to 16 days compared to the fruit that underwent the other two postharvest stored at similar storage periods. However, the significantly highest antioxidant activity was observed in treatments and was stored at similar storage periods. However, the signiﬁcantly highest antioxidant fruit exposed to red LED light and stored for 12 days. In general, red LED light exposed fruit stored for 4 to activity was observed in fruit exposed to red LED light and stored for 12 days. In general, red LED 16 days and fruit exposed to blue LED light and stored for 12 days showed higher antioxidant activity light exposed fruit stored for 4 to 16 days and fruit exposed to blue LED light and stored for 12 days (Figure 11A). showed higher antioxidant activity (Figure 11A). Agronomy 2020, 10, 1654 20 of 25 Agronomy 2020, 10, x FOR PEER REVIEW 20 of 25 Notably, cv. Hass had similar significantly increased antioxidant activity in fruit exposed to red LED Notably, cv. Hass had similar signiﬁcantly increased antioxidant activity in fruit exposed to red light and stored from 4 days to 16 days compared to the fruits fruit from the other two postharvest LED light and stored from 4 days to 16 days compared to the fruits fruit from the other two postharvest treatments. Likewise, commercial fungicide treated cv. Hass showed significantly lowest antioxidant treatments. Likewise, commercial fungicide treated cv. Hass showed signiﬁcantly lowest antioxidant activity than the fruit from the other two postharvest treatments (Figure 11B). Ascorbic acid showed a strong activity than the fruit from the other two postharvest treatments (Figure 11B). Ascorbic acid showed a positive correlation with antioxidant scavenging activity of cultivars Hass (r = 0. 77, p < 0.05) and Fuerte (r strong positive correlation with antioxidant scavenging activity of cultivars Hass (r = 0. 77, p < 0.05) = 0.72, p < 0.05). and Fuerte (r = 0.72, p < 0.05). (A) (B) Figu Figure re 11. (A 11. ) Antio (A) Antioxidant xidant metabolit metabolite e conten contents ts in avocado cultivar Fue in avocado cultivar rte t F ruerte eated post treated harve postharvest st with LED lights. with (B) Antio LED lights. xidant m (B)eAntioxidant tabolite conte metabolite nts in avocado cu contents ltivar in avocado Hass treated cultivar Hass postharvest with L treated postharvest ED lights. Mean ± with LED lights. Mean standard deviation values for each bar were calculated based on ﬁve fruits. Number standard deviation values for each bar were calculated based on five fruits. Number of replicates per treatment of replicates per treatment n = 5. Mean values of bars marked by dierent letters were signiﬁcantly n = 5. Mean values of bars marked by different letters were significantly different at p < 0.05, according to Fisher’s dierent at p < 0.05, according to Fisher ’s protected LSD test. Blue, blue LED light; Red, red LED light; protected LSD test. Blue, blue LED light; Red, red LED light; Com.T, commercial treatment. Com.T, commercial treatment. Agronomy 2020, 10, x FOR PEER REVIEW 21 of 25 Agronomy 2020, 10, 1654 21 of 25 The performing of PCA analysis was to summarise the interrelated biochemical changes with different LED light treatments and storage periods for both avocado cultivars Hass and Fuerte. The obtaining of the The performing of PCA analysis was to summarise the interrelated biochemical changes with two principal components with their factor loading was with respect to Eigen values > 1. The principal dierent LED light treatments and storage periods for both avocado cultivars Hass and Fuerte. components explained 71.4% (PC1 38.2% and 33.2%) (Figure 12). In PC1, the components coefficients or The obtaining of the two principal components with their factor loading was with respect to Eigen correlation coefficients (r) for the following parameters (variables): - oleic acid, ascorbic acid, and values > 1. The principal components explained 71.4% (PC1 38.2% and 33.2%) (Figure 12). In PC1, antioxidant activity, were positively loaded, and tryptophan was negatively loaded in PC1. In PC2: - 3- the components coecients or correlation coecients (r) for the following parameters (variables): feruloyl quinic acid, coumaric acid hexoside, ready-to-eat ripeness were loaded positively and D- - oleic acid, ascorbic acid, and antioxidant activity, were positively loaded, and tryptophan was manoheptulose, firm fruit were loaded negatively. The PCA analysis confirmed the increase of 3-feruloyl negatively loaded in PC1. In PC2: - 3-feruloyl quinic acid, coumaric acid hexoside, ready-to-eat quinic acid, coumaric acid hexoside in ready to ripe stage Hass and Fuerte avocado fruit exposed under ripeness were loaded positively and D- manoheptulose, ﬁrm fruit were loaded negatively. The PCA blue LED light or treated with commercial fungicide and stored for 12 and 16 days. Both cultivars, stored analysis conﬁrmed the increase of 3-feruloyl quinic acid, coumaric acid hexoside in ready to ripe stage for 8 and 12 days under red light, confirmed increased antioxidant activity, D- manoheptulose, tryptophan Hass and Fuerte avocado fruit exposed under blue LED light or treated with commercial fungicide and and skin epicatechin concentration and firm fruit. stored for 12 and 16 days. Both cultivars, stored for 8 and 12 days under red light, conﬁrmed increased antioxidant activity, D- manoheptulose, tryptophan and skin epicatechin concentration and ﬁrm fruit. Figure 12. Principal component analysis (PCA) showing correlation loadings of fruit phenolic compounds; epicatechin in the pericarp; tryptophan, D-mannoheptulose sugar, ascorbic acid, oleic Figure 12. Principal component analysis (PCA) showing correlation loadings of fruit phenolic compounds; acid, and antioxidant activity in the mesocarp; and fruit ripeness related to the LED light treatments in epicatechin in the pericarp; tryptophan, D-mannoheptulose sugar, ascorbic acid, oleic acid, and antioxidant comparison to the commercial treatment. F-QA, 3-feruloyl quinic acid; C-AH, coumaric acid hexoside; activity in the mesocarp; and fruit ripeness related to the LED light treatments in comparison to the D, days; H, cv. Hass; F, Cv. Fuerte. commercial treatment. F-QA, 3-feruloyl quinic acid; C-AH, coumaric acid hexoside; D, days; H, cv. Hass; F, Cv. 4. Discussion Fuerte. In the fruit industry, there is a growing interest in protecting the fruit against postharvest decay using elicitors or resistance inducers to stimulate the natural compounds responsible for resistance against the postharvest pathogens, as this will promote sustainable plant protection against pathogens Agronomy 2020, 10, 1654 22 of 25 and reduce the loss of fruit during marketing. Use of LEDs is becoming a popular and handy tool for sustainable agricultural practices [20]. Antimicrobial properties of light are gaining more attention in fungi, as they are developing resistance against the conventionally used fungicides. Blue LEDs elicited resistance against green mould (Penicillium digitatum) decay in citrus (nonclimacteric fruit) by altering the phenolic proﬁle, especially the scoparone content [21]. However, in our study, red LED light caused signiﬁcant reduction in anthracnose incidence in both cultivars Fuerte and Hass, although the impact was higher in cv. Hass, primarily by eliciting resistance by upregulating the PAL genes and increasing the epicatechin concentration (Figure 2A). A similar increase in PAL activity and PAL gene expression induced by postharvest treatments (e.g., thyme oil vapours) demonstrated the control of anthracnose decay in avocados [22]. Antifungal diene compound (AFD; 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12, 15-diene) is responsible for the resistance of unripe avocado fruit against the anthracnose pathogen [23]. The AFD compound declines as the fruit ripens due to the activity of lipoxygenase enzyme and facilitates the entry of the pathogen into the fruit (host). Alternatively, emission of ethylene further facilitates the infection process of C. gloeosporioides. The concentration of epicatechin, a product of the phenylpropanoid pathway, regulates the activity of lipoxygenase and the levels of AFD compound [24–26]. Furthermore, upregulation of PAL gene expression was found to stimulate the biosynthesis of epicatechin content in the skin of avocado [22,23]. Results presented herein indicate that both cultivars (Fuerte and Hass) exposed to red LED for 12 day, via activation of the phenylpropanoid pathway, revealed PAL gene expression and epicatechin content in the skin (Figure 3A,B and Figure 4A,B). This research reported this by demonstrating the low regulation of LOX genes in fruit exposed to LED light, the higher concentration of epicatechin in the fruit skin, and the observed reduction in anthracnose-infected fruits (Figure 2B). Accumulation of tryptophan and the biosynthetic pathway guide the production of dierent secondary metabolites [27]. Fundamentally, tryptophan biosynthesis and the enzymes are induced by the stress reaction caused by biotic elicitors [27]. There is reportedly an interdependence of the shikimate pathway and the key phenylalanine-derived phenolics and phytoalexin via phenylalanine ammonia lyase in dierent fruits and vegetables [28,29]; similarly, red LED lights stimulated the accumulation of tryptophan amino acid in this study (Figure 6E,F). Sensitivity or response of the fruit or plant and the pathogen regarding the control of anthracnose during storage on the market shelf towards a speciﬁc monochromatic light is more likely due to the sensitivity of their light receptors than the energy or the wavelength of the speciﬁc light [30]. This could be the reason for the observed dierences in response towards the dierent LED light treatments. Blue LED light was found to accelerate the ripening in bananas (climacteric fruit), while red LED light delayed the ripening [31]. A similar trend was observed in both avocado cultivars (Fuerte and Hass) in this study (Figure 3); however, blue LED light showed a remarkable impact in ripening cvs. Fuerte and Hass on days 8 and 16 respectively. Huang et al. [31] investigated the impact of dierent LED lights and wavelengths on climacteric pattern and reported the blue light was the most eective in inducing the onset of the climacteric peak, but the red LED light slowed the ripening. The accelerated ripening in fruit exposed to blue LED light and reduction of epicatechin content in the avocado fruit skin could have aggravated the anthracnose of decay and the entry of C. gloesoporioides into the fruit (Figure 3A,B). The C7 D-mannoheptulos correlated with time of ripening [16] and avocado (cv. Hass) grown under the red nets demonstrated higher concentration of D-mannoheptulos and the fruits took time to reach ready stage in our previous study [32]. Detected in all avocado cultivars, except cv. Bacon, during ripening were p-coumaric and ferulic acids (hydroxycinnamic acids) [33]. Fruit ripening of climacteric fruits such as tomato and kiwi showed an intense modiﬁcation of nonvolatile secondary metabolites in the phenolic proﬁle [34]. Some derivatives of phenolic compounds, such as coumaric and ferulic acid, reportedly increased linearly during development, and accumulation continued in the fully ripe fruit [34]. Likewise, our results showed an increase in coumaric acid hexoside and 3-feruloyl quinic acid related to the number of fruit reaching ready-to-eat stage ripeness exposed to blue LED Agronomy 2020, 10, 1654 23 of 25 light or commercial fungicide treatment (Figure 3A,B). Variability in avocado metabolites occurs due to dierent photoreceptors that sense each light, since the blue light is absorbed by the phototropin and cryptochrome and red light is absorbed by the phytochrome. Phytochromes mediate the red/FAR light related responses in plants and biosynthesis of phenolic compounds (phytochemicals). In our previous research, red net grown cv. Hass avocados showed higher PAL activity and accumulation of epicatechin and phenolic acid components [19]. The ascorbic acid content (vitamin C) was stimulated in cabbage stored under blue LED lights, which improved the ascorbic acid accumulation. However, based on the results presented here, in avocado cultivars Fuerte and Hass, the increase of ascorbic acid is favoured by the red LED light (Figure 10A,B). Furthermore, blue LED light was found to promote the synthesis of all free amino acids, except glutamic and aspartic acids, during postharvest ripening of tomatoes from green to yellow to red [11]. Likewise, in this study, blue and red LED light inﬂuenced the oleic acid biosynthesis in both avocado cultivars (Figure 9A,B). It is noteworthy that the production of antioxidants, ascorbic acid, and phenolic compounds and amino acid biosynthesis occur under more stressful conditions [35]. 5. Conclusions Application of the LED light technology to avocado at postharvest stage can be a useful tool to address the demand for fruits that have reached the ready-to-eat stage without compromising the fruit quality. Applying the blue LED light in cold storage can accelerate fruit ripening, whereas employing the red LED light can induce fruit resistance against the anthracnose postharvest disease in both avocado cvs. Fuerte and Hass for up to 12 days on the market shelf at 10–12 C. Further studies on the application of red and blue LED lights as a combined spectrum are imperative. Author Contributions: S.M. performed the experiment, was responsible for the data collection, conducted formal analysis and the analysis of phenolic compounds, conducted statistical analysis, and wrote the ﬁrst draft. 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Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions

Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions

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Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions

Stimulation of Light-Emitting Diode Treatment on Defence System and Changes in Mesocarp Metabolites of Avocados Cultivars (Hass and Fuerte) during Simulated Market Shelf Conditions

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