Thesis of doctoral (PhD) dissertation POMOLOGICAL EVALUATION OF APRICOT CULTIVARS AND THE ROLES OF POSTHARVEST APPLICATION OF SALICYLIC ACID AND METHYL JASMONATE ON STRESS RESISTANCE Ahmed Ezzat Gaballa Kassem Supervisor: Prof. Dr. Imre Holb, MTA Doctor UNIVERSITY OF DEBRECEN KERPELY KÁLMÁN DOCTORAL SCHOOL OF CROP PRODUCTION, HORTICULTURE AND REGIONAL SCIENCES Debrece 2014 1 1. Introduction The apricot (Prunusarmeniaca L.) is one of the most important fruit species grown in the world, as apricot fruit is highly appreciated by consumers. Consumers cherish the flavour and aroma of high quality apricots, with the sugar content being one of the most appreciable quality characteristics (Ruiz and Egea, 2008). Apricot fruit is usually stored in cold storage and many apricot cultivars can be stored for 4-6 weeks and these cultivars are able to keep an acceptable level of fresh firmness and other quality attributes. However, once the fruit is taken out from cold chamber the fruit start to deteriorate (Stanley et al., 2010). Apricot fruits during cold storage were reported to show chilling injury symptoms and a high percentage of fruit decay, such as mealiness development, loss of juiciness and/or gel breakdown (Stanley et al,. 2010). Apricot fruit starts to lose its physical and chemical qualities directly after harvest and through the storage period. Fruits start to lose their water content reaching 12% after 28 days at 1 oC. This is accompanied with fruit softening, increased fruit acidity and reduction in soluble solid content (SSC) (Ezzatet al., 2012). Storage at low temperature to retard tissue respiration is still the most effective postharvest method for extending the shelf-life. But most of cold stored fruit present the chilling injuries (CI)symptoms, and subsequently fruit loss most of qualities parameters. The use of chemical compounds to potentiate the natural defense of plants represents another alternative, potentially promising way to disease control (Kessmannet al., 1994). Many reports have shown that induced disease resistance in plants by biotic and abiotic elicitors is a very effective method for restricting the spread of fungal infection (Drobyet al., 2002; Qin et al., 2003). Two signaling pathways have been described by Thaleret al. (1999), one involving salicylic acid (SA) and another involving jasmonic acid (JA), which participates in the expression of plant resistance to pathogens and insect herbivores. SA is thought to be a key compound in the regulation of resistance to fungal, bacterial and viral pathogens and provides a signal for 2 expression of PR-proteins and other potential protective compounds (Ryalset al., 1996). However, exogenous application of JA has been demonstrated to induce systemic acquired resistance (SAR) in plants by stimulating many of the systemic metabolites, similar to that which occurs from challenge with pathogens or insects (Kessmannet al., 1994). The importance of the phytohormones SA and JA as critical signals in induced resistance response in plants is recognized (Bostock, 1999). These signal molecules are involved in some signal transduction systems, which induce particular enzymes catalyzing biosynthetic reactions to form defense compounds such as polyphenols, alkaloids or pathogenesis-related (PR) proteins (Tamariet al., 1995; Van Loon, 1995). SA has been extensively used for quality improvement in a number of crops (Peng and Jiang, 2006). Research argued the role of phenolic compounds such as SA in physiological or biochemical processes including ion uptake, membrane permeability, enzyme activity, heat production, growth development (Arberg, 1981).SA significantly reduced the quality loss in peaches (Wang et al., 2006),tomato (Ding et al.,2001), sweet peppers (Funget al.,2004), and loquat fruits (Caiet al.,2005). SA and its derivatives are widely used to enhance pre- and postharvest quality of fruit such as by controlling firmness of harvested peaches during storage, (Wang et al., 2006) and banana fruits during ripening (Srivastava and Dwivedi, 2000 ). Thus, SA has a remarkable ability to maintain the fruit quality during storage life of fruits. Several natural volatile compounds, such as methyl jasmonate (MeJA), were reported to maintain fruit quality. For instance, MeJA reduced the development of chilling injury symptoms in mango (González-Aguilar et al.,2000). Treatment of tomato fruit with low concentration of MeJA or methyl salicylate (MeSA) substantially enhanced their resistance to chilling injury and decreased the incidence of decay during low-temperature storage (Ding et al., 2001; Wang et al., 2006). Phenolic compounds are secondary metabolites that have important contribution to plant-derived food quality as they affect fruits, appearance, flavour and health-promoting properties. Their content in foods is modulated by many factors that influence phenolic stability, biosynthesis and 3 degradation. In their biosynthesis, the key step is catalyzed by the enzyme phenylalanine ammonia-lyase (PAL), which can be induced by various stress conditions (Dixon and Paiva, 1995). Two vital antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) play significant roles in scavenging free radicals produced as a result of metabolic processes. SOD converts OH− into H O which is converted 2 2 into H O and O by catalase (CAT)(Sala, 1998). Higher peroxidase (POD) 2 2 activity resulted in lower browning incidence in treated peach fruits compared with control (Wills et al.,1998). As a consequence, a general requirement is to raise or at least to maintain the acceptable levels of qualities and chemical characters of apricot during a cold storage period and shelf-life. The aims of this study were to investigate  The effect of 0, 1, and 2 mmol L-1 SA concentrations on „Flavor Cot‟, „Jumbo Cot‟, and „Bergeron‟ apricot cultivars and to understand the mechanism of SA in enhancement the storability and fruit quality attributes.  The effect of 2 mmol L-1SA and/or 0.2 mmol L-1MeJA on various fruit quality limits of apricot fruit cultivar Bergarouge.  The effect of 2 mmol L-1 SA and 0.4 mmol L-1MeJAon induced resistance to Monilinialaxa on apricot fruit cv. „Bergarouge‟. 2. MATERIALS AND METHODS 2.1. Effect of three SA concentrations on fruit three apricot cultivars during cold storage 4 3.1.2 3.1.1 Fruit sample and tested cultivars Fruits of apricot cultivars Jumbo Cot, Flavor Cot and Bergeron were hand harvested in a commercial orchard in Boldogkőváralja, Hungary at ripe stage and selected for uniform size, colour and examined to exclude all visual defects. 3.1.3 Chemical treatments The harvested fruits of each cultivar were divided into three groups. Fruits were dipped into a solutions of 0.5, 1 and 2 mmol L-1 SA for 15 minutes as well as control fruits were submerged into distilled water, for the cold storage treatment, fruit was stored at 1 oC and 90% humidity. The examinations were done on days 7, 14, 21 and 28. Each treatment was replicated three times and experiments were repeated twice. 3.2 Effect of SA and/or MeJA on variousfruit quality parameters , of fruit of apricot cultivar ‘Bergarouge’ 3.2.1 Fruit sample Fruits of apricot cultivar „Bergarouge‟ were hand harvested in a commercial orchard in Boldogkőváralja, Hungary at ripe stage and selected for uniform size, colour and examined to exclude all visual defects. 3.2.2 Chemical treatments The harvested fruits were divided into three groups. Fruits were dipped into a solution of 0.2 mmol L-1MeJA and 2 mmol L-1 SA for 15 minutes as well as control fruits were submerged into distilled water. Then for each treatment, fruits were divided into 2 further groups. The first group, as the cold storage treatment, was stored at 1 oC and examinations were done on days 7, 14 and 21. The second group, as the shelf-life treatment, was stored at 1 oC and 95% humidity for 15 days then placed at room temperature (25 oC) and fruits were examined at 4 and 8 days. Each treatment was replicated three times and experiments were repeated twice. 5 3.3. Effect of SA and/or MeJA on induce resistance to Monilinialaxa on apricot fruit cultivar Bergarouge 3.3.1 Isolation of fungi M.laxa were isolated from decayed plum fruit. The fungi were maintained on PDA at 4 °C. Spores of M. laxa were obtained from 2-week-old cultures incubated at 25 oC by flooding the cultures with sterile distilled water containing 0.05% (v/v) Tween 80. The suspensions of spores were filtered through four layers of sterilized cheese cloth. The concentrations of spores were adjusted to (1 x 10-3spores mL-1) with the aid of a haemocytometer. 3.3.2 Mycelial growth The concentrations of (0.5, 2 and 5 mmol L-1) of SA and (0.1, 0.4 and 0.7 mmol L-1) of MeJA were used to study the effects on mycelial growth of monilinialaxa. The effects were assayed by the method of Yao and Tian (2005a). Each concentrate of SA and MeJA solution mixed with molten PDA-agar to give a total volume of 20 mL per petri plate (diameter: 90mm). After the agar had solidified, 5mm disks of M. laxa were placed in the center of each petri plate. Plates were incubated at 25 oC.Colony diameter was determined 6 days after inoculation. Each treatment was replicated three times and the experiment was repeated twice. Mycelial growth of M. laxa on PDA was expressed as growth rate, which was calculated according to the following formula: Growth rate (%) = (colony diameter after inoculation−5mm)/5mm×100. After the determination of the appropriate concentration the experiment was repeated as 2 mmol L-1 SA and 0.4 mmol L-1 MeJA solution mixed with molten PDA-agar and the growth rate was measured after 2, 4, 6 and 8 days of incubation. 6 3.3.3 Sample preparation Fruits of cv. Bergarouge were hand harvested in the orchard of North-Cot Ltd located in Boldogkőváralja, Hungary at ripe stage and selected for uniform size, colour and examined to exclude all visual defects. Priminaly samples were taken from the fruit directly after harvest and undergone for firmness measurements, analysis of totalphenol content, antioxidant capacity, SOD, POD, PAL activities and liginin content and the results was expressed as zero time. 3.3.4 Exploratory experiment The measurement of the appropriate concentration of SA and MeJA, 30 fruits were dipped into solution of (0.5, 2 and 5 mmol L-1) of SA and (0.1, 0.4 and 0.7 mmol L-1) of MeJA for one hour as inducing treatments and water treated fruit were control. Fruit were sterilized with 2% (v/v) sodium hypochlorite for 4 minutes then washed with tap water and dried by air. Each chemical treated group was divided into two inoculation treatment. One group was wounded (a uniform hole 3mm deep and 3 mm wide) with a sterile borer. After 2 hours, the fruit were inoculated by M. laxa suspension. The other was not wounded, after 8 days; the fruit were examined for lesion diameter (mm) and fruit disease incidence (%). After set the tested concentrations the same experiment was repeated with 360 fruit. Divided into three chemical treatments (2 mmol L-1 SA, 0.4 mmol L-1 MeJA and water) then each group was divided into two inoculation treatments (inoculated and non inoculated). There were three replicates for each treatment; fruits were put in 200mmx130mmx50mm plastic boxes at humidity about (95%) at temperature 25 oC. 3.3.5 Effect of salicylic acid on disease severity of apricot fruit The number of the infected fruit in non-inoculated treatment and the average lesion diameter was measured as increasing diameter over the wound of experiment after 2, 4, 6, and 8 days at 25 oC the diameter of wounds were 7 considered as zero mm. Apricot fruits showing surface mycelial developing symptoms were considered as decayed fruit. Fruit disease incidences were estimated by the mean proportion of fruit that showed 1 mm decay at fruit surface. Fruit firmness (N) was measured by MagnessTazlor penetrometer (model FT011, QA Supplies LLC, Italy) directly after harvested as zero time and periodically after 2, 4. 6. and 8 days of treatments. 3.4. Measurements of the fruit pomological characters Measurements for weight loss, fruit firmness, SSC, acidity SSC/acidity ratio and JuicepH were performed on 30 fruits in three replicates immediately after harvest as day zero. Then 30 treated fruits per replicates were examined for each measure according to cold storage and shelf-life treatments. The following parameters were determined: 3.4.1 Weight loss (%) as different between the weight at zero day and the weight at assessment days. 3.4.2 Fruit firmness as penetration force (Newton) by Magness Tazlor penetrometer (model FT011, QA Supplies LLC, Italy). 3.4.3 Juice SSC (degrees Brix) by portable digital refractometer (Model 53007 TR, TR-Turoni Inc Forli, Italy) at 25 oC. 3.4.4 Juice acidity (%) by portable digital acidity meter (Model 53101 TR, TR-Turoni Inc Forli, Italy). 3.4.5 SSC/acidity ratio was calculated. The prepared juice of the sample was used for chemical analysis. 3.4.6 pH measurements were performed using a (84432 HANNA instruments®, Germany) pH meter. 8 3.5. Examination of chilling injury, fruit decay, membrane electrolyte, mealinessdevelopment and lose of juiceness Chilling injury and fruit decay were invistigated in both cold storage and shelf-life treatments by using 30 fruitsper replicates. 3.5.1 The degree of chilling injury (CI) was visually invistigated on the fruit surface following a double cut parallel to the axial diameter. The extent of flesh browning was divided into the following classes: 0, no browning; 1, extensive browning covering <25% of the cut surface; 2, extensive browning covering ≥25% but <50% of cut surface; 3, extensive browning covering ≥50% but <75% of cut surface; 4, extensive browning covering ≥75% of cut surface. From this, a CI index was expressed as: CI index = [(flesh browing class)×(number of fruit at the given flesh browing class)]/(4×total number of fruit in the treatment). 3.5.2 Fruit decay (FD) was assessed as symptomps of superficial browning on the fruit surface. The severity of the symptoms was assessed visually according to the following scale: 0, no browning; 1, browning ≥25% of the fruit surface; 2, browning ≥25% but <50% of the fruit surface; 3, browning ≥50% but <75% of the fruit surface; 4, browning ≥75% of the fruit surface. From this, FD index was expressed as: FD index = [(superficial browning class)×(number of fruit at the given superficial browning class)]/(4×total number of fruit in the treatment). 3.5.3 Electrolyte leakage was measured according to the method of Zhao , (2009). 3 mm thick of mesocarp tissue were excised from equator part of 5 fruits. Disks were put into aqueous 0.1Mmannitol under constant shaking. The conductivity of 9 the solution (L1) was measured with a conductivity meter. Solutions were boiled for 10 min and then cooled to 20 °C. The conductivity of tissues (L2) was measured. The percentage of electrolyte leakage was calculated using the following formula: %Electrolyte leakage (L1/L2)x 100. 3.5.4 mealiness and juiciness, each fruit was tasted three trained assessors independently and rated. Mealiness was considered to be a woolly or lumpy texture and fruit were scored on a scale from 0 (no mealiness) through to 3 (high mealiness). Juiciness was considered to take into account the amount of free fluid released from the sample during chewing and fruit were scored for juiciness on a scale from 0 (no juice) through to 3 (very juicy). 3.6. Chemical measurements 3.6.1 Total amount of soluble phenols Total amount of soluble phenols were determined using Folin-Ciocalteu‟s reagent (Singleton and Rossi, 1965). The content of soluble phenols was calculated from a standard curve obtained from different concentrations of gallic acid. 3.6.2 The total antioxidant capacity The total antioxidant capacity related to ascorbic acid was determined spectrophotometrically using the FRAP (Ferric Reducing Antioxidant Power) (Benzie and Strain, 1996).It is based on the reduction of the Fe3+- TPTZ complex to the ferrous form at low pH. This reduction is monitored by measuring the absorption change at 593nm. Results are expressed as mg equivalents of ascorbic acid (mg AA g-1 FW). 10