EFFECT OF ORGANIC AND BIO-FERTILIZATION ON VEGETATIVE GROWTH, YIELD, AND FRUIT QUALITY OF ‘VALENCIA’ ORANGE TREES

INTRODUCTION

The word citrus is derived from the Latin cedrus that comes from the Greek kedrus, which refers to the kedromelon or cedar-apples (citron) (Jahoda, 1976). Citrus fruit are members of Rutaceaefamily, which include orange, grapefruit, lemon, lime, tangerine and mandarin, pummelo, and kumquat. It is generally accepted that pummelo, mandarin, and citron are the founding true species of all citrus varieties existing today (Spiegel- Royand Goldschmidt, 1996). The targeted cultivar of this study is ‘Valencia’ orange Citrus sinensis (L.) Osbeck (Hodgson, 1967).

Citrus is commercially grown primarily in regions with mild winters located between 20^o and 40^o of latitude in both hemispheres (McKnight and Hess, 2000). Citrus is second after grapes in planting and production of perennial fruits worldwide (Spiegel- Roy and Goldschmidt, 1996). Egypt is considered the sixth largest world producer after Brazil, China, USA, EU, and Mexico. Egypt ranked the sixth in world citrus and orange and the third world production in mandarin  (FAO, 2017)

        Fruit quantity and quality are affected by several factors including cultivar, rootstock, climate, soil, pests, irrigation, and nutrition. Therefore, supplying sufficient nutrition and using sound irrigation scheduling techniques should be high-priority management practices for every grower for expected yield and fruit quality. The most important management practices include irrigation, in addition to nitrogen, phosphorus, potassium, and magnesium nutrition. Some micronutrients, like boron and copper, can also affect fruit quality, but only if they are deficient in the soil and tree. In general, when any nutrient element is severely deficient, fruit yield and quality will be negatively affected (Zekri et al., 2003). The use of organic and bio-fertilizers has been increased in recent years for their positive effects on soil and plant growth and productivity, in addition to the lower cost and less hazardous effects, compared to chemical fertilizers (Shiralipour et al., 1992).

Organic fertilizers and humic acid significantly decreased fruit nitrate and nitrite content, and improved yield and fruit quality of the treated vines and soybean (Saleh and Ahmed, 1988; Farag, 2006).

Organic matter content of compost is high and often improves soil physical, chemical, and biological characteristics. Compost provide a stabilized form of organic matter that improves nutrient content, water holding capacity, cation exchange capacity, soil pore space, and aggregate stability (Shiralipour et al., 1992). Also, (Aseri et al., 2008) found that the use of bio-fertilizers significantly improve yield and fruit quality of pomegranate in India. It also enhanced the concentration of various nutrients, and soil microbial activity. Bio-fertilization is also considered a positive alternative to chemical fertilization to enhance citrus yield and fruit quality, because it are safe for human, animal, and environment. Using biofertilizers in organic food production was accompanied with the reduction of environmental pollution. it increase N fixation and enhance nutrients uptake, as well as the stimulation of natural hormones biosynthesis and the production of antibiotics (Subba-Rao et al., 1993).

Therefore, the aim of this study was to provide the best combined application of mineral, organic, and bio- fertilization that could be used to gradually replace regular mineral fertilization programs and to improve the vegetative growth, productivity, and fruit quality of ‘Valencia’ orange trees.

MATERIALS AND METHODS

This study was carried out during 2013/ 2014 and 2014 / 2015 seasons on 40 years old ‘Valencia’ orange, Citrus sinensis,(L) osbek budded trees on sour orange rootstock, Citrus aurantium, and grown in a private citrus orchard located at Meat lait Hashem in AlMahalla AlKubra, district Algharbia, Governorate, Egypt. Trees were planted at 5×5 m apart with a total number of 168 trees/Faddan in a light clay soil.

Common agricultural practices during both seasons:

The irrigation method is the surface irrigation of river water :

Fertilization :

Three different types of fertilizers were used;

1-      NPK (ammonium nitrate [33.5% N], potassium sulphate [48.5% K₂O], and superphosphate [15.5% P₂O₅]) were added each season, as follow;

• · Both superphosphate (100 kg/faddan) and sulfur (200 Kg/faddan) were scattered around the tree shade in late winter.

         N fertilizer was added three times:

• · During the first week of March (early spring) of both seasons, trees received 200 kg/feddan ammonium nitrate and 100 kg/feddan potassium sulfate [48.5%].
• · Another 150 kg/feddan of ammonium nitrate was added in May after fruit set.
• · Fertilization was repeated again in July of both seasons by adding 200 kg/feddan ammonium nitrate and 100 kg/feddan potassium sulfate/tree.

2- Organic fertilizer as compost (mixture of rice straw and animal manure) was added before irrigation by scattering it around the tree trunk (15 kg/tree/season) during the first week of March (early spring).  

3- Bio-Fertilizer (TS) [Bio-enriched fluid of Bayutk Company consists of Bacillus Circulance, Bacillus Poylmyxa, Candida spp., Trichoderma  spp., and  Bacillus Megatherium]was add around the tree trunk after 24 hours of irrigation,during the first week of March (early spring) at a level of 100, 150, and 200 ml per tree. This fertilization program was applied in both seasons in the same dates.

- Weeds were mechanically removed in March, April, and May, and chemically controlled using 48.5% Herbozd herbicide in June, July and August.

-   The following micronutrients were sprayed in May and June; amino acids (7.42%), iron (1.2%), zinc (0.8%), manganese (1%), boron (0.1%), and molybdenum (0.1%). 

-  Tree stem was sprayed, in mid-September, for silver leaf whitefly (Bemisia argentifolii ) control using a mixture of 10% Lambda pesticide (1.25 Kg/ 600 liters water) and Malathion pesticide (1 liter/600 liters water).

Experiment

Forty eight trees were selected for this experiment, and grouped in 8 treatments. Each treatment was represented by 6 trees in 3 replicates, with 2 trees each. Fertilization treatments can be summarized, as follow:

T1= Full amount NPK (control), T2= Full amount compost, T3= Full amount compost +100 ml TS, T4= Full amount compost +150 ml TS, T5= Full amount compost + 200 ml TS, T6= Half amount NPK + half amount compost +100 ml TS, T7= Half amount NPK + half amount compost +150 ml TS, T8= Half amount NPK + half amount compost +200 ml TS.

Soil and compost analysis

Soil samples were collected at (30 - 60 cm) depth from the experimental orchard. Compost samples were also collected, and both type of samples were sent for analysis at the Soil Sciences Department, Faculty of Agriculture, Kafrelsheikh University. Samples were air-dried and sieved using a 2.0 mm sieve. Chemical characteristics were carried out using 1:5 sample to water solution. Total nitrogen content was determined using micro-kjeldahl method as described by (Pregle, 1945). Phosphorus percentage was determined colorimetrically, as described by (Murphy and Riley, 1962). Potassium percentage was also determined using the flame photometric method described by (Brown and Lilleland, 1974). Micronutrients, such as Mn, Zn, Cu, and Fe were determined using Perkin Elmer atomic absorption spectrophotometer according to (Wilde et al.,  1985). Soil and compost analysis results are shown in Table 1.

Data recorded as follows :

Determination of NPK  & micronutrients in leaves:

To determine leaf mineral content, forty leaves were collected in September from non-fruiting shoots that were previously tagged during the spring growth cycle (Jones and Embleton, 1960). Leaves were washed with

tap water followed by distilled water, and leaf fresh weight was calculated. Leaves were oven dried at 70°C until constant weight. Leaf dry weight was calculated, and then dry leaves were finely ground. Samples of  0.1 g from each leaf sample were wet digested using a 5:1 mixture of concentrated H₂SO₄ + perchloric acid (HCLO₄) to determine total nitrogen using the micro-kjeldahl method. Nitrogen, phosphorus, and potassium percentage was determined using the same methods used in soil samples. The amount of micronutrients, such as Mn, Zn, Cu, and Fe were determined according to the methods described by (Cottenie et al., 1982).

Growth aspects:

1. Shoot number and length: Four main branches; one toward each direction, approximately uniform in length and diameter, were selected on each tree at early spring of both seasons. All growing spring shoots were labeled and monitored monthly from April 1^st to November 1^st for average shoot number and length per main branch.

2.Tree size (m²): This was calculated according to Morse and Robertson (1987) as follow equation:

Tree size = 0.5236 × HD² ;

Where H = tree height, and D = tree diameter.

3. Leaf number: Number of leaves on the selected four branches was counted, and the mean number was calculated out (Chou, 1966).

4. Leaf area (cm²): Six months after the end of spring growth, forty leaves were collected in September during both seasons. Leaves collection started at the fourth leaf on the branch from all four directions of the tree, and leaf area was calculated according to Chou(1966) as follows equation:

                        Leaf area= 2/3 leaf length × leaf width.

5. Chlorophyll content: By the end of spring growth cycle in September, fresh leaf samples were collected from all four directions of each tree. Leaf collection started at the fourth leaf from the top of non-bearing shoots only. Leaves were washed three times with tap water, and then washed using distilled water to measure total chlorophyll, and both chlorophyll (a) and (b). Values were calculated as µg/cm² according to Moran (1982). Chlorophyll (a) and (b) were calculated by the following equation:

Chlorophyll (a) or (b) [µg / cm²] =  Reading (µg/ml) × Extracted  volume (ml)               Leaf area (cm²)

Total chlorophyll = Chlorophyll (a) + Chlorophyll (b).

Fruiting and fruit quality

1. Fruit set (%):  Fruit set was calculated two weeks after full bloom in April during both seasons using the following equation;

          Fruit set (%)  =   Total fruit number x100

                                    Total flowers number

The percentage of remaining fruit was calculated again after June drop using the following equation:

Final Fruit set (%)  = Number of remaining fruit after June drop x 100 

                  Total flowers number

2. Total Yield: Total yield (kg), average fruit weight (g), and fruit number per tree were calculated at harvest time (first week of May in both seasons).

3. Peel thickness:  The average thickness of the peel (mm) was measured according to Kwan, (2011).  

4. Juice Volume: Four random fruits are taken from each treatment and then squeezed with a hand-squeezer. Juice was collected in a flask, and juice volume was record and then seeds were removed.

5. Total soluble solids (TSS): It was measured using a refractometer, according to A.O.A.C.(1995).

6. Total acidity: Juice samples was filtered and used to determine total acidity using the titration method against NaOH (0.1 N) in the presence of phenol phethalein, as an indicator, according to (Chen and Mellenthin (1981) to calculate citric acid (mg) per100 ml juice.

7. Ascorbic acid (Vitamin C) content: This was determined in filtered juice samples and expressed as mg/100 ml juice, as described by A.O.A.C.(1995) using 5ml juice sample and 5ml of oxalic acid solution (2%), and then titrated against 2,6-dichlorophenolendophenol indicator dye to the end point (the appearance of pink color) to calculate vitamin C.

Statistical analysis

Data were analyzed using M-Stat program in a randomized complete block design (RCBD) as described by Snedecor and Cochran (1980), and means were compared using least significance difference (LSD) at P≤0.05 (Little and Hills, 1972).

RESULTS AND DISCUSSION

Shoot number, shoot length (cm), and tree size (m³): 

Results in Table (2) indicate that the application of half amount NPK and half amount compost in combination with 100, 150 or 200 ml TS significantly affected both shoot number and length of ‘Valencia’ orange trees , a well as , tree size in comparison to the control during both seasons.  Anyhow , application of full amount of compost enriched with 100, 150 or 200 ml TS has slightly affected all vegetative growth characters . In this regards, control recorded the lowest values of all three parameters compared to other treatments during the two seasons .

Previous reports revealed that organic fertilization positively affected vegetative growth traits of ‘Satsuma’ mandarin (Eghaball and Power, 1994), sweet orange (Mourafilha et al., 1998), acid lime (Hiwarale et at., 2004), ‘Washington’ navel orange (El-Kobbia, 1999), sour orange (El-Morshedy, 1997), ‘Le- Conte" pear seedlings (Eissa et al., 2007a; Ismail et al., 2007), guava (Yadav et al., 2012), ‘Dashehari’ mango (Mohit and Kumar, 2013), and ‘Picual’ olive (Safar and Ahmed, 2012).  

Results showed a positive effect of combined application of NPK, organic, and bio- fertilization, also compared to the control. The interaction between organic and bio- fertilizers was evident, and significantly improved shoot length, shoot number per tree, and hence tree size.

Such findings were in parallel with those reported on ‘Balady’ mandarin (Ebrahiem and Mohamed, 2000), ‘Balady’ guava (Osama et al., 2016), ‘Sukary’ mango (Faissalet al., 2015b), ‘Washington’ navel orange (Abdelaal et al., 2010), mango and avocado (Abd-Rabouo, 2006), olive (Faissal and EL-Dawwy, 1999), and grapes (Ahmed et al., 1997). Bio-fertilization provides suitable amount of nitrogen to the plant. Nitrogen is a main component of protein, protoplasm, chlorophyll, and DNA. It plays an important role in cell division, and therefore improves vegetative growth and increases tree size throughout increasing shoot number and length (Oparka, 1993).

Leaf number and leaf area (cm²):

Data of Table (3) clarify significant effect related to the combined application of half amount of NPK and half amount of compost, with either 100, 150 or 200 ml TS. The effect of these treatments has extented to leaf area and number compared to the control which recorded the lowest leaf number and area in both seasons. These results were closed to previous which findings indicated that biofertilization had significantly increased

Table 3: Effect of different fertilizer treatments on Leaf number and area of  ‘Valencia’ orange trees during 2013/2014 and 2014/2015 seasons.

shoot length and leaf area on various fruit crops, such as ‘Satsuma’ mandarin (Motskobili, 1984) and mango and Avocado (Abd-Rabouo, 2006). Organic fertilization has also affected leaf number and area in sour orange seedlings (El-Morshedy, 1997), ‘Washington’ navel orange (Abd El-Naby et al., 2004), apricot (Kotez and Joubert, 1992), grapes (El-Shenawy and Fayed, 2005a), and peaches (Fayed, 2005a).

The addition of organic fertilizer to the biofertilizer gave the highest effect compared to the biofertilizer alone. This was reflected on the increased number of shoots and leaves, and leaf area, as reported on olive (Hegazi et al., 2007), ‘Le Conte’ pear (Fawzi et al., 2010), and ‘Superior Seedless’ grapes (Shaheen et al., 2013).

Chlorophyll content 

Like leaf number and area, total chlorophyll and chlorophyll (a) & (b) content were significantly increased during both seasons half amount of NPK and organic fertilization mixed with biofertilizer at either 150 or 200 ml TS, as indicated in Table (4) compared to all other treatments during both seasons. Chlorophyll (b) gave similar trent in this respect. Similar results were reported by El-Morshedy, (1997), ‘Washington’ navel orange (Helail et al., 2003a), and (Abd El-Samad et al., 2006) ‘Valencia’ orange. Similar results were reported on ‘Canino’ apricopt (Kabeel, et al., 2005) and red peach (Fayed, 2005b). They found that the Gradual increase in organic fertilization rate in form of chicken manure led to a gradual increase in chlorophyll (a) and (b) content of sour orange trees.

Leaf NPK percentage

The application of half amount NPK and half amount compost in combination with 100, 150 or 200 ml TS had positively affected the percentage of NPK in ‘Valencia’ orange leaves, compared to the control during both seasons. Also, the application of full amount of compost and 200 ml TS had a pronounced effect on both N and P levels compared to the control during both seasons (Table 5).

 In addition, it was noticed that the mixture of half amount NPK and half amount compost enriched with the highest level of TS (200 ml) had more pronounced effect on NPK percentage compared to all other treatments during both seasons. Previous investigations indicated also that the addition of biofertilizers to each or both of the mineral and organic fertilizers significantly increased NPK percentage in ‘Valencia’ orange leaves. This results are agreed with previous findings of Abd El-Samad et al. (2006) on ‘Valencia’ orange, Mansour (1998) on ‘Anna’ apples trees, Ahmed et al. (1997) on ‘Red Roomy’ grapevines, Kabeel et al. (2005) on apricot, and Amro et al. (2014) on ‘Hayany’ date palm.

Results were also revealed an increase in leaf NPK percentage with a combined application of organic and bio- fertilizers only. These results are confirmed with previous findings of Tayeh (2003) on ‘Washington’ navel

Table 4: Effect of different fertilizers treatments on leaf chlorophyll content (µg/cm²) of ‘Valencia’ orange trees during 2013/2014 and 2014/2015 seasons.

Table 5: Effect of different fertilizers treatments on NPK percentage in ‘Valencia’ orange leaves during 2013/2014 and 2014/2015 seasons.

orange, Helail et al. (2003a) on ‘Washington’ navel orange, Hegazi et al. (2007) on ‘Picual’ olive trees, Fayed (2005c) on Apples, and (Fayed 2005a) on peaches.

Leaf Fe, Zn, and Mn content

Leaf content of Fe, Zn, and Mn varied from one season to another, but the only pronounced effect on all three nutrients during both seasons was noticed with the combined application of half amount NPK, half amount compost, and 200 ml TS, as shown in (Table 6). Other treatments have a similar trend with those reported by leaf NPK content compared with control one. 

Results are in the same context of previously reported findings of organic fertilization effect on sour orange trees (El-Morshedy, 1997), ‘Clementine’ mandarin (Abed El-Hamied, 2014), and ‘Balady’ guava (Osama et al., 2016).  In regards to biofertilization, our results were in the

same context of previous results of Mahmoud and Mahmoud (1999) on peach seedlings and Sharma and Bhutani (2000) on apples seedlings. The combined application of organic and bio- fertilization significantly increased nutrients content compared to organic fertilization only (Fayed, 2005b and Ahmed et al., 2013).

Fruit set (%)

Application of half amount NPK, half amount compost, and 200 ml TS significantly improved fruit set compared to all other treatments and control one which recorded the lowest fruit set percentage in both seasons (Table 7). Compost treatment only as with biofertilizer TS showed intermediate effect . Such trend was clear in the two seasons.

Results are in line with previous reports that indicated a positive effect of fertilization regimes, which include organic and bio- fertilizers, on fruit set percentage of orange trees (El-Kobbia, 1999; Ebrahiem and Mohamed, 2000 ; Helail et al., 2003b) and ‘Meet-Ghamr’ peach ‘(Kabeel, 2004).

Total yield (Kg/tree) and its components.

All treatments significantly increased total yield per tree as fruit number and weight compared to the control during both seasons (Table 7). The most pronounced effect was recorded for half amount NPK, half amount compost, and 150 or, 200 ml TS, compared to the control and all other treatments. Similarly to the previously mentioned parameters, yield results are actually confirming the pronounced effect of all three fertilizers combined (Hepler, 1982).

Results showed some increase in fruit number and weight with the higher levels of biofertilization in the fertilizers mixture. This was

Table 6: Effect of different fertilizers treatments on leaf micronutrient contents of ‘Valencia’ orange trees during 2013/2014 and 2014/2015 seasons.

confirmed by the previous findings of Tayeh (2003) on ‘Valencia’ orange, (Fayed, 2005b) on peach, (Mansour, 1998) on ‘Anna’ apple, (Ahmed et al.,

1997) on ‘Red Roomy’ grapes, (Osman, 2003) on date palm, and (El-Shenawy and Fayed, 2005b) on "Crimson Seedless" grapes.

Peel thickness (mm):

Results of both seasons confirm the significant effect of combined application of the three different fertilizers represented by half amount of NPK, half amount of compost, with either100 or 150 ml TS. Control treatment recorded the lowest fruit peel thickness.

 Although, peel thickness may not be preferable for customers; however, it is considered an advantage for shipping.  This may mean that control fruit are the best for quick consumption, unless some other treatments that insignificantly differ from the control may add some value to the fruit.  Previous findings stated that fruit peel thickness of ‘Newhall’ navel orange was linearly increased with increased dose of organic fertilization (El-Kobbia, 1999). Organic and bio- fertilization reported to be positively affected peel thickness of the fruit (Hepler, 1982).

Juice volume (ml³)

Like peel thickness, the application of half amount NPK, half amount compost with either 100 or 150 ml TS significantly increased juice volume compared to the control during both seasons. However, half amount NPK, half amount compost, and 200ml TS recorded the highest juice content in comparison to the control and all other treatments during both seasons. Control fruit contained the lowest juice content (Table 8). This may give an indication about the importance of biofertilization in fertilizers mixture.

Results are in agreement of the previous findings reported by El-Kobbia, (1999) on ‘Washington’ navel orange, and Ebrahiem and Mohamed (2000) on ‘Balady’ mandarins.

Table 8: Effect of different fertilizer treatments on some fruit physical characteristics of ‘Valencia’ orange trees during 2013/2014 and 2014/2015 seasons.

Total soluble solids (TSS %) and acidity percentages :

Application of half amount NPK, half amount compost with 200 ml TS has significantly affected the percentage of fruit TSS in comparison to the control during both seasons (Table 9). Other treatment showed intermediate effect like other vegetative and fruit parameters. control treatment the lowest TSS percentage.

The role of organic and bio-fertilizers in improving TSS was reported by Mansour, (1998), on apples, Helail et al. (2003b),on ‘Washington’ navel orange and Kabeel (2004; Fayed (2005b) on peaches. Nitrogen and potassium play an important role in the process of carbohydrate storage and

Table 9: Effect of different fertilizers treatments on some fruit chemical traits of ‘Valencia’ orange trees during 2013/2014 and 2014/2015 seasons.

the synthesis of protein and starch, and eventually increase fruit TSS and overall fruit quality (Hepler, 1982).

The results of the present study are in agreement with (El-kobbia, 1999) and (Abdelaal et al., 2010) on ‘Washington’ navel orange, (Mansour, 1998) on ‘Anna’ apple, and (Kassem and Marzouk, 2002) on ‘Flame’ Seedless grapes.

Vitamin C (mg/100 ml juice)

Trees treated with full amount compost and 200 ml TS, or received half amount NPK, half amount compost with150 or 200 ml TS showed a significant improvement in vitamin C in comparison to the control, which recorded the lowest vitamin C, during both seasons (Table 9). Same findings

were reported on lemon trees (Antonio et al., 2002), guava (Faqir et al., 2000), and olive (Fayed, 2010).

Conclusively, it can be concluded that the best results were recorded with the combination of all three fertilizers, especially with middle and high rates of biofertilization (half amount NPK, half amount compost, enriched with,150 or 200 ml TS). This confirm the role of organic and biofertilization on plant growth, development, productivity, and fruit quality, compared to the sole use of NPK fertilizers (control), or even organic fertilizers (full amount of compost). The most pronounced effect of the three combined fertilizers was noticed with high rates of biofertilizers (half amount NPK, half amount compost, and 200 ml TS).

Results for sure do not nullify the important role of mineral NPK fertilization, and just confirming that minimizing NPK level to half amount with combination of organic and bio- fertilizers was so effective. It should also be noticed that application of half amount NPK, half amount compost, and 200 ml TS increased peel thickness and fruit acidity, and this increase in both parameters considered unfavorable for the consumer. In most cases, all other treatments show slightly significant effect on vegetative and fruit characteristics in comparison to the control.

So, in this case it would be mentioned that half amount NPK, half amount compost with150 or 200 ml TS could be recommended to improve tree growth, productivity, and fruit quality, but the disadvantage of peel thickness and fruit acidity may be noted based on the consumer acceptability.