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The influence of different applications of vermicompost tea on the quality of pomegranate fruits

This investigation is clarifying the effect of vermicompost tea by foliar application or by adding it to the soil, either separately or in combination form, on the physical and chemical properties of pomegranate (cv. Manfalouty) fruits during two successive seasons, 2019 and 2020. Trees were treated with the vermicompost tea application by spraying fruits and leaves or adding them to the soil at a distance of 50 cm from the trunk of the tree, or a combination thereof, three times a year; the first one in the middle of July (after fruit set), the second after a month of the first application, and the last one after a month of the second. The results demonstrated that all treatments achieved the increment and improvement of all parameters compared with the control, while the treatment that included a combination of foliar application and adding to the soil gave the best effectiveness compared with the other treatments in the two successive seasons. It was also noted that the physical and chemical properties of fruits were better in the second season compared with the first, which was due to the accumulation of the effect of vermicompost in the soil. So the recommendation of this study is to use vermicompost either spray the trees or add to the soil. Both had a positive impact on fruits, while the best effect is to use a combination of both.


Introduction
The pomegranate (Punica granatum) is one of the most important and oldest fruit crops, having been grown since antiquity. Trees thrive in a variety of climates, including tropical and subtropical locations, as well as the Mediterranean region, particularly in Morocco, Spain, Egypt, and California's arid districts. Egypt is in the northern hemisphere's temperate zone, close to the equator, with arid or semi-arid climates suited for pomegranate production (Mirzapour and Khoshgoftar Manesh, 2013). Pomegranate belongs to the family (Punicaceae). It includes many cultivars such as Manfaloty, El-Hameed, Panaty, El-Sokry, Hejazi, Nab-El-Gamal, and Wonderful. Manfalouty is one of the most famous commercial cultivars cultivated in Egypt. It grows in various soil types, from light to sandy soils, if orchards are well-irrigated, except for alkaline soils, calcareous, or saline (Kitren and Louise, 2011). Recently, pomegranate has become more important to researchers who engage themselves in neurological, pharmacological, and pharmaceutical research and new drug development for their various effects and multiple bioactivities, such as antioxidants, antivirals, hypolipidemic, antibacterial, anti-diarrheal, anti-neoplastic, helminthic effects, and anti-diabetics. Pomegranate fruit is considered an abundant source of sugars and minerals, as well as some phenolic compounds that act as natural antioxidants and vitamin C (Zaouaya et al., 2012). The pomegranate tree's fruits, bark, and leaves have long been used as traditional medicine in various cultures, dating back at least to the Egyptian Papyrus of Ebers, circa 50 B.C. (Hulya Orak, 2009). The objective of fertilizing by organic or chemical fertilizers is to enhance tree vegetative and root growth and, therefore, boost the yield as long as the fertilizer is applied as a balanced fertilizer in the proper amount at the right time and from reputable sources. (Tanari et al., 2019). Chemical fertilizers have become increasingly popular in recent years despite their harmful effects on the environment, human and plant health, and high costs, because they have a good effect on world food production because they work as fast nutrition for plants, causing them to grow more quickly and efficiently. Because of the passive effect of chemical fertilizers, nowadays a major target is focused on minimizing the intensive amounts of mineral fertilizers (Abd Al-Hadi and Abd AL-Adeem, 2005).
Organic agriculture is a method that avoids or minimizes the use of synthetic fertilizers, growth regulators, livestock feed additives, and pesticides. Bio-fertilization, animal manures, crop residues, crop rotations, green manures, legumes, mineral-bearing rocks, off-farm organic wastes, mechanical cultivation, and components of biological pest management are used as much as possible in organic agricultural systems to retain soil production. The fundamental issue in the organic production system, however, is yield decline (Lind et al., 2003). The influence of organic manure application is noticeable to a great extent on both the nutrient sources for plants and the physical and chemical properties of the soil in the long term (EL-Kouny et al., 2004;Abbas et al., 2006;Tirol-Padre et al., 2007). Some researchers demonstrated that combining sources both organic and chemical, often known as integrated nutrient management, is a method of raising agricultural yields and productivity in a sustainable manner (Mahajan et al., 2008). Although organic manure combined with mineral fertilizers boosted crop development and yield in the short term, organic manure alone improved the fruit quality, nutritional content, and yield of date and grape. (Marzouk and Kassem, 2011).
Recently, researchers worldwide have been focusing on studying vermicompost applications and their influences on plants and soil (Adhikary, 2012). Edwards and Burrows (1988) described vermicompost as a peat-like substance resulting from a non-thermophilic process that includes bio-degradation and stability of organic materials through interactions between microorganisms and earthworms. It's rich in macro and micronutrients, growth hormones, enzymes including proteases, amylases, lipase, cellulose, and chitinase, immobilized microflora, and vitamins, and it's made by earthworms. Even after the enzymes have been removed from the worms, they continue to degrade organic material (Barik et al., 2011).
It is characterized by its water-holding capacity, drainage, high porosity, aeration, and microbial activity. It's also possible to enhance soil organic carbon, exchangeable calcium, phosphates, nitrates, and a variety of other nutrients for plants (Orozco et al., 1996;Garcia-Gil et al., 2000;Bulluck et al., 2002;Jindo et al., 2016). Vermicompost has been shown to have a positive impact on multi kinds of crops, such as flowering plants, ornamentals, legumes and cereals, field crops, and vegetables (Chan and Griffiths, 1988;Edwards and Burrows, 1988;Mba, 1996;Subler et al., 1998;Atiyeh et al., 2000). Vermicompost contains many nutrient substances containing N, P, K, Fe, Mg, Ca, Mn, Zn, S, B, and Cu. It improves plant nutrition as well as the amount of chlorophyll in the leaves, photosynthesis, and the nutrient content of various plant components (roots, shoots, and fruits). Vermicompost contains a sufficient amount of humic acid for plant health since it promotes the synthesis of phenolic compounds like flavonoids and anthocyanins, which may improve plant quality and function as a disease and insect preventative. (Theunissen et al., 2010). This research aims to study the effect of vermicompost tea by both foliar applications and adding it to the soil alone form, or in combination form, to improve the physical and chemical properties of pomegranate (Punica granatum Manfalouty) and for reducing the use of chemical fertilizers and replacing them with organic fertilizers resulting from plant and animal waste to avoid all of the following: Longterm damage caused by chemical fertilizers to the plant and soil -Reducing the cost of fertilizing agricultural fields as chemical fertilizers are expensive -Providing consumer food safety.

Materials and Methods
The experiment was carried out over two continuous growing seasons (2019 and 2020) to study the effect of vermicompost application on pomegranate trees of the Manfalouty cultivar, which was cultivated at the Pomology Department, Faculty of Agriculture, Assiut University. 25 pomegranate trees 15 years old were selected to be as uniform as possible, spaced at 5x5 m apart, and grown in clay soil. The tested trees were healthy, as uniform in vigor as possible, and divided for this study. Irrigation, pest management, and other conventional agricultural methods were applied to all of the trees, and fertilizer (FYM @ 20 kg/tree) was supplied to all the trees (including control) in December. Before beginning the study, both the tested soil and the vermicompost tea used in the experiment were analyzed. Tables 1 and 2 areThe trees selected were treated with the following applications three times a year on the same tree. The first one is in the middle of July (after the fruit set), the second after the month of the first application, and the last one after the month of the second.
Soil supplement of vermicompost (one liter of vermicompost for every 5 liters of water/tree).
Combination between spray and soil supplement treatment/tree. Each treatment sprayed five trees and was taken three replicates only for each treatment, and two fruits from each tree were harvested.

Prepare vermicompost tea
10 liters of water were added for each kilogram and a half of vermicompost and placed in a water-permeable cloth bag. 25 grams of molasses -5 grams of magnesium sulfate -1 gram of monopotassium phosphate -citric acids for reaching pH 6-10 grams of humic acidventilate for 48 hours, then take it from the solution and use it directly.

Yield weight
When fruits reached the ripening stage in each season (approximately in the middle of October), they were harvested separately from each tree, and their weight was calculated by kilogram per tree.

The percentages of fruit cracking, fruit sunburn, and marketable fruit
The number of fruits cracked (FCP), sunburned (FBP), and marketable fruit (MFP) was determined for each treatment at the time of harvest as follows: - TNF *NBF is the number of sunburned fruits. *TNF is the total number of fruits. *NCF is the number of cracked fruits.

Leaf constituents
At harvest date, leaves were randomly collected from the middle part of non-fruiting shoots of each replicate tree in both seasons to determine total chlorophyll and N.P.K content. Total chlorophyll content was determined with a SPAD-502-meter (Minolta Camera Co., Osaka, Japan). N.P.K Contents: Leaf samples were first cleaned with tap water, then distilled water and non-ionic detergent, before being dried in an air oven at 70°C and manually ground with a mortar and pestle. One gram of powder was burned at 550°C for 25 minutes in a muffle oven. The resulting white ash was then dissolved in 10 ml of 2 N HCl and distilled water adjusted to a volume of 100 ml for macro-and micronutrient analysis (Chapman and Pratt, 1961). A flame photometer was used to determine potassium content, and a spectrophotometer was used to determine phosphorous content. The Kjeldahl technique was used to determine total nitrogen content (Olsen et al.,1954 andJackson, 1973).

Fruits properties
Two fruits were taken randomly from the yield of each tree (3 replicates) and made as a composite sample for physical and chemical determinations.

Physical properties:
The weight of each fruit, arils and peel of fruit were determined in grams by using an analytical balance. Juice volume of arils was determined in ml by the Graduated cylinder.

Total anthocyanins
The determination of the total anthocyanin was realized by the method proposed by Di Stefano et al.,1989. The absorbance was measured at 540 nm after the samples were diluted with a solution of 70/30/1 (v/v/v) ethanol/water/HCl (concentrated). The overall anthocyanin contents were represented as malvidin-3-glucoside equivalents due to the lack of a malvidin-3glucoside standard. TA540 nm (mg/L) = A540 nm16.7d *Where A540 nm is the absorbance at 540 nm and d is the dilution.

Statistical analysis
The experiment was set up as a three-replication randomized full blocks design. SPSS software was used to do an analysis of variance (ANOVA). The LSD test was used to determine to mean differences. (least significant difference) at P < 5% based on Gomez and Gomez (1984) in Statistics 8.1 (Analytical Software, 2008).

Leaf constituents, including total chlorophyll, N, P, and K content
The results in Table 3 demonstrated that all treatments had a positive effect on the total chlorophyll, N, P, and K percentages in leaves and gave significant differences compared with the control in both seasons. The values of total chlorophyll had insignificant differences between treatments in both seasons. The highest value of chlorophyll was recorded in leaves that were sprayed with vermicompost (65.97, 66.97) and the lowest value was recorded in control (56.03, 56.03) in both seasons, respectively. There were no significant differences between T 2 and T 4 values on N% of leaves compared with T 3 values in both investigative seasons, respectively. The best values of N % were found in T 2 (1.95 & 2.02%) and T 4 (1.98, 2.03%), while the least value was found in T 1 (1.55, 1.58%). There were minor differences between T 2 with both T 3 and T 4 in the percentage of P of leaves. While there were significant differences between T 3 and T 4 . The maximum effect was recorded on T 4 (0.65, 0.69 %) and the minimum value was recorded on T 3 (0.57;0.58 %). The control one gave the lowest value (0.31, 0.34%) in both seasons, respectively. In the first season, there was no difference among treatments on the percentage of K of leaves whereas in the second season there were significant differences between T 4 and T 2 compared with T 3. T 4 gave the highest value (1.75, 1.78%) and the control treatment gave the lowest value (1.29, 1.33%) in the two successive seasons.

Physical properties: fruit weight, peel weight, arils' weight, juice volume, juice weight.
The results in Table 4 described, that all treatments increased the fruit weight compared with the control one by significant differences.
In the first season, there was an insignificant difference between T 2 and T 4 (535, 541.7 gm), respectively, compared with T 3 , which had a lower value (503 gm). whereas, in the second season, T 4 (574.2 gm) had the highest value  Table 4 illustrated that all treatments had a significant effect on arils' weight compared with the control. In the first season, the highest value was found in T 2 (236.7 gm), while in the second season, T 4 had the highest one (265.8 gm). In both seasons, the lowest value was in the control (135 and 150 gm, respectively). In the same table, results proved that all treatments had a positive effect on the juice volume and juice weight and there was an insignificant difference between them and there were significant differences between all treatments with the control in two investigated seasons, respectively. T 4 recorded the highest amount of juice (64, 76.2 ml) and the control recorded the lowest amount (40,45.1 ml) in both seasons, respectively. In the same way, T 4 had the highest value in juice weight (66.82,77.8 g) compared with the control had the lowest value (41.7, 47 g).  Table 5 had results illustrating the effect of treatments on yield properties, and they gave a positive effect compared with the control in two investigative seasons. The results in this table showed the effect of treatment on fruit cracking %. In the first season, data proved, there were significant differences between T 4 with both T 3 and the control, and there was a trivial difference between T 2 and T 4 . the lowest rate was found in T 4 (2.90%), while, the highest rate was found in the control (7.73 %). In the second season, there were significant differences between treatments and control and there were significant differences between T 4 and both T 2 and T 3 . The lowest rate was found in T 4 (3.23%) and the highest rate was found in the control (7.70 %). Regarding fruit sunburn % the data proved that there were significant differences between all treatments and the control. The minimum rate was found in both T 2 (7.60,7.00 %) and T 4 (5.77, 5.50 %) with insignificant differences between them and control, which gave the maximum rate (15.93, 16.93%) in two successive seasons, respectively. About the marketable fruit % in the same table data demonstrated that, in the first season, there was no difference between T 2 (88.73%), and T 4 (91%); they gave the highest rate of marketable fruit compared with the control (76.27 %) and T 3 (80.13 %), which gave the lowest rate. Moreover, in the second season, there were significant differences between treatments and the control. T4 recorded the maximum (92.67 %) value compared with the other treatments and the control, which gave the lowest value (73.27%). Results in this table proved that all treatments gave significant differences in yield compared with the control in each season, respectively. The high rate was recorded in T 4 (114.67, 113.50 kg) and the low rate was obtained in T 1 (53.33, 53.33kg) in both successive seasons.  Table 6 explained the effect of treatments on the chemical properties of aril's juice and compared them with the control during two successive seasons. Data reported that there weren't any differences between treatments and the control in the first season, and they had the same effect on TSS. In the second season, there were trivial differences in value between treatments, including the control, but there was a significant difference between T 1 and T 3. The best value of TSS was found in T 1 (14%) while the lowest value was found in T 3 (13.2%). Data illustrated the treatments gave significant values of acidity percentage compared with the control in the first season. The highest percentage was obtained with the control (1.07 %), and the lowest percentage was found in all treatments (0.99 %). On the other hand, there was no difference between treatments and the control in the second season, and all treatments had the same effect on acidity% as the control.

Chemical properties: TSS %, Acidity%, TSS/acidity, Total anthocyanin. The results in
Furthermore, the value found in T 1 (12.8%) in the first season had the lowest effect on TSS/acid ratio compared with the other treatments, while in the second season there was no difference in effect between the control and treatments on TSS/acid ratio. In the same table, the results demonstrated that T 2 & T 3 had the maximum effect on anthocyanin in two seasons. In the first season, T 4 (0.056 mg/100gm) gave the minimum value of anthocyanin compared with the other treatments, and the control. Whereas in the second season T 1 &T 4 (0.06,0.061mg/100gm) gave the minimum values of anthocyanin compared with other treatments T 2 and T 3 (0.09, 0.098mg/100gm).

Discussion
When compared to the control, all treatments had a considerable impact on the physical and chemical attributes of the fruit. It's most likely because of the hormones or humate concentration in the vermicompost, as well as a variety of plant nutrients. It was also noted that the physical and chemical properties were better in the second season compared with the first, which was due to the accumulation of the effect of vermicompost in the soil (Baligar et al., 2001;Arancon et al., 2003;Roy et al., 2006). Earthworm excrement is rich in micronutrients, macronutrients, and humus, and is known as vermicompost. It can improve the soil's physical qualities, as well as the health of the soil and crop productivity (Azarmi et al., 2008;Adhikary, 2012). The major components that characterize the agronomical potential of soil are its physical features, such as tilth, texture, and soil structure. Water availability, nutrient uptake and movement, root penetrability, potential rooting volume, and soil aeration will all be influenced by these characteristics (Roy et al., 2006). Soil structure has an impact on soil moisture content as well as chemical qualities like cation exchange capacity (CEC), or the soil's ability to store positively charged ions (Delgado and Gómez, 2016). Therefore, vermicompost is very beneficial for soil, as increase the soil organic substance (SOM) composition, which in turn helps in maintaining good soil aggregation, improving soil aeration, increasing nutrient availability, and protecting against soil erosion (Baligar et al., 2001;Roy et al., 2006). In addition, vermicompost contains plant nutrients like N, P, K, Fe, Ca, Mg, S, B, Cu, Zn, and Mn, which contribute to the nutritious content of various plant components like roots, shoots, and fruits (Theunissen et al., 2010). The living microorganisms found in vermicompost tea encourage both nutrient uptake, plant growth, and disease resistance (Scheuerell and Mahaffee, 2002;Ingham, 2005;Hargreaves et al., 2009). So, the application of a watery extract of vermicompost (vermicompost tea) has been proven to improve crop yield, plant health, and nutritive components in the plant (Gamaley et al., 2001;Pant et al., 2009). Vermicompost can also promote the establishment of nitrogen-fixing bacteria in the rhizosphere, which increases N availability by releasing biologically fixed nitrogen through close contact between ingested particles and soil. They discovered that after applying the vermicompost, the soil NH4 and NO3 levels improved instantly (Mackay et al., 1982;Singh and Varshney, 2013). The application of vermicompost had the same effect as the application of inorganic fertilizers, indicating that vermicompost is a viable alternative to chemical fertilizer application (Singh et al., 2008).  Arancon et al. (2003) proved that the enhancements in crop growth and fruit yields could be attributable to a partially considerable increase in soil microbial biomass following vermicompost application, resulting in greater hormones or humate content in the vermicompost treatment. All of the above was taken into consideration to illustrate and demonstrate the results. About the result of leaf constituents including total chlorophyll, N, P, and K content all treatments gave a positive effect compared with the control this reason is due to the content of vermicompost which had several plant nutrients which progress the nutritional value of various plant parts such as roots, shoots, and fruits (Theunissen et al., 2010).

Conclusion
This research studied the effect of foliar application of vermicompost tea, applying vermicompost tea to the soil and using it either as an individual or in combination form. It was proven that it could increase and improve the physical and chemical properties of pomegranates (Punica granatum Manfalouty). Moreover, the effect of vermicompost tea is the same as that of inorganic fertilizers, so using it is a promising natural resource that can be used as an alternative to synthetic fertilizers for crop development, reducing chemical fertilizer application and hence reducing both the environmental pollution and the cost of the fertilizer. The research recommended that all treatments, especially those that include the combination of foliar and application of vermicompost tea, be used to the best effect for the quality and improvement of pomegranates. In this experiment, it was observed that spraying trees with vermicompost alone had a quick and effective effect on the quality of pomegranates. The addition of vermicompost to the soil alone had a slight effect on pomegranates in the first season, while it had a clear effect in the second season due to the cumulative effect of vermicompost tea. However, the combination of spraying and application had the best effect on the quality of pomegranates.