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Biodegradable Mulch Film Technology and Harpin Protein for Quality Enhancement of Anna Apples During Cold Storage



M.E. Tarabih and E.E. Eleryan
 
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ABSTRACT

Background and Objective: Growers of Anna apple have been facing many production problems that hinder them and represent a main obstacle towards obtaining high quality of organic fruits. Therefore, the main goal of this work was to evaluate the possibility of producing organic fruits, extend color and enhance resistance of fruits during storability. Materials and Methods: The present investigation was conducted during three successive seasons 2016, 2017 and 2018 (the first season was experimental) on Anna apple trees to evaluate the effectiveness of soil biodegradable mulch films (BMF) and sprinkle fruits by harpin protein to enhance color and texture of fruits. As well as extend storability and enhanced systemic resistance of fruits during 90 days of cold storage (0°C±1 and 90-95% RH) to be available for longer period. Results: The results indicated that, covering the soil with (BMF) mulch produced higher organic matter at the end of experiment. All biodegradable films (BMF) treatments improved ratios of nutrient contents Ca, P and K in the fruits. In this respect, harpin protein at 40% with mulching soil with biodegradable films (BMF) led to produce higher fruit yield at harvest and reduce postharvest loss weight, respiration rate, electrolyte leakage and pathogen decay. Further, it significantly affected enhanced vitamin C content, anthocyanin, phenolic, antioxidant activity and reduced the decline in titratable acidity while, reduced percent of TSS and total sugar. Conclusion: From above mentioned results, it concluded that, soil biodegradable mulch films (BMF) with sprinkle fruits by harpin protein at 40% appear to be a good solution for the obtaining high quality and storability of organic fruits.

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  How to cite this article:

M.E. Tarabih and E.E. Eleryan, 2020. Biodegradable Mulch Film Technology and Harpin Protein for Quality Enhancement of Anna Apples During Cold Storage. Asian Journal of Crop Science, 12: 97-108.

DOI: 10.3923/ajcs.2020.97.108

URL: https://scialert.net/abstract/?doi=ajcs.2020.97.108
 
Copyright: © 2020. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Apple “Malus domestica L.” is one of the most important horticultural deciduous fruit crops in Egypt. The total area cultivated with apple is 72264 feddans produced 798574 t according to FAO1. The organic product creation has been consistently expanding lately due to the fantastic returns for cultivators. Cultivators considering changing from traditional to organic production can confront difficulties with soil nutrition managemen2. Effective ways to increase the production and marketing of organic apples are crucial for farmers who have to deal with pests and diseases without using the traditional tools available to inorganic farmers. Maintaining a healthy orchard floor is the key to preventing weeds and keeping soil healthy. Using organic mulch increase the soil organic matter and can control soil temperature fluctuation under mulch. In this respect, the mulch directly affect the climate around the plant by adjusting the budget for surface irradiation and reducing water loss in the soil, which leads to more soil moisture3.The main benefits of mulching technique with organic or inorganic materials are early crop production, higher yields, better product quality, more efficient water use, reduced leaching of fertilizers, reduced soil and wind erosion, reduced herbicide applications3. Therefore, the use of plastic films in agriculture has had a serious drawback in producing massive amounts of waste.

Biodegradable mulch films (BMF) represents a real challenge for enhancing sustainable and environmentally friendly agricultural activities4. Biodegradable mulches (BMF) offer an environmentally sustainable alternative to conventional polyethylene (PE) mulch. BMF was developed as a substitute for polyethylene mulch (PE) and was designed to be decomposed into the soil after use by microorganisms5. Common bio-based polymers used in BMF include polylactic acid (PLA), starch, cellulose and polyhydroxy alkanoates (PHA)3. Polymers used in BMF contain ester bonds or are polysaccharides, which are amenable to microbial hydrolysis6.Mulches enhanced fruit quality such as red coloration of cv. 'Gala Mondial' apples by improving flavonoids and anthocyanins. Flavonoids increased up to 52.4% in the fruit peel and anthocyanin content improved up to 66% compared to control. While, reflective mulch did not affect chlorophyll and carotenoid content in the 'Gala' fruit peel7. The biodegradable polymers have two different formulations, those which the chemical structure enables direct enzymatic degradation (e.g. starch or cellulose) and those that undergo photo-oxidation or thermo- oxidation upon exposure to UV light or heat8. Starch is an environmentally friendly material as it is a renewable resource derived from corn and other crops.

Harpin is a heat-stable, glycine-rich protein produced by Erwinia amylovora that is involved in its pathogenesis of apple. Morphological and cellular changes in apple fruit treated with harpin suggest that it may trigger or intensify cellular responses in apples9. Harpin can provoke a hypersensitive response in non-host plants. This response is characterized by a rapid localized cell death at the site of the invasion for which reason it can act as a chemical elicitor10. Moreover, this protein activates reactive oxygen species(ROS) burst, salicylic acid (SA) and the jasmonate (JA)/ethylene signal transduction pathways that confer systemic acquired resistance (SAR) to different plants10. Harpin has been applied as an effective postharvest treatment to prevent decay in apples11and pears12. In addition, field applications demonstrated its usefulness for controlling pathogen-borne diseases in pear13. Harpin was described as a stimulant capable of activating enzymes such as phenylalanine ammonialase (PAL) from the biosynthesis pathway of polyphenols. Examples of this have been found in several fruits; for instance, in postharvest-treated peaches with a subsequent increase in total phenols14.

The main goal of this work was to evaluate the effective methods of soil biodegradable mulch film and sprinkle Anna apples by harpin protein to produce organic fruits, extend color and enhance systemic resistance of fruits during storability.

MATERIALS AND METHODS

The present investigation was conducted during the three successive seasons 2016, 2017 and 2018 (the first season was experimental) on Anna apple fruits trees to evaluate the effectiveness of harpin protein with or without mulching soil for keeping quality of Anna apple fruits under cold storage. Fruits were picked from trees about eight years old, healthy and as uniform as possible in vigor and shape which, grown in sandy soil (4×4 m) apart at private orchard located at Cairo-Alexandria desert road, Egypt. The trees subjected to the recommended cultural practices in completely randomized design and divided into 5 groups, three replicates for each and two trees for a replicate.

Prior to executing the experiment, Soil samples were randomly taken from depth (0-30 cm) then, the soil physical and chemical properties were analyzed as showed in Table 1-2. Also, the main properties of the soil were evaluated at the end of experiment after two seasons of study.

Table 1: Soil physical characteristics prior to initiation of the experiment
Soil depth (cm)
Sand (%)
Clay (%)
Silt (%)
Texture
pH
EC (dS m1)
0-30
92.6
3.2
4.2
Sandy
8.2
0.65


Table 2: Soil chemical characteristics prior to initiation of the experiment
Soil Soluble cations (meq L1) Soluble anions (meq L1)
depth  
(cm)
Ca++
Mg++
Na+
K+
HCO3
Cl
SO4
CO3
0-30
1.65
0.7
2.55
0.2
2.8
2.1
0.53
0

In the present study, selected trees were mulching on both sides by using biodegradable (BMF) mulch films on February. Biodegradable mulch films (BMF) (starch based) Mater-Bi® film was silver with 20 μm thick.

Treatments:

•  Control (without treated or mulched)
Spraying fruits with harpin 20%
Spraying fruits with harpin 20%+mulching soil with biodegradable films (BMF)
Spraying fruits with harpin 40%
Spraying fruits with harpin 40%+mulching soil with biodegradable films (BMF)

The harpin protein was applied within two applications:

First application 15 April: 2 weeks after full bloom (WAFB)
Second application 15 June: 7-10 days before harvest

Harpin protein (Messenger, Plant Health Care de Mexico, D.F.) dissolved in ultrapure water by reverse osmosis (RO). Tween-20 (0.1%) as a wetting agent was added at the rate of 40 cm/100 L water to the foliar solution in order to obtain best penetration results.

Mature apple fruits were picked when the red color reached over 50%, fruit firmness was about 11-12% according to Drake and Kupferman15. Fruits were picked at random in the early morning and packed in plastic boxes. The selected fruits were transported directly to the fruit handling laboratory and the defective fruits were almost equal in size and free from other pathogen injury.

For storage study, treated fruits were stored in perforated carton boxes in one layer about 5 kg (3 boxes for each replicate). All boxes were stored at 0°C±1 and 90-95% relative humidity (RH) for 90 days. Fruits were examined at initial time and after 30, 60 and 90 days during cold storage. Fruit quality assessment was recorded as described below for different chemical and physical properties.

Assessments of fruits at harvest
Total yield (kg1 tree): The mature fruits of each tree were weighted and then the average fruit yield per each tree was estimated.

Fruit nutrient contents: Samples of 10 fruits were taken from each tree for analysis nutrient. Content of Ca, P and K in the fruits were determined by atomic absorption spectrophotometry (AAS Shimadzu 7000 AA), as shown in the instructions specified in the ISO 11047 method, after they were extracted with nitrate (HNO3) and sulfuric acid (H2SO4) according to Evenhuis and Dewaard16.

Quality assessments of fruits during cold storage
Weight loss (%): It was determined according to the following equation:

Image for - Biodegradable Mulch Film Technology and Harpin Protein for Quality Enhancement of Anna Apples During Cold Storage

Fruit decay (%): It was determined according to the following equation:

Image for - Biodegradable Mulch Film Technology and Harpin Protein for Quality Enhancement of Anna Apples During Cold Storage

Respiration rate (mL CO2 kg1 h1): Respiration rate was measured by gas analyzer (Model 1450-Servomex 1400) according to Saquet et al.17, the airtight glass jars (4 L) were used to fruit incubation under the same storage circumstances for 24 h, respiration rate was measured as mL of CO2 kg1 fruits h1.

Electrolyte leakage (%): It was determined using the method described byLiu et al.18 with a slight modification. Twelve disks of the eight fruits were collected using a cork borer (8 mm diameter) and washed in distilled water three times. The disks were soaked in a glass tube containing 20 mL distilled water and incubated in a water bath shaker at 25°C for 2 h. The initial electric conductivity (C0) was measured using a conductivity meter. Then the glass tube was boiled for 30 min, cooled in room temperature and total electric conductivity (C1) was taken. Three biological replications were used for each treatment. Electrolyte leakage rate was calculated using the following equation:

Image for - Biodegradable Mulch Film Technology and Harpin Protein for Quality Enhancement of Anna Apples During Cold Storage

Fruit firmness (Ib inch2): It was determined on the two opposite sides of fruit using a hand Effegi- Penetrometers and the average was estimated as lb inch2 according to AOAC19.

Vitamin C (mg /100 mL juice): Vitamin C was measured by the oxidation of ascorbic acid with using 2, 6-dichlorophenolindophenol solution and 2% oxalic acid as a substrate then the results were indicated as mg ascorbic acid per 100 mL juice according to AOAC19.

Total soluble solids (TSS%): It was measured using drops of apple juice using a Carl-Zeiss hand refractometer according to AOAC19.

Titratable acidity (TA%): It was determined in fruit juice by titration with 0.1 N sodium hydroxide (NaOH), using phenolphthalein as an indicator and calculated as citric acid according to the method described in AOAC19.

Total sugars (100 μg mL1 of glucose): The total sugars were determined on the dried raw fruit of each treatment using 18% phenol and 96% sulphuric acid, the absorption was recorded with spectrophotometer at 490 nm, according to the method described by Sadasiyam and Manickam20.

A standard curve was prepared by plotting the known concentrations of glucose solution (100 μg mL1 of glucose) against respective optical density (OD) value of each. From the standard curve, the amount of total sugars actually present in the sample is determined.

Anthocyanin content (mg/100 g FW): The anthocyanin for the peel of the fruit was estimated by of pH differential method21 using a UV spectrophotometer. Absorption was measured at 533 and 700 nm in buffers with a pH of 1.0 and 4.5 with a molar extinction coefficient of 29.600. The results were expressed as mg cyanidin-3-glucoside equivalents per 100 g1 of fresh apple peel for triple extracts.

Total phenolic content (mg GAE 100/g FW): The total phenol content in the apple fruits was determined using the method of Slinkard and Singleton22 and the absorbance was measured at 760 nm. Results are expressed as milligrams of gallic acid equivalents (GAE)/100 g fresh weight (FW) using gallic acid as a standard peel for triplicate extracts.

Antioxidant activity (mg AEAC g1 FW): The antioxidant capacity with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was performed as reported by Brand-Williams et al.23 with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH).The absorbance at 517 nm was recorded to determine the concentration of the remaining DPPH. Ascorbic acid was used as a standard and DPPH radical scavenging activity were expressed in mg of ascorbic acid equivalents antioxidant activity (AEAC) per gram fresh weight for triplicate extracts.

Statistical analysis: Data of both seasons of the study were analyzed using analysis of variance (ANOVA). Differences among treatment means were statistically compared using Duncan’s multiple tests at a level 0.05, using the CoStat V6.4 program.

RESULTS

The effect of biodegradable (BMF) mulch films on soil properties and the harpin protein applications on storability of Anna apple fruits were discussed as follow during three successive seasons 2016, 2017 and 2018 (the first season was experimental).

Soil properties after treatment: Data in Table 3 showed that, soil organic matter was affected by biodegradable (BMF) mulch. Since, covering the soil with (BMF) mulch produced higher organic matter (2.50%) at the end of experiment. Moreover, Soil available nitrogen, phosphorus and potassium were enhanced by biodegradable (BMF) mulch than the control at the end of experiment, respectively.

Table 3: Evaluation of main properties of soil at the end of experiment after two seasons of study
Treatments Organic matter (%) Nitrogen (mg kg1) Phosphorus (mg kg1) Potassium (mg kg1)
Control 1.47 18.20 6.90 62.50
Biodegradable films (BMF) 2.50 24.50 11.50 96.20


Image for - Biodegradable Mulch Film Technology and Harpin Protein for Quality Enhancement of Anna Apples During Cold Storage
Fig. 1(a-d): (a) Total fruit calcium content, (b) Phosphorus content, (c) Potassium content and (d) Total yield (kg1 tree) in “Anna” apple at harvest during 2017and 2018 seasons

Fruit nutrient contents: Nutrient contents in fruits and their ratios varied between treatments during the two years as showed (Fig. 1a-c). The Ca, P and K content in fruits varied significantly between treatments, in the two years of assessment. In this respect, all biodegradable films (BMF) treatments improved ratios of nutrient contents in fruits than the other treatments under the study. The highest contents of calcium, phosphorus and potassium were found in fruits treated with harpin 40%+mulching soil with biodegradable films (BMF) during the both seasons, respectively.

While, control produced the lowest contents of calcium, phosphorus and potassium as compare to all treatments under the study during the both seasons, respectively.

Total yield (kg1 tree): Data in Fig. 1d obviously reveal that all treatments applied significantly improved fruit yield per tree rather than the control. Since, harpin at 40%+mulching soil with biodegradable films (BMF) were significantly very effective in enhancing fruit yield, which gave the highest values during both seasons, respectively. Untreated ones produced the minimum yield during both seasons, respectively.

Storability assessments
Weight loss of Anna apple fruits: Data in Table 4 presented the effect of the different conducted treatments on weight loss percentage of Anna apple fruits in 2017 and 2018 seasons. Weight loss percentage increased gradually after 90 days of cold storage. Moreover, all treatments showed lower significant weight loss percent compared with the control treatments which achieved the higher significant percent of weight loss (12.09 and 11.30%) after 90 days of cold in both seasons, respectively. On the other hand, spraying fruits with harpin 40%+mulching soil with biodegradable films (BMF) showed the lowest significant weight loss values (5.19 and 4.70%) during cold storage in the both seasons, respectively.

Decay of Anna apple fruits (%): From Table 4 data presented the influence of the different applied treatments on decay percentage of Anna apple fruits in both seasons. Decay percentage increased gradually with the prolongation of cold storage period. Moreover, all treatments showed lower significant percent of decayed fruits compared with the untreated. In this respect, spraying fruits with harpin 40%+mulching soil with biodegradable films (BMF) showed the lowest significant decay percentage (4.13 and 4.00%) after 90 days of cold storage compare with all treatments or the untreated ones in the both seasons, respectively.

Table 4: Weight loss (%) and decay (%) of Anna apple fruits after 90 days of cold storage in 2017 and 2018 seasons
Days in cold storage
Weight loss (%) Decay (%)
Treatments
Initial
30
60
90
Initial
30
60
90
Season 2017
Harpin 20%
0.00p
2.17l
4.93g
8.00b
0.00k
0.00k
3.19g
6.26c
Harpin 20%+BMF
0.00p
1.76n
3.10j
5.50e
0.00k
0.00k
3.04h
5.90d
Harpin 40%
0.00p
1.96m
3.20i
7.85c
0.00k
0.00k
2.08i
4.59e
Harpin 40%+BMF
0.00p
1.65o
2.89k
5.19f
0.00k
0.00k
1.79j
4.13f
Control
0.00p
4.73h
6.14d
12.09a
0.00k
5.91d
16.84b
27.94a
Season 2018
Harpin 20%
0.00h
2.15fgh
4.85cde
7.80b
0.00j
0.00j
3.02g
6.10c
Harpin 20%+BMF
0.00h
1.53gh
2.73efg
4.90cde
0.00j
0.00j
3.00g
5.85d
Harpin 40%
0.00h
5.13cde
3.08efg
7.22bc
0.00j
0.00j
2.05h
4.23e
Harpin 40%+BMF
0.00h
1.26gh
2.16fgh
4.70de
0.00j
0.00j
1.70i
4.00f
Control
0.00h
4.10def
5.65bcd
11.30a
0.00j
5.88d
14.43b
26.06a
Means followed by the same letters are not significantly different by Duncan multiple range test at 0.05 levels


Table 5: Respiration rate (mg CO2 kg-1 h-1) and electrolyte leakage (%) of Anna apple fruits after 90 days of cold storage in 2017 and 2018 seasons
Days in cold storage
Respiration rate (mg CO2 kg1 h1) Electrolyte leakage (%)
Treatments
Initial
30
60
90
Initial
30
60
90
Season 2017
Harpin 20%
13.30g
12.90j
13.79e
14.72b
9.50a
18.30l
24.53e
26.90c
Harpin 20%+BMF
13.30g
13.10hi
13.54f
14.67b
9.50a
18.00m
24.14g
26.66d
Harpin 40%
13.30g
13.13h
13.27g
14.22c
9.50a
17.72n
22.93i
24.304f
Harpin 40%+BMF
13.30g
13.10hi
13.21gh
14.03d
9.50a
17.70n
20.35j
23.40h
Control
13.30g
13.00ij
13.73e
16.84a
9.50a
18.75k
29.75b
32.97a
Season 2018
Harpin 20%
13.01f
12.26g
13.10f
14.31c
8.93p
17.93j
23.86c
21.89e
Harpin 20%+BMF
13.01f
12.19g
13.10f
14.24c
8.93p
17.23k
23.14d
21.21g
Harpin 40%
13.01f
12.21g
13.70e
13.80de
8.93p
16.30o
21.83f
19.82h
Harpin 40%+BMF
13.01f
12.20g
13.83d
13.72e
8.93p
16.53n
16.65m
18.57i
Control
13.01f
12.05h
14.82b
15.24a
8.93p
17.15l
26.61b
29.34a
Means followed by the same letters are not significantly different by Duncan multiple range test at 0.05 levels

Moreover, control showed the highest significant decay percentages (27.94 and 26.06%) after 90 days of cold storage period in both seasons, respectively.

Respiration rate (mg CO2 kg1 h1): As presented in Table 5, significant differences in respiration rates were recorded in response to storage periods and treatments investigated in this study. Regardless of storage period, all treatments in both seasons significantly inhibited respiration rate compared to control. Presented results showed that respiration rates were slowly declined after 30 days of cold storage and this declination was followed by consistently increase respiration rates for all the treatments till 90 days at the end of the investigated storage period.

The overall mean results of respiration rate showed that spraying fruits with harpin 40%+mulching soil with biodegradable films (BMF) recorded lower delay of respiration rate (14.03 and 13.72 mg CO2 kg1 h1) after 90 days of cold storage as compared with all treatments or the untreated ones in the both seasons, respectively. Moreover, control showed the highest significant of respiration rate (16.84 and 15.24 mg CO2 kg1 h1) after 90 days of cold storage in both seasons, respectively.

Electrolyte leakage (%): Another index of cell injury is ion leakage, which is a commonly used technique to assess cell damage or viability. Data fromTable 5showed clearly that, ion leakage increased during the progress of storage periods and showed higher values than initial.

Table 6: Fruit firmness (lb inch-2) and vitamin C (mg/100 mL juice) of Anna apple fruits after 90 days of cold storage in 2017and 2018 seasons
Days in cold storage
Firmness (lb inch2) Vitamin C (mg/100 mL juice)
Treatments
Initial
30
60
90
Initial
30
60
90
Season 2017
Harpin 20%
11.34a
10.82c
9.23h
8.88j
8.91a
8.39c
7.48f
6.50i
Harpin 20%+BMF
11.34a
10.63d
9.00i
8.85j
8.91a
8.37c
7.50f
6.50i
Harpin 40%
11.34a
11.02b
9.90e
9.48g
8.91a
8.51b
7.81e
6.90h
Harpin 40%+BMF
11.34a
11.04b
9.92e
9.58f
8.91a
8.58b
7.89e
6.91h
Control
11.34a
10.00e
8.90j
7.13k
8.91a
8.18d
7.14g
6.05j
Season 2018
Harpin 20%
12.00a
10.93e
9.69j
8.82m
9.16a
8.86cd
7.98h
6.90l
Harpin 20%+BMF
12.00a
11.10d
9.84i
9.00l
9.16a
8.81d
7.98h
6.90l
Harpin 40%
12.00a
11.50c
10.14g
9.94h
9.16a
8.98b
8.14g
7.23k
Harpin 40%+BMF
12.00a
11.64b
10.31f
10.01h
9.16a
8.90c
8.32f
7.49j
Control
12.00a
10.10g
9.18k
7.94n
9.16a
8.49e
7.71i
6.63m
Means followed by the same letters are not significantly different by Duncan multiple range test at 0.05 levels

All applied treatments produced less percent of electrolyte leakage as compare to the control. In this respect, the untreated apples had the highest electrolyte leakage percentage (32.97 and 29.34%) after 90 days of cold storage in both seasons, respectively. While, harpin 40%+mulching soil with biodegradable films (BMF) presented the lowest percent of electrolyte leakage (23.40 and 18.57%) than all treatments after 90 days of cold storage in both seasons, respectively.

Firmness (lb inch2): Data from Table 6 showed clearly that, fruit firmness was reduced as storage period advanced during the both seasons. It also confirmed that, all treatments used significantly reduced the decline in fruit firmness than the control through the two seasons. In this respect, fruit firmness for the control treatment showed a lower fruit firmness (7.13 and 7.94) lb inch2after 90 days of cold storage in both seasons, respectively. Furthermore, treated fruits with harpin 40%+mulching soil with biodegradable films (BMF) presented a higher fruit firmness (9.58 and 10.01 lb inch2) after 90 days of cold storage in the two seasons, respectively.

Vitamin C (mg 100 mL1 juice): The amount of ascorbic acid decreased continuously with storage time as showed in Table 6. Moreover, all treatments used significantly delayed the decrease of vitamin C than the control after 90 days of cold storage. The contents of vitamin C in control treatments declined to (6.05 and 6.63 mg/100 mL juice) after 90 days at cold storage during both seasons, respectively. The higher amounts of vitamin C contents were obtained with harpin 40%+mulching soil with biodegradable films (BMF) during the entire storage period. The contents of vitamin C in this treatment were (6.91 and 7.49 mg/100 mL juice) after 90 days of cold storage during both seasons, respectively.

Total soluble solid (TSS %): From Table 7 data presented that, TSS percentage of Anna apple fruits was significantly increased in all treatments applied with the progress of cold storage in both seasons. Moreover, all applied treatments showed decrement in TSS percentage as compare to control during cold storage in both seasons, respectively. Since, the higher significant TSS percent were obtained at control treatment (16.51 and 16.06%) after 90 days of cold storage in both seasons, respectively.

While, harpin 40%+mulching soil with biodegradable films (BMF) presented the lower significant TSS (15.75 and 15.09%) in both seasons, respectively.

Titratable acidity (%): Results in Table 7 showed that TA (%) of Anna apple fruits was significantly reduced in all treatments applied with the progress of cold storage in both seasons. Moreover, all applied treatments showed somewhat increment in titratable acidity as compare to control throughout this study after 90 days of cold storage in both seasons. In this respect, harpin 40%+mulching soil with biodegradable films (BMF) led to significant increment in TA percentage (0.49 and 0.54%) after 90 days of cold storage in the two seasons, respectively. While, control treatment gave lower significant acidity (0.46 and 0.49%) after 90 days of cold storage in the two seasons, respectively.

Anthocyanins (mg/100 g FW): In the study, statistical analyses showed that total anthocyanin content of Anna apple fruits were significantly reduced during 90 days of cold storage (Table 8). All treatments applied maintain the content of anthocyanin in fruits skin than the control under cold storage.

Table 7: TSS (%) and titratable acidity (%) of Anna apple fruits after 90 days of cold storage in 2017 and 2018 seasons
Days in cold storage
TSS (%) Titratable acidity (%)
Treatments
Initial
30
60
90
Initial
30
60
90
Season 2017
Harpin 20%
13.41k
14.44j
15.18f
15.92b
0.73a
0.69c
0.57f
0.48k
Harpin 20%+BMF
13.41k
14.41j
15.20f
15.80cd
0.73a
0.69c
0.58e
0.48k
Harpin 40%
13.41k
14.51i
15.05g
15.82c
0.73a
0.70b
0.52i
0.49j
Harpin 40%+BMF
13.41k
14.54i
15.02g
15.75d
0.73a
0.70b
0.56g
0.49j
Control
13.41k
14.62h
15.35e
16.51a
0.73a
0.67d
0.53h
0.46l
Season 2018
Harpin 20%
13.04h
13.90g
14.70d
15.53b
0.77a
0.69cd
0.61g
0.50k
Harpin 20%+BMF
13.04h
13.84g
14.60d
15.50b
0.77a
0.70c
0.61g
0.50k
Harpin 40%
13.04h
13.88g
14.10ef
15.15c
0.77a
0.71b
0.64f
0.53j
Harpin 40%+BMF
13.04h
13.90g
14.14e
15.09c
0.77a
0.72b
0.64e
0.54i
Control
13.04h
14.02f
15.50b
16.06a
0.77a
0.69d
0.58h
0.49l
Means followed by the same letters are not significantly different by Duncan multiple range test at 0.05 levels


Table 8: Anthocyanin (mg/100 mL juice) and total sugar (%) of Anna apple fruits after 90 days of cold storage in 2017 and 2018 seasons
Days in cold storage
Anthocyanin (mg/100 mL juice) Total sugar (%)
Treatments
Initial
30
60
90
Initial
30
60
90
Season 2017
Harpin 20%
18.53a
18.09b
17.19fg
16.80i
14.16f
14.01g
14.41cd
14.61b
Harpin 20%+BMF
18.53a
18.52a
17.52e
17.04h
14.16f
14.06fg
14.34de
14.50c
Harpin 40%
18.53a
18.05b
17.23f
16.84i
14.16f
14.04g
14.45c
14.61b
Harpin 40%+BMF
18.53a
18.12b
17.78d
17.14g
14.16f
14.05fg
14.30e
14.49c
Control
18.53a
17.89c
17.05h
16.24j
14.16f
14.00g
14.49c
14.90a
Season 2018
Harpin 20%
18.89a
18.30c
18.00d
17.01g
14.68b
14.24cd
14.69b
14.90ab
Harpin 20%+BMF
18.89a
18.49b
18.08d
17.20f
14.68b
13.99d
14.62b
14.84ab
Harpin 40%
18.89a
18.24c
18.01d
17.02g
14.68b
14.25cd
14.66b
14.88ab
Harpin 40%+BMF
18.89a
18.53b
18.07d
17.30e
14.68b
14.36c
14.62b
14.80ab
Control
18.89a
18.00d
17.09g
16.48h
14.68b
14.21cd
14.87ab
15.00a
Means followed by the same letters are not significantly different by Duncan multiple range test at 0.05 levels

The results showed that, the treatment of harpin 40%+mulching soil with biodegradable films (BMF) maintained anthocyanin in fruit skin (17.14 and 17.30 mg 100 g1 FW) after 90 days under cold storage comparison with other treatments used during both seasons. However, control treatment presented lower values of anthocyanin in fruit skin (16.24 and 16.48 mg 100 g1 FW) after 90 days of cold storage through the both seasons under the study.

Total sugars (%): Considering to the effect of total sugar, data in Table 8 revealed that, total sugars were increased gradually according to the progress of cold storage during both seasons. Since, fruits of control samples had significantly the highest level of total sugars values after 90 days of cold storage (14.90 and 15.0%) in the first and second seasons, respectively. Conversely, harpin 40%+mulching soil with biodegradable films (BMF) presented the lower significant sugar percent (14.49 and14.80%) after 90 days of cold storage under the two seasons, respectively.

Total phenols (mg GAE/100 g FW): Results presented in Table 9 showed that total phenols were increased gradually according to the progress of cold storage during both seasons. Since, fruits of control samples presented significantly the lowest values in total phenol content of Anna apple fruits compared with all treatments used after 90 days of cold storage (103.33 and 112.00 mg eq. gallic acid 100 g1) in both seasons, respectively.

The higher values of total phenol were obtained by sprinkle harpin 40%+mulching soil with biodegradable films (BMF) ranged (109.66 and 115.0 mg eq. gallic acid 100 g1) after 90 days of cold storage in both seasons, respectively.

Antioxidant activity (mg AEAC g1 FW): Results presented in Table 9 showed that all investigated treatments increment antioxidant activity compared to the control after 90 days of cold storage during both seasons.

Table 9: Total phenols (mg eq. gallic acid 100 g-1) and antioxidant activity (μg mL-1) juice of Anna apple fruits after 90 days of cold storage in 2017 and 2018 seasons
Days in cold storage
Total phenols (mg eq. gallic acid/100 g) Antioxidant activity (mg AEAC g1 FW)
Treatments
Initial
30
60
90
Initial
30
60
90
Season 2017
Harpin 20%
99.00de
99.00e
102.00cde
105.33abcd
0.55ab
0.52bc
0.50bcd
0.48cd
Harpin 20%+BMF
99.00de
101.33cde
101.66cde
106.33abc
0.55ab
0.51bc
0.53abc
0.54abc
Harpin 40%
99.00de
102.33cde
102.00cde
108.33ab
0.55ab
50.00bcd
0.49cd
0.49cd
Harpin 40%+BMF
99.00de
103.33bcde
103.00bcde
109.66a
0.55ab
0.50bcd
0.54abc
0.58a
Control
99.00de
100.66cde
103.00bcde
103.33bcde
0.55ab
0.54abc
0.51bc
0.45d
Season 2018
Harpin 20%
105.33cd
105.66cd
107.33bcd
112.33abc
0.59abc
0.55cd
0.55cd
0.60abc
Harpin 20%+BMF
105.33cd
108.33abcd
108.00abcd
113.00ab
0.59abc
0.57bc
0.59abc
0.63ab
Harpin 40%
105.33cd
108.66abcd
110.33abcd
114.00ab
0.59abc
0.58abc
0.58abc
0.60abc
Harpin 40%+BMF
105.33cd
107.33bcd
109.66abcd
115.00a
0.59abc
0.58abc
0.58abc
0.64a
Control
105.33cd
109.00abcd
110.66abcd
112.00abcd
0.59abc
0.51d
0.50d
0.57bc
Means followed by the same letters are not significantly different by Duncan multiple range test at 0.05 levels

It also showed that harpin 40%+mulching soil with biodegradable films (BMF) resulted significant increment in antioxidant activity compared with all treatments used or the control after 90 days of cold storage (0.58 and 0.64 mg AEAC g1 FW) in both seasons, respectively. Moreover, control treatment attained the minimum significant values of antioxidant activity in apple fruits compared with all treatments used after 90 days of cold storage (0.45 and 0.57 mg AEAC g1 FW) in both seasons, respectively.

DISCUSSION

In this study, covering the soil with biodegradable (BMF) mulch enhanced its organic matter. Moreover, Soil available Nitrogen, phosphorus and potassium were improved by biodegradable (BMF) mulch at the end of experiment. Mulches of organic origin typically enter a relationship with the soil, increasing the activity of the enzymes which break down plant residues. Mulches developed from biomass are increasingly used to cover the soil around the plants which improve soil properties involved the organic matter, available phosphorus and potassium. The organic matter improves environmental conditions for soil via, preventing of water and wind erosion, inhibiting drastic variation in humidity and temperature24. A higher amount of available phosphorus in the soil in mulched plots was determined by Sinkeviciene et al.25. Organic mulches probably have much more potassium in their structure and regulation of soil temperature and moisture which helped increase soil available potassium26.

Biodegradable plastic mulches (BDMs) have been developed as substitutes to PE mulch films and are designed to be tilled into soil after use where resident microorganisms degrade the plastic. Biodegradable mulch 0.35 mm thick with a surface weight of 40 g m2, consisting of 16% corn starch and 84% natural fibre, allowed about 15% of radiation in the range 450-850 mm to pass, exhibited very good water permeability and thermal stability under field conditions. Its degradation rate in soil at 10-25°C and 65% humidity was the highest compared to mulches based on natural polyvinyl alcohol and polyacrylate27. Physical properties of the inorganic mulch depend on the type of polymer and the manufacturing process, as well as the additives during production (stabilizers, stimulants, conveyors, dyes and fillers)28. Since, mulching soil with biodegradable films (BMF) with sprinkle harpin at 40% were significantly very effective in enhancing fruit yield, which gave the highest values during both seasons, respectively. Maintaining high moisture content of the soil during the cucurbit vegetation period allows advancement and prolongation of the cropping season, leading to an increase in yield29. All biodegradable films (BMF) treatments improved ratios of nutrient contents in fruits than the other treatments under the study. The fruits showed a decreased dry matter content, with a similar amount of soluble sugars compared to the fruit from the control site30.

In this study, harpin at 40% and mulching soil with biodegradable films (BMF) presented the lowest percent of electrolyte leakage after 90 days of cold storage. Harpin is a protein isolated from Erwinia amylovora bacteria which cause fire blight on fruit trees. It is thought that this protein has several cellular impacts on various processes or factors such as plant’s resistance31activation of reactive oxygen types32 transportation of effector proteins in plant cytoplasm33 and depolarization of cell membrane. The product commercially called Messenger Gold which consists of 1% harpin and stimulates growth is generally used on fruits in order to increase the resistance against diseases. Previous studies have shown that postharvest treatment with harpin reduced disease in many harvested crops, such as apple11. Spraying fruits by harpin 40% and mulching soil with biodegradable films (BMF) showed the lowest significant decay percentage and recorded lower delay of respiration rate after 90 days of cold storage. The treatment with harpin on pear fruits can induce activities of POD, PAL, 4CL and GLU and content of TPC, all of which play important roles in disease resistance12. The lignin accumulations were enhanced by harpin treatment irrespective of the side considered. Lignin is extremely resistant to microbial degradation and thus constitutes one of the most effective barriers against pathogenic invasion34. The results showed that, the treatment of harpin at 40% with mulching soil by biodegradable films (BMF) presented a higher fruit firmness in fruit skin and maintained anthocyanin after 90 days of cold storage. The harpin treatment induced lignification of cell walls, promoting the combination of lignin with the cell wall and consequently enhancing protection of the cell wall35. The treatment of harpin spraying four times could reduce postharvest disease in fruits. The control for postharvest disease was clearly correlated with the activation of defense enzymes and metabolites as well as enhancement of lignin accumulation. In this study, harpin at 40% with mulching soil by biodegradable films (BMF) presented the lower significant TSS percentage while, showed somewhat increment in titratable acidity as compare to control treatment after cold storage. Conversely, harpin at 40% with mulching soil by biodegradable films (BMF) presented the lower significant sugar percent while, showed higher values of total phenol and significant increment in antioxidant activity.

Harpin are affected slightly fruit quality by enhancing vitamin C content and the ratio between soluble solids and titratable acidity. Harpin treatment increased flesh firmness and vitamin C content of fruit 10 days after storage. However, it significantly decreased titratable acidity (TA) content of fruit after storage. Moreover, it enhanced the TSS/TA ratio of fruit after storage. Therefore, scores of tastes in fruit treated with harpin were improved compared with control36.

Harpin protein, originally isolated fromErwinia amylovora, is one of the plant activators that have become common in biological agriculture practices. When applied, harpin protein binds to plant receptors that activate several biochemical pathways related to growth and resistance enhancement by way of a systemic acquired resistance pathway (SAR)37. It is reported that an improved accumulation of phenolics may be one of the mechanisms that help control pathogen growth38. Harpin has been reported to stimulate the activity of phenylalanine ammonia lyase enzyme, a key to the biosynthesis of polyphenolic compounds such as gallic, caffeic, chlorogenic and some other acids39. The accumulation of phenolic compounds is associated with a hypersensitive response stimulated by genes involved in the synthesis of secondary metabolites such as phenolic compounds that may have antimicrobial activity. Phenylpropanoid metabolism has been reported to be involved in fruit-pathogen interactions40. The biosynthesis and accumulation of anthocyanins in the skin cells start from ripening to harvest and are mainly subject to genetic control however, climatic conditions and cultural practices, including the use of external plant growth regulators, often influence gene expression and activation of the biosynthetic enzymes.

Harpin application on grapes during the growing period, they trigger the expression of hundreds of genes related to the plant disease resistance, among which those involved in the biosynthesis of bioactive compounds such as anthocyanins41. Applying harpin protein (HrP) to grapes improved berry skin color by stimulating the biosynthesis of anthocyanins and enhancing their content in grape skin.

Harpin has been reported to stimulate the activity of the enzyme phenylalanine ammonia lyase, which is the key to the biosynthesis of polyphenols41.

High correlations between phenolic content and antioxidant capacity (DPPH and FRAP) so, phenolic compounds may be the most important phytochemicals responsible of induce accumulation antioxidant capacity38.

CONCLUSION

The biodegradable mulch film technology appears to be a good solution to replace the traditional polyethylene mulch and get a high-quality organic fruit. Sprinkle harpin protein 40% with mulching soil with biodegradable films (BMF) led to obtain high quality of organic fruits with the desired coloration and enhance systemic resistance of fruits during storability.

SIGNIFICANCE STATEMENT

This study discovered the possible effect of the soil biodegradable mulch films (BMF) and sprinkle fruits by harpin protein that can be beneficial to produce organic fruits, extend color and enhance systemic resistance of fruits during storability of Anna apples. This study will help the researchers to uncover the critical areas of maintaining quality of organic fruits and enhance systemic resistance accompanied by changes in various metabolic and physiological processes during storability that many researchers were not able to explore. Thus, a new theory of soil (BMF) with harpin protein are promising examples that are beginning to be adopted on a commercial scale which may be arrived at retain quality of apple fruits during storability.

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