Research Article
 

Pyridoxine Improving Effect on Yield, Chemical and Nutritional Value of Egyptian Clover Plant



M. F. El Karamany, Mervat Sh. Sadak, Gehan Sh. Bakhoum, Hamed A.A. Omer and Bakry A. Bakry
 
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ABSTRACT

Background and Objective: Vitamins are natural substances that are important in tiny quantities to sustain the normal growth and development of the plant. Thus this study was carried out to study the physiological effect of pyridoxine on increasing yield quantity and quality of Egyptian clover plant. Materials and Methods: In sandy soil at the Research and Production Station, National Research Centre, Nubaria Province, Behaira Governorate, two field experiments were carried out during two consecutive winter seasons to study the effects of pyridoxine treatment on the yield and nutritional value of Egyptian clover. Results: Spraying Egyptian clover with pyridoxine significantly increased yield attributes, crude protein, Ash contents, however, crude fibre content decreased by 50 or 75 mg L1. Pyridoxine levels significantly decreased nitrogen, free extract contents. Pyridoxine with 100 mg L1 significantly increased gross, digestible, metabolizable and net energy, total digestible nutrients, digestible crude protein, neutral detergent fibre, acid detergent fibre, acid detergent lignin and cellulose contents in comparison with control and the other treatments. Cut stages affect the significantly chemical composition and their contents of both energetic and nutritive values and cell wall constituents. Moreover, there were significant interactions between pyridoxine supplementation and berseem cut stages on chemical composition and their contents of energetic and nutritive values and cell wall constituents. Also, photosynthetic pigments, carbohydrates constituents, indole acetic acid, phenolic and flavonoids contents were increased gradually with increasing pyridoxine concentrations. Conclusion: In conclusion, different pyridoxine treatments have a promotive effect on increasing the forage yield of the berseem plant in addition to its nutritive values.

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  How to cite this article:

M. F. El Karamany, Mervat Sh. Sadak, Gehan Sh. Bakhoum, Hamed A.A. Omer and Bakry A. Bakry, 2022. Pyridoxine Improving Effect on Yield, Chemical and Nutritional Value of Egyptian Clover Plant. Asian Journal of Plant Sciences, 21: 654-666.

DOI: 10.3923/ajps.2022.654.666

URL: https://scialert.net/abstract/?doi=ajps.2022.654.666
 
Copyright: © 2022. 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

Egyptian clover (Trifolium alexandrinum L.) is an annual, cool-season forage crop grown in diverse conditions of Egypt. It gives several cuttings during its growing season and supplies nutritious and juicy forage for animals1. Normally four to six cuttings of berseem are taken2. It is used as animal feed either green or in hay form when seasonal conditions permit3,4. It is famous for farmers due to its rapid growth, an excessive wide variety of harvests and clean forage manufacturing with correct high-satisfactory and quantity5,6. The production rate of Egyptian clover depends on sowing date, climatic conditions, soil fertility, shrub height, number of crops and variety2,3. Therefore, one of the most important goals of Egyptian crops is to increase forage production, especially Egyptian clover, by maintaining its properties as a vegetation cover and soil fertility4. It also plays an important role in increasing the intake of dairy products and animal protein. In the same context. Arif et al.7 cleared that Egyptian clover (Trifolium spp.) is one of the most important forage legumes and is known as the Forage King. Often used as green feed for all livestock. Dried Egyptian clover is also an important poultry feed. Due to its desirable properties, it has been suggested that it may be an inexpensive additional source of protein in cattle feed8.

Salem et al.9 also reported that Egyptian clover (Trifolium alexandrinum L.) can grow as the first forage plant under Egyptian conditions. The cultivated area is approximately 1.07 million ha/year. In addition, Egyptian clover is an important forage plant because it contains high levels of protein, phosphorus, calcium and various amino acids (valine, leucine, methionine, lysine, tryptophan, arginine and others) as well as 0.75 kg of energy units, provided in every kilogram of hay, so clover improves the structure of the soil. Moreover, the presence of bacteria in the roots of clover increases nitrogen in the soil (100-150 kg of nitrogen is produced per hectare of soil, which is equivalent to 400-500 kg of ammonium chloride). In addition, Egyptian clover plays an important role not only in reducing soil erosion and salt formation but also in cleaning the soil from weeds3.

Egypt is suffering from the loss of cropland due to erosion and desertification. Therefore, it is very important to increase the area of the agricultural land. The newly reclaimed sandy soil on the outskirts of the Nile has received a lot of attention in Egypt. Reclaimed sandy soils are affected by a combination of nutrient deficiencies and lack of available water as well as biological stresses such as temperature fluctuations and increased lighting10.

So, to increase the resistance of plants to these destructive conditions, unusual methods are used. These strategies include selecting resistant varieties, using optimal growing methods and using natural compounds (amino acids, vitamins and antioxidants) through various treatments such as soaking seeds or treating leaves. Therefore, the use of various substances or vitamins that control growth has an important effect on the growth and yield of plants and affects various biochemical and physiological processes of plants. Amino acids are one of the precursors of these growth-promoting substances11.

Vitamins are natural substances that are essential in trace quantities to sustain the normal growth and proper development of all organisms and they function as coenzyme systems and thus play an essential role in the regulation of metabolism. Vitamins are known as limiting factors in plant development12. The diverse treatment of vitamins led to an effective role of plant bioregulators, which then influence various biochemical processes and protect the plant from the negative influences of abiotic stress13. Pyridoxine (vitamin B6) is an essential coenzyme which incorporated in a wide range of physiological processes, among them glycogen metabolism and biosynthesis of amino acids. Pyridoxine can act as a co-enzyme for numerous metabolic enzymes14 and it is shown to be a potent antioxidant15,16. It improved the efficiency of photosynthetic carbon reactions and increased dry matter production. In the meantime, treatment of different plants with pyridoxine enhanced cell division, improved growth and differentiation and increased nutrient uptake17. In seedling growth, exogenous treatments with optimal pyridoxine concentrations were beneficial via increasing availability of water and nutrient. Pyridoxine is highly recommended for use in various plants. It has been established that pyridoxine enhances the growth of the root system which helps in higher nutrient uptake and leads to higher economic yield18.

So, the objective of this search is to investigate the influence of pyridoxine in different amounts (0, 50, 75 and 100 mg L1 of water) of supplementation on the yield of the crop in different stages of cutting forage of Egyptian clover, which is cultivated under sandy soil for its Increase the harvest as an added value in animal feed and improve its chemical, physiological, biochemical processes, composition and nutritional values.

MATERIALS AND METHODS

Study area: The study was conducted in co-operation work among Department of Field Crops, Institute of Agriculture and Biological Researches, National Research Center, Dokki, Cairo, Egypt and Department of Animal Production, Institute of Agriculture and Biological Researches, National Research Center, Dokki, Cairo, Egypt.

The purpose of this study was to influence the amount of Egyptian clover (crop yield) and chemicals by including pyridoxine in various amounts (0, 50, 75 and 100 mg L1 water) of supplements. It was to find out investigate composition at various cutting stages (4 cuts).

In the winter of 2019/2020 and 2020/2021, two field experiments were conducted at the Research and Production Station of National Research Centre (NRC), Al-Nubaria District, Al Behaira Governorate, Egypt. The experimental soil before sowing was analyzed according19. The analysis of the experimental soil was: Soil texture: Sand 91.2%, Silt 3.7%, Clay 5.1%, pH 7.3, Organic matter 0.3%, CaCO3 1.4%, E.C. dS/m 0.3, Soluble N, ppm 8.1, Available P 3.2 ppm, Exchangeable K, 20 ppm.

Experimental soil was ploughed twice and divided into plots 3×7 m, then made rows 20 cm2 between. Egyptian clover cultivar (Meskawy) was inoculated with the appropriate (Rhizobium trifolii ) in a commercial product produced by the Ministry of Agriculture, Egypt.

The recommended agricultural practices were applied. Pre-sowing, 150 kg/feddan of calcium super-phosphate (15.5% P2O5) was applied to the soil. Nitrogen was applied after emergence in the form of ammonium nitrate 33.5% at a rate of 75 kg/feddan in five equal doses before the 1st, 2nd, 3rd and 4th irrigation.

Potassium sulfate (48.52% K2O) was added in two equal doses of 50 kg/feddan, before the 1st and 3rd irrigations. Irrigation was carried out using the new sprinkler irrigation system where water was added every 5 days.

The applied substance pyridoxine used in the present study was supplied from sigma chemical.

The plants were sprayed twice with pyridoxine at (50, 75 or 100 mg L1) while control plants were sprayed with distilled water during vegetative growth at 30 and 45 days after sowing.

Four cuts were taken from each of the two seasons. The first cut was obtained 60 days post-seeding date, the second cut was obtained after 50 days from the first one, while the third one was taken after 40 days from the second cut and the fourth was taken after 40 days from the third cut.

Fresh forage yields: Fresh forage yield of Egyptian clover determined in m2 for each of the subsequent four cuts, in each experimental plot recorded and estimated in ton/feddan. This was done for each of the two growing seasons.

Biochemical determinations: Photosynthetic pigments (chlorophyll a and b, carotenoids and total pigments) in fresh leaves were determined as the method described by Lichtenthaler and Buschmann20, total carbohydrates was determined according21, total soluble sugars were extracted by the method22 and extracted by the method21, polysaccharides were determined according21, phenolic content was measured as the method described23 and flavonoids contents were determined by the method24.

Different samples of unsprayed Egyptian clover and sprayed Egyptian clover that sprayed by pyridoxine solution at different levels mentioned above were collected at different cuts and chemical analyses were determined for their contents of moisture, Dry Matter (DM), Organic Matter (OM), Crude Protein (CP), Crude Fibre (CF), Ether Extract (EE), Nitrogen-Free Extract (NFE) and Ash using methods that described25.

On other hand, cell wall constituents include Neutral Detergent Fibre (NDF), Acid Detergent Fibre (ADF), Acid Detergent Lignin (ADL), hemicellulose and cellulose were evaluated in both unsprayed Egyptian clover and sprayed Egyptian clover as the method26,27.

Energetic values composed of Gross Energy (GE), Digestible Energy (DE), Metabolizable Energy (ME) and Net Energy (NE), in addition to nutritive values of both Total Digestible Nutrients (TDN) and Digestible Crude Protein (DCP) were also calculated according to different equations that concluded by NRC28.

Analytical procedures: Chemical analysis of unsprayed Egyptian clover and Egyptian clover includes moisture, Ash, Crude Protein (CP), Crude Fibre (CF) and Ether Extract (EE) contents were determined according to Talreja et al.25.

Crude protein determination involved the use of routine Kjeldahl nitrogen assay (N×6.25).

Meanwhile, Nitrogen-Free Extract (NFE) or carbohydrate content was determined by the difference using the following equation:

NFE content = 100-(Moisture+CP+CF+EE+Ash)

On the other hand, cell wall constituents including Neutral Detergent Fiber (NDF), acid detergent fibre and Acid Detergent Lignin (ADL) were determined according to Samreen et al.26 and Vishwakarma and Dubey27. However, hemicellulose and cellulose contents were calculated by difference as follows:

Hemicellulose = NDF-ADF
Cellulose = ADF-ADL

Gross energy (Kcal Kg1 DM) was calculated according to Liu et al.29.

where, each g crude protein = 5.65 Kcal, g fat = 9.40 Kcal and g (crude fibre and carbohydrate) = 4.15 Kcal.

Digestible energy (Kcal kg1 DM) was calculated according to NRC28.

Where:

Digestible Energy (DE) = Gross energy×0.76

Metabolizable energy (Kcal kg1 DM) was calculated according to NRC28.

Where:

Metabolizable Energy (ME) = Digestible energy×0.82

Net energy (Kcal kg1 DM) was calculated according to NRC28 as follows:

Net Energy (NE) = Metabolizable energy×0.56

Total digestible nutrients (%) was calculated according to NRC28.

Where:

Image for - Pyridoxine Improving Effect on Yield, Chemical and Nutritional Value of Egyptian Clover Plant

Digestible crude protein (%) was calculated according to NRC28.

Where:

Digestible crude protein (%) = 0.85 X1-2.5

where, X1 = Crude protein (%) on DM basis.

Statistical analysis: Except for data of indole acetic acids the others collected data of chemical composition that includes (Moisture, Dry Matter (DM), Organic Matter (OM), Crude Protein (CP), Crude Fibre (CF), Ether Extract (EE), Nitrogen-Free Extract (NFE) and Ash), cell wall constituents includes (Neutral Detergent Fibre (NDF), Acid Detergent Fibre (ADF), Acid Detergent Lignin (ADL), Hemicellulose and Cellulose), energetic values include (Gross Energy (GE), Digestible Energy (DE), Metabolizable Energy (ME) and Net Energy (NE)) and nutritive values includes Total Digestible Nutrients (TDN) and Digestible Crude Protein (DCP), fresh forage yields, photosynthetic pigments includes (chlorophyll a and b, carotenoids and total pigments), total carbohydrates, total soluble sugars, polysaccharides, phenolic and flavonoids contents were statistically analyzed as two factors-factorial analysis of variance using the general linear model procedure of Salkind30 was used to examine the significance between means.

The following model was used as the following:

Yijk = μ+Li+Cj+(LC)ij+eijk

Where:

Yijk = Observation
μ = Overall mean
Li = Effect of levels of pyridoxine solution sprayed for i = 1-4, 1 = 0 pyridoxine, 2 = 50 mg pyridoxine/L water, 3 = 75 mg pyridoxine/L water and 4 = 100 mg pyridoxine/L water
Cj = Effect of Egyptian Clover (EC) Cut stages (C) for j =1-4, 1= First cut stage of EC, 2 = Second cut stage of EC, 3 = Third cut stage of EC and 4 = Fourth cut stage of EC
(LC)ij = Interaction between pyridoxine supplementation levels and EC cut stages
eijk = Experimental error

Meanwhile, data of indole was statistically analyzed as one-way analysis of variance using the general linear model procedure of Boghdady31 that used to examine the significance between means, using the following model:

Yij = μ+Li++eij

Where:

Yij = Observation
μ = Overall mean
Li = Effect of levels of pyridoxine solution sprayed for i = 1-4, 1 = 0 pyridoxine, 2 = 50 mg pyridoxine/L water, 3 = 75 mg pyridoxine/L water and 4 = 100 mg pyridoxine/L water
eij = Experimental error

RESULTS

Changes in fresh forage yield: Table 1, 2 cleared that, sprayed Egyptian clover forage with various pyridoxine levels (50, 75 or 100 mg L1) realized a significantly (p<0.05) increasing in yield quantity compared with control plants. Meanwhile, cut stages were also significantly (p<0.05) affected by sprayed Egyptian clover forage with pyridoxine solution with various levels as mentioned above (50, 75 or 100 mg L1).

Table 1: Main consequences of nutritional remedies on forage yield
Pyridoxine treatment levels (mg L1)
Items 0
50
75
100
SEM
Forage yield (ton/feddan) 2.946d
3.117c
3.701b
4.539a
0.115
Egyptian clover cut stages
First cut
Second cut
Third cut
Fourth cut
SEM
Forage yield (ton/feddan) 2.968d
3.530c
3.870a
3.786b
0.115
a-dMeans in the same row within each treatment having different superscripts differ significantly (p<0.05) and SEM: Standard error of the mean


Table 2: Interactions between pyridoxine treatment levels and Egyptian clover forage cut stages on forage yield
Pyridoxine treatment (mg L1)
Treatments 0
50
75
100
First cut 2.223n
2.350m
3.123k
4.424d
Second cut 3.122k
2.867l
3.600g
4.530c
Third cut 3.320i
3.400h
4.100e
4.660b
Fourth cut 3.120k
3.300j
3.982f
4.740a
SEM 0.115
a-nMeans in the same row having different superscripts differ significantly (p<0.05) and SEM: Standard error of the mean


Table 3: Chemical analysis of pyridoxine treatment on energetic and nutritive values and cell wall constituents
Pyridoxine treatment levels mg L1 water
Items
0
50
75
100
SEM
1-Chemical analysis
Moisture
9.39b
9.10c
10.16a
10.14a
0.26
Dry Matter (DM)
90.61b
90.90a
89.84c
89.86c
0.26
Chemical analysis on DM basis
Organic Matter (OM)
88.01a
86.81c
87.52b
88.03a
0.18
Crude Protein (CP)
17.09d
17.83c
18.53b
18.64a
0.15
Crude Fiber (CF)
21.04b
20.45d
20.88c
22.04a
0.45
Ether Extract (EE)
3.03b
2.31d
2.85c
3.46a
0.14
Nitrogen-Free Extract (NFE)
46.85a
46.22b
45.26c
43.89d
0.48
Ash
11.99c
13.19a
12.48b
11.97c
0.18
2-Energetic and nutritive values
Gross Energy (GE)
4068b
3992d
4059c
4114a
10.83
Digestible Energy (DE)
3092b
3034d
3085c
3127a
8.22
Metabolizable Energy (ME)
2535b
2488d
2530c
2564a
6.73
Net Energy (NE)
1420b
1393d
1417c
1436a
3.79
3-Nutritive values (%)
Total Digestible Nutrients (TDN)
69.79b
68.48d
69.63c
70.59a
0.19
Digestible Crude Protein (DCP)
12.03d
12.66c
13.25b
13.34a
0.13
4-Cell wall constituents
Neutral Detergent Fibre (NDF)
42.75b
42.36d
42.64c
43.40a
0.3
Acid Detergent Fibre (ADF)
28.62b
28.09d
28.48c
29.53a
0.41
Acid Detergent Lignin (ADL)
5.09b
4.99d
5.06c
5.26a
0.08
Hemicellulose*
14.13c
14.27a
14.16b
13.87d
0.12
Cellulose**
23.53b
23.10d
23.42c
24.27a
0.34
a-dMeans in the same row within each treatment having different superscripts differ significantly (p<0.05), SEM: Standard error of the mean, *Hemicellulose: NDF-ADF and **Cellulose: ADF-ADL

Moreover, the above-mentioned treatments caused significant (p<0.05) increases in Egyptian clover forage yield quantity compared with control plants (Table 2). Furthermore, the foliar treatment of pyridoxine accompanied with cut stages show significant

increases compared with their corresponding controls throughout cut stages of Egyptian clover forage yield.

Changes in chemical attributes: Table 3 showed that treating Egyptian clover with 75 and 100 mg L1 pyridoxine significantly decreased DM content compared to untreated plants, while, 50 mg L1 significantly increased DM content in comparison with the other treatments.

Table 4: Forage cuts and chemical analysis on energetic and nutritive values and cell wall constituents
Egyptian clover forage cut stages
Items
First cut
Second cut
Third cut
Fourth cut
SEM
1-Chemical analysis
Moisture
10.70a
9.88b
9.35c
8.85d
0.26
Dry Matter (DM)
89.30d
90.12c
90.65b
91.15a
0.26
Chemical analysis on DM basis
Organic Matter (OM)
88.15a
87.60b
87.21d
87.41c
0.18
Crude Protein (CP)
17.40d
17.85b
19.11a
17.72c
0.15
Crude Fibre (CF)
21.77b
17.57d
20.26c
24.81a
0.45
Ether Extract (EE)
3.32a
2.16c
3.35a
2.81b
0.14
Nitrogen-Free Extract (NFE)
45.66b
50.02a
44.49c
42.07d
0.48
Ash
11.85d
12.40c
12.79a
12.59b
2-Energetic and nutritive values
Gross Energy (GE)
4094a
4017d
4082b
4041c
10.83
Digestible Energy (DE)
3111a
3053d
3102b
3071c
8.22
Metabolizable Energy (ME)
2551a
2504d
2544b
2515c
6.73
Net Energy (NE)
1429a
1402d
1425b
1411c
3.79
3-Nutritive values (%)
Total Digestible Nutrients (TDN)
70.23a
68.91d
70.03b
69.32c
0.19
Digestible Crude Protein (DCP)
12.29d
12.68b
13.74a
12.56c
0.13
4-Cell wall constituents
Neutral Detergent Fibre (NDF)
43.23b
40.47d
42.23c
45.22a
0.3
Acid Detergent Fibre (ADF)
29.29b
25.46d
27.91c
32.06a
0.41
Acid Detergent Lignin (ADL)
5.21b
4.50d
4.96c
5.73a
0.08
Hemicellulose*
13.94c
15.01a
14.32b
13.16d
0.12
Cellulose**
24.08b
20.96d
22.95c
26.33a
0.34
a-dMeans in the same row within each treatment having different superscripts differ significantly (p<0.05), SEM: Standard error of the mean, *Hemicellulose: NDF-ADF and **Cellulose: ADF-ADL

Different treatments of pyridoxine occurred significantly (p<0.05) increasing in their CP content, the highest CP content was obtained from 100 mg L1 pyridoxine (18.64%).

Despite, CF contents were significantly (p<0.05) reduced by treating Egyptian clover forage with 50 or 75 mg L1 pyridoxine (20.45 and 20.88%) as compared with control and 100 mg L1 water (21.04 and 22.04% CF, respectively). Pyridoxine foliar treatment with 100 mg L1 caused a significant (p<0.05) increase in EE content of Egyptian clover compared to control and the other two levels. The corresponding values of EE content were (3.03, 2.31, 2.85 and 3.46%) for 0, 50, 75 and 100 mg pyridoxine/L water, respectively. All spraying levels of pyridoxine (50, 75 and 100 mg L1 water) significantly (p<0.05) decreased their contents of NFE (46.22, 45.26 and 43.89%) in comparison with the control (46.85% NFE). In addition, treating Egyptian clover with pyridoxine with (50 or 75 mg L1 water) significantly increased their content of Ash compared to control, meanwhile, 100 mg pyridoxine was noticed at the same range of Ash content with the control.

Table 4 revealed the changes in chemical attributes of Egyptian clover forage all over different cut stages. Results showed significant (p<0.05) differences between the first, second, third and fourth cut stages. Results show the progressive increases in DM throughout the first, second, third and fourth cuts (89.30%, 90.12, 90.65 and 91.15% DM), respectively. Organic Matter (OM) content was significantly (p<0.05) decreased throughout the different cut stages of Egyptian clover forage (second, third and fourth cuts) compared to control (first cut). Crude Protein (CP), Ether Extract (EE) and Ash contents showed significantly (p<0.05) increases in the third cut stages compared to the others cut stages. Meanwhile, Nitrogen-Free Extract (NFE) content showed a significant (p<0.05) increase in the second cut in comparison with the others cuts. Also, Crude Fibre (CF) content showed a significant (p<0.05) increase in the fourth cut in comparison with the others cuts.

For the interaction of different levels of pyridoxine treatments and cut stages, Table 5 and 6 show the promotive effect of different concentrations of pyridoxine (50, 75 and 100 mg L1) in DM, OM, CP, EE, CF and NEF as compared with the corresponding controls in the four cuts first, second, third and fourth cuts in most treatments except, Ash content of the first cut, OM and EE of the second and third cuts and DM, OM, CF and NFE of the fourth cut different pyridoxine levels decreased plant contents (Table 5).

Changes in energetic and nutritive values: Table 3 revealed the enhancing effect of treating Egyptian clover forage with 100 mg L1 water caused significant (p<0.05) increases in Gross Energy (GE), Digestible Energy (DE), Metabolizable Energy (ME), Net Energy (NE) and Total Digestible Nutrients (TDN) contents compared with control and the other treatments (50 and 75 mg L1).

Table 5: Interactions between pyridoxine treatment levels and Egyptian clover forage cut stages (chemical analysis, energetic and nutritive values and cell wall constituents
Egyptian clover forage cuts
First cut
Second cut
Third cut
Fourth cut
0
50
75
100
0
50
75
100
0
50
75
100
0
50
75
100
Items
Pyridoxine treatment levels mg L1 water
SEM
1-Chemical analysis
Moisture
9.71f
7.85k
10.73c
14.51a
10.52d
8.71j
9.56g
10.72c
9.79e
10.45d
11.00b
6.16m
7.54l
9.37h
9.33h
9.16i
0.26
Dry Matter (DM)
90.29h
92.15c
89.27k
85.49m
89.48j
91.29d
90.44g
89.28k
90.21i
89.55j
89.00l
93.84a
92.46b
90.63f
90.67f
90.84e
0.26
Chemical analysis on DM basis
Organic Matter (OM)
86.18m
88.02e
87.57g
90.82a
88.73c
86.34l
88.40d
86.92j
87.73f
87.06i
86.58k
87.47h
89.41b
85.81n
87.53gh
86.89j
0.18
Crude Protein (CP)
16.00l
16.60j
18.20f
18.81c
17.42h
17.29e
17.91g
18.79c
18.53d
19.54b
19.75a
18.61d
16.41k
17.89g
18.24f
18.34e
0.15
Crude Fibre (CF)
18.10l
22.67f
22.58g
23.73d
17.15n
16.71o
18.68i
17.74m
21.34h
18.26k
18.56j
22.87e
27.56a
24.16b
23.70d
23.82c
0.45
Ether Extract (EE)
3.96a
1.82j
3.65c
3.86b
3.28f
1.03m
1.10l
3.24gh
3.62c
3.31ef
3.27fg
3.19h
1.25k
3.09i
3.36e
3.54d
0.14
Nitrogen-Free Extract (NFE)
48.12d
46.93f
43.14k
44.42i
50.88b
51.31a
50.71c
47.15e
44.24j
45.95g
45.00h
42.80l
44.19j
40.67o
42.23m
41.19n
0.48
Ash
13.82b
11.98j
12.43h
9.18n
11.27l
13.66c
11.60k
13.08e
12.27i
12.94f
13.42d
12.53g
10.59m
14.19a
12.47gh
13.11e
0.18
2-Energetic and nutritive values
Gross Energy (GE)
4024i
3997j
4099d
4254a
4116b
3897k
3995j
4059h
4109c
4080f
4061gh
4077f
4022i
3992j
4082f
4067g
10.83
Digestible Energy (DE)
3058g
3038h
3115c
3233a
3128b
2962i
3036h
3085f
3123b
3101d
3086ef
3099d
3057g
3034h
3102d
3091e
8.22
Metabolizable Energy (ME)
2508g
2491h
2554c
2651a
2565b
2429i
2490h
2530f
2561b
2543d
2531ef
2541d
2507g
2488h
2544d
2535e
6.73
Net Energy (NE)
1404g
1395h
1430c
1485a
1436b
1360i
1394h
1417f
1434b
1424d
1417f
1423d
1404g
1393h
1425d
1420e
3.79
3-Nutritive values (%)
Total Digestible Nutrients (TDN)
69.03g
68.58h
70.32c
72.98a
70.61b
66.86i
68.53h
69.64f
70.50b
70.00d
69.66ef
69.95d
69.01g
68.49h
70.02d
69.77e
0.19
Digestible Crude Protein (DCP)
11.10l
11.61j
12.97f
13.49c
12.31h
12.20i
12.72g
13.47c
13.25d
14.11b
14.29a
13.32d
11.45k
12.71g
13.00f
13.09e
0.13
4-Cell wall constituents
Neutral Detergent Fibre (NDF)
40.82l
43.82f
43.76g
44.51d
40.19n
39.90o
41.20i
40.58m
42.94h
40.92k
41.12j
43.95e
47.03a
44.80b
44.49d
44.57c
0.3
Acid Detergent Fiber (ADF)
25.94l
30.11f
30.02g
31.07d
25.07n
24.67o
26.47i
25.61m
28.89h
26.09k
26.36j
30.29e
34.57a
31.47b
31.05d
31.16c
0.41
Acid Detergent Lignin (ADL)
4.59j
5.36f
5.35f
5.54d
4.43l
4.35m
4.68h
4.53k
5.14g
4.62i
4.67h
5.40e
6.19a
5.62b
5.54d
5.56c
0.08
Hemicellulose*
14.88d
13.71j
13.74i
13.44l
15.12b
15.23a
14.73g
14.97c
14.05h
14.83e
14.76f
13.66k
12.46o
13.33n
13.44l
13.41m
0.12
Cellulose**
21.35l
24.75f
24.67g
25.53d
20.64n
20.32o
21.79i
21.08m
23.75h
21.47k
21.69j
24.89e
28.38a
25.85b
25.51d
25.60c
0.34
a-oMeans in the same row having different superscripts differ significantly (p<0.05), SEM: Standard error of the mean, *Hemicellulose: NDF-ADF and **Cellulose: ADF-ADL

Meanwhile, spraying Egyptian clover by 50 or 75 mg L1 significantly (p<0.05) decreased all energetic values (GE, DE, ME and NE) and nutritive value of TDN as compared to control plants. Meanwhile, Digestible Crude Protein (DCP%) were increased with different levels of pyridoxine treatment to Egyptian clover forage. The most effective concentration was 100 mg L1 pyridoxine as it gave 13.34%, in DCP% compared with 12.03% of control, 12.66% and 13.25%, for 50 and 75 mg treating plants.

Table 4 revealed show that values of Gross Energy (GE), Digestible Energy (DE), Metabolizable Energy (ME), Net Energy (NE) and Total Digestible Nutrients (TDN) in Egyptian clover forage decreased significantly in the second, third and fourth cut stages in comparison with the first cut. Meanwhile, digestible crude protein showed an increase in the second, third and fourth cut stages compared to the first cut stage.

Table 5 and 6, the presented data showed the interactive effect of different pyridoxine levels and cut stages. Exogenous treatment of 50, 75 and 100 mg L1 pyridoxine significantly increased the various energy and nutritional values studied in the first and fourth cut, meanwhile, in the second and third cuts, it caused significant decreases in most of the studied parameters as compared with their corresponding values of controls (Table 5).

Changes in cell wall constituents: Table 3 showed that exogenous treatment of Egyptian clover with 100 mg L1 significantly (p<0.05) increased NDF, ADF, ADL and cellulose contents in comparison with the control plants. Meanwhile, the above-mentioned level (100 mg L1) significantly (p<0.05) decreased the hemicellulose content of Egyptian clover forage.

Data of different cell wall constituents of Egyptian clover as affected by different cut stages are presented in Table 4. The obtained results stated that Neutral Detergent Fibre (NDF), Acid Detergent Fibre (ADF), Acid Detergent Lignin (ADL) and cellulose showed significant (p<0.05) increases in fourth cut stages over the other cut stages of Egyptian clover forage. On the other hand, hemicellulose content showed a significant (p<0.05) decrease in the fourth cut stages compared to the other cut stages.

Table 5 and 6 showed that there were significantly (p<0.05) interactions between pyridoxine levels and berseem green forage cut stages on all parameters of chemical analysis, energetic and nutritive values and cell wall constituents.

Table 6: ANOVA for chemical analysis on energetic and nutritive values and cell wall constituents
Main effects
Items
Pyridoxine treatment levels
Egyptian clover cuts
Interaction
1-Chemical analysis
Moisture
*
*
*
Dry Matter (DM)
*
*
*
Chemical analysis on DM basis
Organic Matter (OM)
*
*
*
Crude Protein (CP)
*
*
*
Crude Fibre (CF)
*
*
*
Ether Extract (EE)
*
*
*
Nitrogen-Free Extract (NFE)
*
*
*
Ash
*
*
*
2-Energetic and nutritive values
Gross Energy (GE)
*
*
*
Digestible Energy (DE)
*
*
*
Metabolizable Energy (ME)
*
*
*
Net Energy (NE)
*
*
*
3-Nutritive values (%)
Total Digestible Nutrients (TDN)
*
*
*
Digestible Crude Protein (DCP)
*
*
*
4-Cell wall constituents
Neutral Detergent Fiber (NDF)
*
*
*
Acid Detergent Fiber (ADF)
*
*
*
Acid Detergent Lignin (ADL)
*
*
*
Hemicellulose*
*
*
*
Cellulose**
*
*
*
*Significant (p<0.05), *Hemicellulose: NDF-ADF and **Cellulose: ADF-ADL


Table 7: Main consequences of remedies on photosynthetic pigments (μg g-1 fresh wt.) of Egyptian clover plant under sandy soil conditions
Pyridoxine treatment levels mg L1 water
Egyptian clover cut stages
Items
0
50
75
100
SEM
First cut
Second cut
Third cut
Fourth cut
SEM
Total carbohydrates (%)
15.48c
16.20b
16.64a
16.75a
0.13
15.52c
16.09b
17.32a
16.15b
0.13
Total soluble sugars (%)
3.89d
4.34c
5.06a
4.59b
0.09
3.93d
5.03a
4.68b
4.22c
0.09
Polysaccharides (%)
11.59c
11.86b
11.58c
12.16a
0.10
11.59c
11.06d
12.64a
11.93b
0.10
Chlorophyll a
1077c
1165b
1198a
1162b
20.09
962d
1249b
1281a
1109c
20.09
Chlorophyll b
625c
639b
651a
653a
3.12
634c
654b
661a
619d
3.12
Carotenoids
304c
312b
318a
318a
1.54
310c
319b
323a
301d
1.54
Total pigments
2006d
2116c
2167a
2133b
23.43
1906d
2222b
2265a
2029c
23.43
Phenolic compounds
77.39d
84.29c
92.79a
85.34b
2.03
71.40d
75.73c
88.88b
103.81a
2.03
Flavonoids
33.85d
36.86c
40.59a
37.32b
0.89
31.22d
33.12c
38.87b
45.40a
0.89
a-dMeans in the same row within each treatment having different superscripts differ significantly (p<0.05) and SEM: Standard error of the mean


Table 8: Interactions between pyridoxine treatment levels and Egyptian clover cut stages on photosynthetic pigments (μg g-1 fresh wt.) of the Egyptian clover plant under sandy soil conditions
Egyptian clover cuts
First cut
Second cut
Third cut
Fourth cut
0
50
75
100
0
50
75
100
0
50
75
100
0
50
75
100
Items
Pyridoxine treatment levels mg L1 water
SEM
Total carbohydrates (%)
14.54f
15.30e
16.25c
16.08c
15.59d
15.78d
16.20c
16.78b
16.72b
17.50a
17.58a
17.46a
15.17e
16.21c
16.54b
16.66b
0.13
Total soluble sugars (%)
3.45f
3.52f
4.60d
4.16e
4.27e
4.83c
5.78a
5.24b
4.20e
4.68cd
5.19b
4.65cd
3.63f
4.32e
4.65cd
4.29e
0.09
Polysaccharides (%)
11.00f
11.78cd
11.65cde
11.92c
11.32e
10.95f
10.42g
11.54de
12.52ab
12.82a
12.39b
12.81a
11.54de
11.89cd
11.89cd
12.37b
0.10
Chlorophyll a
927h
955gh
993f
972fg
1150de
1273c
1308ab
1263c
1256c
1275bc
1317a
1277bc
974fg
1155de
1173d
1134e
20.09
Chlorophyll b
612k
624i
637g
665bc
638g
651e
668b
659d
643f
663c
672a
667b
606l
619j
629h
620j
3.12
Carotenoids
299h
304g
311e
324bc
311e
317d
326ab
322c
313e
324bc
328a
325b
295i
302g
307f
302g
1.54
Total pigments
1838h
1883g
1941f
1961f
2099d
2241bc
2302a
2244bc
2212c
2262b
2317a
2269b
1875g
2076de
2109d
2056e
23.43
Phenolic compounds
63.15m
70.39k
78.77h
73.27j
69.08l
73.90j
83.29g
76.65i
83.20g
90.39f
98.47d
83.44g
94.14e
102.48c
110.62a
107.98b
2.03
Flavonoids
27.62m
30.78k
34.45h
32.04j
30.21l
32.32j
36.48g
32.52i
36.38g
39.53f
43.06d
36.49g
41.17e
44.82c
48.38a
47.22b
0.89
a-mMeans in the same row having different superscripts differ significantly (p<0.05) and SEM: Standard error of the mean


Table 9: ANOVA for photosynthetic pigments (μg g-1 fresh wt.) of Egyptian clover plant under sandy soil conditions
Main effects
Items
Pyridoxine treatment levels
Egyptian clover cuts
Interaction
Total carbohydrates (%)
*
*
*
Total soluble sugars (%)
*
*
*
Polysaccharides (%)
*
*
*
Chlorophyll a
*
*
*
Chlorophyll b
*
*
*
Carotenoids
*
*
*
Total pigments
*
*
*
Phenolic compounds
*
*
*
Flavonoids
*
*
*
*Significant (p<0.05)

Table 5 and 6 showed that different concentrations of pyridoxine treatment of Egyptian clover caused significant increases in different cell wall constituents in the first, second and third cuts, meanwhile, decreased them in the fourth cut as compared with untreated control plants throughout the cut stages.

Changes in some biochemical and physiological attributes
Changes in photosynthetic pigments: The role of foliar treatment of pyridoxine with different concentrations (50, 75 and 100 mg L–1) on various photosynthetic pigments constituents of Egyptian clover forage are tabulated in Table 7. Foliar treatments of various levels of pyridoxine caused significant (p<0.05) increases in chlorophyll a and b, carotenoids and total pigments) throughout first, second, third and fourth cuts. The increases in different photosynthetic pigments were concurrent within pyridoxine concentrations increases. 75 mg L1 followed by 100 mg L1 gave the highest increases in the studied pigments compared with other treatments in the studied cut stages (Table 7).

Regarding cut stages, the studied photosynthetic pigments constituents (chlorophyll a and b, carotenoids and total pigments showed gradual and significant increases throughout the studied cut stages from the first cut to the second cut to the third cut then decreased in the fourth cut but still more than first cut.

The interactive effect of different pyridoxine treatments and the four tested cut stages showed the promotive effects of pyridoxine treatments throughout the four cut stages (Table 8) as they caused significant increases in chlorophyll a and b, carotenoids and total pigments).

Changes in carbohydrates constituents: The effect of various treatments of pyridoxine treatment on Egyptian clover is shown in Table 7. The obtained results showed the promotive effects of the used concentrations (50, 75 and 100 mg L1) on improving total carbohydrates (%), Total Soluble Sugars (TSS%) and polysaccharides (%) of Egyptian clover cultivated in sandy soil. These increases were significantly and gradually in general concurrent with increasing pyridoxine concentrations compared to the control (untreated plants).

The changes in carbohydrate components throughout the four tested cuts of the Egyptian clover forage plant are tabulated in Table 7. Total carbohydrates (%) and TSS (%) were increased significantly in the second, third and fourth cuts as compared with those of the first cut. While polysaccharides content in the first cut was reduced significantly in the second cut than increased in the third and fourth cuts over the first cut (Table 7).

Concerning the interactive effects of exogenous treatments of pyridoxine different levels and cut stages the results in Table 8 show that different pyridoxine treatments (50, 75 and 100 mg L1) increased significantly various carbohydrates components of Egyptian clover forage throughout the different cut stages as compared with untreated controls in all the tested cut stages.

Changes in phenolic and flavonoids contents: Also, the tabulated results obtained in Table 7 stated that different pyridoxine levels used as foliar treatments (50, 75 and 100 mg L1) on Egyptian clover plants cultivated in sandy soil caused significant increases in both total phenol and flavonoids contents. Increasing in phenolic and flavonoid are gradually in all cut stages.

Table 9 showed the significant (p<0.05) increases in phenolic and flavonoids contents of Egyptian clover as affected by the interactions between pyridoxine treatment levels and the four cut stages.

DISCUSSION

The obtained results of the promotive effect of pyridoxine treatments on forage yield (ton/feddan) Table 1. These promotive effects of pyridoxine are in harmony with those obtained by Zamanipour15 on tomato, Boghdady31 on Egyptian lupine and Nassar et al.32 on sesame plant. Barakat17 found that application of pyridoxine on wheat plants enhanced cell division, increased the root growth and nutrient uptake enhanced efficiency of photosynthetic surface and increased dry matter production. The efficiency of pyridoxine on the growth, yield and quality of berseem was similar to those results investigated by Nassar et al.32 and Younis et al.33. The treated plants were characterized by the longer main stem which developed more primary leaves number of high specific weight. The positive effect of vitamin B6 (pyridoxine) was confirmed on the plant's development. In addition, vitamin B could act as an antioxidant in improving plant production34. It was confirmed that pyridoxine treatments improve the growth of the root system35 which causes increases in different nutrient absorption and improved economic productivity36.

The promotive effect of pyridoxine on the Egyptian clover forage photosynthetic pigments components are presented in Table 7. These effects might be caused by the increased activities of various enzymes associated with the biosynthesis of these pigments or the preservation of chromoproteins37. Foliar spraying of lupine plants with pyridoxine vitamin with different concentrations improved all fractions of photosynthetic pigments, especially in plants subjected to salt stress. Our obtained results of pyridoxine are in good harmony with those obtained by Hamada and Khulaef38, they concluded that treatment of bean plants with 100 ppm pyridoxine stimulated biosynthesis of photosynthetic pigments fractions and net photosynthetic rate. Moreover, Soltani et al.39 confirmed the increased contents of photosynthetic pigments of Calendula officinalis L. by treatments of vitamins, they referred these increases to the role of vitamin B as co-enzymes in the enzymatic reactions in carbohydrates, fats and protein metabolism and used in respiration and photosynthesis. Also, Nassar et al.32 on sesame plant. Moreover, the increases in different photosynthetic pigments components were reflected in increasing different physiological parameters as carbohydrates constituents of Egyptian clover plants. These increases in carbohydrate constituents could have resulted from the key effect of vitamins on chlorophyll biosynthesis which in turn increased the biosynthesis of carbohydrates and phenolic as well as flavonoids contents. These results are in agreement with those noticed by Rady et al.40, El-Metwally and Sadak41 and Younis et al.33 on different plant species using vitamins treatments. In addition, An increase in total phenols and flavonoids (Table 7) could reduce or inhibit IAA oxidase enzyme activity, thereby increasing IAA levels leading to improved Egyptian clover growth and yield42.

Regarding the effect of forage yield throughout the four cuts, the obtained results are in harmony with those obtained earlier by Juskiw43, Gaballah44, Patel and Rajagopal45, Yucel et al.46 and Shahrajabian47. In this content, Soleymani and Shahrajabian48 and Thalooth et al.49 reported that the highest fresh yield was recorded for the 2nd cut followed by the1st cut then the 3rd one and dry weight (g/m2) increased from the 1st to the 2nd cut up to 3rd cut. Moreover, El-Karamany et al.50 noted that the production of dry forge increased from 1st-2nd and 3rd harvest after treatment with bioorganic+mineral fertilizers (1.1432.026 and 3.093 ton/fd.) followed by the mineral fertilization treatments that produced 0.934, 1.838 and 2.746 ton/fad., for the successive three cuts of Egyptian berseem clover. Moreover, this is in agreement with the results obtained by El Karamany et al.50, Omer et al.51, Abdel-Magid et al.52 and Salama53, who noticed that clover hay on a dry matter basis (in average) contained 92.00, 87.17, 13.40, 26.03, 4.03, 43.71, 43.20, 30.06, 5.54%, 4153 and 2661 kca kg1 DM of DM, OM, CP, CF, EE, NFE, Ash, NDF, ADF and ADL, Gross Energy (GE) and Digestible Energy (DE), respectively.

From the results of our study, we found that all pyridoxine levels realized significant increases in Egyptian clover yield quantity as well as their crude protein content while crude fibre content was significantly decreased by spraying Egyptian clover by 50 or 75 mg L1 compared with control and 100 mg L1. Furthermore, all levels of pyridoxine significantly decreased the contents of nitrogen-free extract. Therefore, we recommend from our study, the cultivation of Egyptian clover and treatment with peroxide in sandy soils. So we can be concluded that pyridoxine can be safely used without adversely affecting the cultivated plants and this has led to an increase in feed yield and an improvement in nutritional value.

CONCLUSION

This study discovers the promotive role of exogenous pyridoxine as natural vitamin on plants especially on Egyptian clover that can be beneficial for increasing yield and yield component as well as improving its nutritive value. Pyridoxine as a natural and safely plant component affect plant growth and productivity without adversely affecting the cultivated plants, through increasing photosynthetic pigments, IAA contents as well as, increasing content of DM, OM, CP, CF, EE, NFE, Ash, NDF, ADF and ADL, Gross Energy (GE) and Digestible Energy (DE). So, this study will help the researcher to uncover the critical areas of pyridoxine as a natural to improve plant growth and productivity under sandy conditions.

SIGNIFICANCE STATEMENT

This study discovers the possible promotive role of vitamins especially Pyridoxine in increasing growth and foraging yield quantity of Egyptian clovers all over the four cuts this study will help the researcher to uncover the critical areas of pyridoxine as a natural to improve plant growth and productivity under sandy conditions.

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