Effect of Micronutrients on the Biochemical Contents of Mulberry (Morus alba L. Moraceae) Leaves

Introduction

In moriculture, quality of mulberry leaves has been considered as a prime feature in good crop production.¹ Better the leaf quality, greater are the possibilities of obtaining good cocoons, resulting in qualitative and quantitative silk production. The biochemical composition of mulberry leaf, viz., carbohydrates, proteins, minerals, fatty acids, amino acids and vitamins satisfy the nutritional requirement of silkworm larvae ensuring their healthy growth, and development of quality silk gland.² Application of fertilizers and biofertilizers have been reported to increase the biochemical contents of the mulberry leaves with regard to its nutritional status.^3-5 Albeit, information on the nutritional status of the biochemical aspects of mulberry leaves through soil application of micronutrients is scanty. The quality and quantity of mulberry leaves can be increased by adopting physiological manipulations.⁶ Micronutrients are involved in numerous metabolic events of mulberry plant viz., photosynthesis, chlorophyll establishment, cell wall development, water absorption and xylem permeability, plant growth and resistance to plant diseases, responsible for quality leaf production.⁷ Further, micronutrients perform the role of cofactors in enzyme stimulation, partake in redox reactions, photosynthesis and respiration, besides playing an indispensable part in the metabolism of carbohydrates and translocation of sugars.^8-12 Therefore, in the present study, the impact of micronutrients on the biochemical contents of mulberry leaves was assessed.

Materials and Methods

Study area

A three year old mulberry garden, free from other plants which received direct sunlight exposure with proper irrigation served for conducting field experiments. This experimental plot was situated at an altitude of 29 m above sea level at Poovancode village, Kanyakumari district, Tamil Nadu, India (8.3031° N, 77.2881° E).

Mulberry cultivation

For the experimental study, MR₂ (Mildew Resistant Variety 2) mulberry plant (Morus alba) was selected. This was developed by the Sericulture Department, Govt. of Tamil Nadu experimental station, Coonoor, Tamil Nadu, India. The mulberry plants were pruned in the month of June, ploughed, FYM applied at 20t/ha/year, and a single dose of nitrogen, phosphorous and potash at 120:120:60 kg/ha/year was hoed in the soil uniformly. All of the above were done prior to the commencement of the experiment. Irrigation at an interval of five day was provided, depending upon the climatic conditions. After twenty days of pruning, the micronutrients were added to the soil. Care was taken to ensure that the experimental plot was protected from plant pests and also in periodical removal of diseased/affected parts of the plant.

Experimental design and treatments

A randomized block design with twelve treatments with spacing of 90x60cm between the plants was chosen for the field experiments. Recommended dose of fertilizers and the macronutrients, viz., nitrogen, phosphorous and potassium (NPK) in the form of ammonium sulphate, super phosphate, potash (120:120:60) was common for all treatments. Each treatment (except control) was supplemented with the required amount of micronutrients, viz., zinc, copper and iron in the form of zinc sulphate, copper sulphate and ferrous sulphate either individually or in combination (Table 1) and were given as soil application. Each treatment replicated thrice, with ten plants per replication was supplemented with the required amount of micronutrients.

Table 1: Treatments used for the present study

Treatment		Micronutrients (individual/combination)
T0	:	Control
T1	:	FeSO4 10Kg/ha
T2	:	Zn SO4 5Kg/ha
T3	:	CuSO4 5Kg/ha
T4	:	CuSO4 5Kg/ha	+	ZnSO4 5Kg/ha
T5	:	CuSO4 5Kg/ha	+	FeSO4 10Kg/ha
T6	:	FeSO4 10Kg/ha	+	ZnSO4 5Kg/ha
T7	:	CuSO4 5Kg/ha	+	ZnSO4 5Kg/ha	+	FeSO4 10Kg/ha
T8	:	CuSO4 10Kg/ha	+	ZnSO4 10Kg/ha	+	FeSO4 20Kg/ha
T9	:	CuSO4 15Kg/ha	+	ZnSO4 15Kg/ha	+	FeSO4 30Kg/ha
T10	:	CuSO4 20Kg/ha	+	ZnSO4 20Kg/ha	+	FeSO4 40Kg/ha
T11	:	CuSO4 25Kg/ha	+	ZnSO4 25Kg/ha	+	FeSO4 25Kg/ha

Five plants in each replication were randomly selected and labelled for recording observations at the 45^th and 60^th day of pruning for its biochemical parameters viz., leaf moisture, leaf moisture retention, chlorophyll ‘a’, ‘b’, total chlorophyll, carbohydrates, proteins, free amino acids and nitrogen.

Biochemical analysis of mulberry leaf

Leaf moisture and leaf moisture retention was estimated through gravimetric method on fresh weight basis.¹³ Chlorophyll ‘a’, ‘b’, and total chlorophyll content of leaves^14-16 were estimated using spectrophotometer at a wavelength of 645 to 663nm, and computed via Arnon’s formulae,¹⁷ and expressed in mg/g of leaf on fresh weight basis. The harvested leaves (at 45^th and 60^th day of pruning) from the plant were oven dried at 70^°C for one hour and powdered. The dried leaf samples were then analysed for carbohydrate,^18-21 total protein,^22,23 total free amino acid²⁴ and nitrogen²⁵ content. 

Statistical analysis

Pooled data affirmed as Mean ±S.D, and subjected to Student’s ‘t’ test to determine significant difference between control and treatment groups. 

Results

Leaf moisture

The leaf moisture was found to be more in apical leaves, followed by middle and bottom leaves. Maximum mean leaf moisture content on the 45^th and 60^th day of pruning was noted in T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) (80.72 ±1.44%) and (81.41 ±3.32%) which increased by 10.30% and 12.39% over control, respectively and its minimum was recorded in T₆ (FeSO₄ 10Kg/ha + ZnSO₄ 5Kg/ha) (74.48 ±1.31%) which increased by 1.77% over control, and in T₄ (CuSO₄ 5Kg/ha + ZnSO₄ 5Kg/ha) (74.8 ±4.44%) with an increase of 3.27% over control, respectively (Table 2; Figure 1). 

Leaf moisture retention

The bottom leaves had higher moisture retention percentage when compared to top and middle leaves. Maximum mean leaf moisture retention percentage on the 45^th and 60^th day of pruning was noted in T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) (70.70 ±7.86%) and (80.67 ±5.66%) which increased by 29.32% and 11.02% over control, respectively and its minimum was recorded in T₅ (CuSO₄ 5Kg/ha + FeSO₄ 10Kg/ha) (59.58 ±1.34%) with an increase of 8.98% over control, and in T₁₁ (CuSO₄ 25Kg/ha + ZnSO₄ 25Kg/ha + FeSO₄ 25Kg/ha) (77.12 ±2.26%) with an increase of 6.13% over control respectively (Table 3; Figure 1). 

Chlorophyll

On the 45^th day of pruning, maximum and minimum chlorophyll ‘a’ content was observed in T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) (1.80 ±0.25mg/g) and T₃ (CuSO₄ 5Kg/ha) (1.61 ±0.08mg/g) which increased by 18.42% and 5.92%, respectively over control; while for chlorophyll ‘b’ it was recorded at T₇ (CuSO₄ 5Kg/ha + ZnSO₄ 5Kg/ha + FeSO₄ 10Kg/ha) (0.58 ±0.05mg/g) and T₁₁ (CuSO₄ 25Kg/ha + ZnSO₄ 25Kg/ha + FeSO₄ 25Kg/ha) (0.42 ±0.01mg/g) which increased by 48.71% and 7.69%, respectively over control. The total chlorophyll content was maximum in T₈ (2.34 ±0.34mg/g) which increased by 21.87% over control; and the minimum value was reported in T₁ (FeSO₄ 10Kg/ha) (2.05 ±0.12mg/g) with an increase of 6.77% over control (Table 4; Figure 1). Whereas, on the 60^th day of pruning, maximum and minimum chlorophyll ‘a’ content was observed in T₈ and T₉ (CuSO₄ 15Kg/ha + ZnSO₄ 15Kg/ha + FeSO₄ 30Kg/ha) (1.91 ±0.10mg/g and 1.91 ±0.08mg/g) and T₁ (1.16 ±0.06mg/g) which increased by 29.05% and 2.03%, respectively over control; while for chlorophyll ‘b’ it was again recorded in T₈ and T₉ (0.64 ±0.12 and 0.64 ±0.13mg/g) with an increase of 18.52% over control and in T₁ and T₂ (ZnSO₄ 5Kg/ha) (0.54 ±0.16mg/g) which was on par with the control. Maximum total chlorophyll content was observed in T₉ (2.56 ±0.18mg/g) which increased by 26.25% when compared to control; and the minimum value was reported in T₁ (2.05 ±0.18mg/g) with an increase of 1.48% over control (Table 4; Figure1). 

Carbohydrate

The soil application of micronutrients increased the sugar content in middle leaves followed by apical and bottom leaves. On the 45^th and 60^th day of pruning, maximum carbohydrate content was recorded in T₁₀ (CuSO₄ 20Kg/ha + ZnSO₄ 20Kg/ha + FeSO₄ 40Kg/ha) (373.41 ±45.65µg/mg) and T₉ (CuSO₄ 15Kg/ha + ZnSO₄ 15Kg/ha + FeSO₄ 30Kg/ha) (236.66 ±62.13µg/mg) which increased by 22.25% and 24.99% respectively, over control, and its minimum was recorded in T₁₁ (CuSO₄ 25Kg/ha + ZnSO₄ 25Kg/ha + FeSO₄ 25Kg/ha) (317.53 ±60.47µg/mg) and T₇ (CuSO₄ 5Kg/ha + ZnSO₄ 5Kg/ha + FeSO₄ 10Kg/ha) (195.66 ±32.46µg/mg) which increased by 3.96% and 3.34% respectively, over control (Table 5; Figure 1). 

Protein

The soil application of micronutrients increased the protein content in middle leaves followed by apical and bottom leaves. On the 45^th and 60^th day of pruning, maximum protein content was recorded in T₉ (CuSO₄ 15Kg/ha + ZnSO₄ 15Kg/ha + FeSO₄ 30Kg/ha) (405.16 ±83.51µg/g) and T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) (385.20 ±3.38µg/g) which increased by 60.56% and 70.69% respectively, over control, and its minimum was recorded in T₂ (ZnSO₄ 5Kg/ha) (312.83 ±15.46µg/g) and T₃ (CuSO₄ 5Kg/ha) (246.16 ±59.07µg/g) which increased by 23.97% and 9.08% respectively, over control (Table 6; Figure 1). 

Free amino acid

In all the treatments, free amino acid content was higher in the apical leaves followed by middle and bottom leaves. On the 45^th and 60^th day of pruning, maximum free amino acid content was recorded in T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) (41.83 ±9.81µg/g) and T₄ (CuSO₄ 5Kg/ha + ZnSO₄ 5Kg/ha) (19.33 ±8.86µg/g) which increased by 112.76% and 123.21% respectively, over control, and its minimum was recorded in T₁₀ (CuSO₄ 20Kg/ha + ZnSO₄ 20Kg/ha + FeSO₄ 40Kg/ha) (20.0 ±2.64µg/g) and T₁ (FeSO₄ 10Kg/ha) (9.66 ±3.88µg/g) which increased by 1.72% and 11.54% respectively over control (Table 7; Figure 1).

Nitrogen

In all the treatments, nitrogen content was higher in the apical leaves followed by middle and bottom leaves. On the 45^th and 60^th day of pruning, maximum nitrogen content was recorded in T₉ (CuSO₄ 15Kg/ha + ZnSO₄ 15Kg/ha + FeSO₄ 30Kg/ha) (4.50 ±0.61%) and T₁₀ (CuSO₄ 20Kg/ha + ZnSO₄ 20Kg/ha + FeSO₄ 40Kg/ha) (3.46 ±0.06%) which increased by 30.81% and 11.93% respectively, over control, and its minimum was recorded in T₁ (FeSO₄ 10Kg/ha) (3.67 ±0.57%) and T₆ (FeSO₄ 10Kg/ha + ZnSO₄ 5Kg/ha) (3.13 ±0.12%) which increased by 6.68% and 0.96% respectively, over control (Table 8; Figure 1).

Table 2: Effect of micronutrients on leaf moisture of mulberry leaf

Treatment	45th day of pruning				60th day of pruning
	Top	Middle	Bottom	Mean	Top	Middle	Bottom	Mean
T0	75.75 ±0.88	72.97 ±2.06	70.82 ±0.09	73.18 ±2.47	73.58 ±0.71	72.57 ±1.29	71.14 ±1.01	72.43 ±1.22
T1	80.57 ±0.21	78.89 ±0.50	77.6 ±2.26	77.39 ±2.72	78.05 ±1.03	76.58 ±0.71	75.13 ±1.02	76.58 ±1.46
T2	80.14 ±1.43	78.97 ±1.32	77.12 ±0.44	78.74 ±1.52	78.22 ±1.40	76.23 ±1.0	72.15 ±2.88	75.53 ±3.09
T3	80.0 ±0.05	78.06 ±2.04	75.23 ±0.16	77.76 ±2.39	80.85 ±2.24	77.71 ±0.61	72.64 ±3.58	77.06 ±4.14
T4	77.79 ±1.4	75.91 ±2.14	73.66 ±0.17	76.12 ±2.57	79.90 ±5.07	72.72  ±1.14	71.78 ±0.68	74.80 ±4.44
T5	79.41 ±2.7	77.80 ±1.14	76.86 ±2.43	78.02 ±1.28	82.90 ±4.58*	76.42 ±0.79	74.47 ±1.37	77.93 ±4.41
T6	75.86 ±1.43	74.34 ±1.11	73.24 ±2.38	74.48 ±1.31	79.22 ±1.74	76.75 ±0.12	73.42 ±2.59	76.46 ±2.91
T7	79.17 ±3.1	77.88 ±1.27	76.14 ±1.43	77.73 ±1.52	83.23 ±3.22*	78.67 ±1.16	75.32 ±3.36	79.07 ±3.97
T8	82.22 ±3.61*	80.62 ±1.19*	79.33 ±1.67*	80.72 ±1.44*	80.78 ±2.98	85.0 ±1.00*	78.45 ±4.63*	81.41 ±3.32*
T9	81.48 ±2.64*	80.22 ±1.17*	78.66 ±2.83*	80.12 ±1.41*	81.12 ±1.02*	79.67 ±0.66	80.62 ±0.07*	80.47 ±0.73*
T10	79.41 ±2.74	77.53 ±1.34	76.32 ±1.16	77.75 ±1.55	81.64 ±2.57*	78.0 ±1.00	76.59 ±0.92	78.74 ±2.60
T11	77.14 ±3.2	75.37 ±1.51	73.45 ±0.76	75.32 ±1.84	82.56 ±2.10*	79.58 ±0.71	75.28 ±2.04	79.14 ±3.65

Values expressed in %; *Significant @ P<0.05 (t-test) 

Table 3: Effect of micronutrients on leaf moisture retention of mulberry leaf

Treatment	45th day of pruning				60th day of pruning
	Top	Middle	Bottom	Mean	Top	Middle	Bottom	Mean
T0	54.16 ±1.86	56.52 ±2.25	53.33 ±2.12	54.67 ±2.91	78.78 ±1.71	76.36 ±4.86	62.84 ±4.32	72.66 ±8.59
T1	60.14 ±5.85*	68.42 ±3.71*	73.68 ±4.43*	67.41 ±6.82*	80.23 ±1.19	78.54 ±1.96	72.65 ±2.18*	77.14 ±3.97
T2	58.31 ±4.51	64.70 ±4.25*	70.58 ±4.27*	64.53 ±6.13*	81.89 ±2.51	78.34 ±0.86	74.43 ±2.59*	78.22 ±3.73
T3	59.32 ±1.82	61.90 ±5.18*	69.23 ±4.06*	63.48 ±5.14*	80.50 ±1.61	78.21 ±1.42	73.38 ±2.21*	77.36 ±3.63
T4	59.28 ±6.46	68.42 ±2.26*	65.22 ±0.64*	64.30 ±4.63*	82.75 ±0.85	81.54 ±3.30	72.63 ±2.67*	78.97 ±5.52
T5	58.55 ±0.38	59.09 ±1.42	61.11 ±1.07*	59.58 ±1.34	80.65 ±2.21	77.52 ±0.49	74.82 ±2.11*	77.66 ±2.91
T6	54.47 ±7.09	62.5 ±5.56*	70.37 ±6.07*	61.78 ±8.97*	81.36 ±0.58	80.53 ±3.39	71.63 ±2.48*	77.84 ±5.39
T7	57.81 ±5.23	65.21 ±1.11*	63.64 ±1.04*	62.22 ±3.89*	79.35 ±1.43	77.32 ±0.18	79.36 ±0.47*	78.67 ±1.17
T8	61.87 ±10.64*	76.92 ±2.53*	73.33 ±1.85*	70.70 ±7.86*	84.12 ±0.25*	83.76 ±3.82*	74.14 ±2.43*	80.67 ±5.66*
T9	63.09 ±0.64*	64.0 ±5.25*	71.43 ±3.71*	66.17 ±4.57*	83.97 ±1.81	81.40 ±2.42	73.87 ±2.98*	79.74 ±5.24
T10	59.79 ±3.83*	65.21 ±2.10*	68.18 ±2.67*	64.39 ±4.25*	82.97 ±2.25	79.78 ±2.32	72.18 ±3.29*	78.31 ±5.54
T11	58.29 ±2.29	61.53 ±6.40	70.59 ±5.03*	63.47 ±6.37*	79.13 ±1.10	77.57 ±0.82	74.67 ±1.41*	77.12 ±2.26

Values expressed in %; *Significant @ P<0.05 (t-test) 

Table 4: Effect of micronutrients on the chlorophyll content of mulberry leaf Click here to view Table

Values expressed in mg/g; *Significant @ P<0.05 (t-test)

Table 5: Effect of micronutrients on the carbohydrate content of mulberry leaf

Treatment	45th day of pruning				60th day of pruning
	Top	Middle	Bottom	Mean	Top	Middle	Bottom	Mean
T0	310.13 ±42.42	371.50 ±3.53	234.67 ±10.77	305.43 ±71.45	232.50 ±19.60	181.5 ±26.16	154.0 ±5.65	189.33 ±37.65
T1	385.21 ±21.21*	355.86 ±63.63	267.57 ±15.09*	336.21 ±61.23*	263.50 ±10.61*	210.0 ±70.71*	158.50 ±2.12	210.66 ±52.50*
T2	355.63 ±21.21*	325.45 ±21.23	280.93 ±16.99*	320.67 ±37.57	250.50 ±16.57	250.0 ±107.27*	160.50 ±0.70	220.33 ±51.81*
T3	310.0 ±0.0	380.19 ±84.85	348.12 ±27.82*	346.10 ±35.13*	269.50 ±16.27*	223.0 ±80.61*	162.50 ±4.94	218.33 ±53.65*
T4	385.72 ±16.0*	362.50 ±3.53	269.13 ±15.97*	339.11 ±61.71*	259.0 ±12.83*	231.50 ±101.01*	155.50 ±3.53	215.33 ±53.61*
T5	390.81 ±14.14*	385.22 ±7.07	259.50 ±14.12*	345.17 ±74.25*	268.50 ±17.68*	270.50 ±96.85*	159.0 ±2.82	232.66 ±63.80*
T6	360.37 ±28.28*	410.84 ±42.42*	264.45 ±16.34*	345.22 ±74.36*	264.50 ±13.34*	268.50 ±57.68*	153.0 ±5.65	228.66 ±65.55*
T7	305.54 ±49.49	375.14 ±48.49	313.32 ±27.18*	331.33 ±38.13	218.50 ±72.83	210.0 ±84.85*	158.50 ±12.0	195.66 ±32.46
T8	345.23 ±49.49*	420.32 ±56.56*	298.46 ±20.11*	354.67 ±61.47*	238.50 ±15.25	234.50 ±106.77*	158.0 ±1.41	210.33 ±45.36*
T9	430.57 ±42.42*	375.25 ±7.07	278.53 ±17.83*	361.45 ±76.95*	269.50 ±16.27*	275.50 ±47.78*	165.0 ±8.48*	236.66 ±62.13*
T10	410.0 ±62.63*	388.0 ±61.35	322.25 ±22.73*	373.41 ±45.65*	237.50 ±12.53	216.0 ±94.65*	163.50 ±2.12	205.66 ±38.06
T11	270.47 ±41.42	385.75 ±21.21	296.39 ±20.93*	317.53 ±60.47	261.50 ±13.44*	219.0 ±72.12*	160.50 ±10.6	213.66 ±50.71*

Values expressed in µg/mg; *Significant @ P<0.05 (t-test)

Table 6: Effect of micronutrients on the protein content of mulberry leaf

Treatment	45th day of pruning				60th day of pruning
	Top	Middle	Bottom	Mean	Top	Middle	Bottom	Mean
T0	268.50 ±19.30	311.50 ±20.59	268.0 ±1.14	252.33 ±75.66	223.0 ±1.41	250.0 ±34.30	204.0 ±32.50	225.66 ±23.11
T1	330.12 ±10.07*	367.50 ±25.50*	332.50 ±13.43*	343.37 ±20.9*	278.0 ±57.30	308.0 ±35.38	238.0 ±20.81	274.66 ±55.67*
T2	321.0 ±57.98*	322.50 ±60.10	295.0 ±21.21*	312.83 ±15.46*	275.0 ±49.49	429.50 ±67.9	158.0 ±15.96	287.50 ±136.14*
T3	328.50 ±19.09*	355.0 ±13.0*	325.12 ±14.31*	336.20 ±16.36*	278.0 ±28.56	282.50 ±29.03	178.0 ±14.27	246.16 ±59.07
T4	334.50 ±16.26*	357.50 ±11.50*	331.62 ±12.19*	341.20 ±14.18*	317.0 ±4.42	333.0 ±21.0	316.0 ±5.65	322.0 ±9.53*
T5	316.50 ±12.02*	338.0 ±13.0	314.37 ±9.01*	322.95 ±13.07*	308.0 ±23.24	321.50 ±48.19	298.0 ±29.10	309.83 ±7.01*
T6	407.0 ±59.39*	414.50 ±48.79*	260.50 ±26.57	360.66 ±86.82*	311.50 ±10.60	341.50 ±17.50	314.0 ±14.11	322.33 ±16.60*
T7	373.50 ±13.42*	381.0 ±17.0*	375.87 ±10.07*	376.79 ±3.83*	315.87 ±11.10	338.0 ±9.0	318.50 ±14.84	324.12 ±12.08*
T8	400.50 ±6.36*	410.0 ±84.85*	375.0 ±91.92*	395.16 ±62.63*	384.12 ±6.89	389.0 ±13.0	382.50 ±9.19	385.20 ±3.38*
T9	408.0 ±93.33*	408.50 ±8.48*	399.0 ±21.83*	405.16 ±83.51*	368.12 ±2.61	398.50 ±3.50	367.50 ±3.53	378.04 ±17.25*
T10	396.50 ±45.30*	403.50 ±16.17*	329.50 ±40.75*	376.50 ±85.54*	356.87 ±2.65	386.50 ±6.50	377.50 ±3.53	373.62 ±15.19*
T11	366.50 ±0.70*	383.50 ±23.33*	343.50 ±33.23*	364.50 ±20.07*	312.50 ±3.53	326.0 ±16.0	313.12 ±2.65	317.20 ±7.62*

Values expressed in µg/g; *Significant @ P<0.05 (t-test) 

Table 7: Effect of micronutrients on the free amino acid content of mulberry leaf

Treatment	45th day of pruning				60th day of pruning
	Top	Middle	Bottom	Mean	Top	Middle	Bottom	Mean
T0	19.50 ±12.02	22.0 ±5.65	17.50 ±6.36	19.66 ±4.25	8.66 ±3.01	2.0 ±1.19	1.0 ±0.01	8.66 ±3.88
T1	29.0 ±18.38*	22.0 ±8.48	35.0 ±9.89*	28.66 ±6.50*	9.66 ±8.54	2.70 ±1.74*	2.90 ±0.82*	9.66 ±3.88
T2	27.50 ±23.33*	33.50 ±14.84*	17.0 ±8.48	26.0 ±8.35*	10.0 ±5.29*	2.60 ±0.02	6.80 ±2.10*	10.0 ±5.08*
T3	36.50 ±17.67*	40.50 ±12.02*	28.0 ±5.65*	35.0 ±6.38*	15.0 ±2.76*	9.0 ±1.63*	2.60 ±1.36*	15.0 ±6.46*
T4	29.0 ±15.55*	35.0 ±7.07*	24.0 ±8.48*	29.33 ±5.50*	19.33 ±1.01*	5.0 ±2.47*	2.00 ±0.03*	19.33 ±8.86*
T5	30.50 ±53.03*	20.0 ±5.65	27.50 ±47.37*	25.83 ±5.34*	13.66 ±1.18*	13.50 ±1.73*	2.10 ±0.06*	13.66 ±8.77*
T6	27.50 ±9.19*	34.50 ±19.09*	41.0 ±9.89*	34.33 ±6.75*	14.33 ±3.50*	14.0 ±1.31*	2.0 ±0.02*	14.33 ±9.75*
T7	25.0 ±1.41*	36.0 ±16.97*	37.0 ±15.55*	41.66 ±19.4*	12.66 ±3.31*	15.0 ±1.32*	1.90 ±1.25	12.66 ±8.18*
T8	30.50 ±0.70*	47.50 ±24.74*	47.50 ±24.74*	41.83 ±9.81*	11.66 ±2.20*	10.0 ±2.73*	2.90 ±0.84*	11.60 ±7.26*
T9	40.50 ±17.67*	41.50 ±16.26*	29.0 ±1.41*	37.0 ±6.94*	16.0 ±6.70*	4.0 ±0.14*	1.80 ±0.07	16.0 ±10.22*
T10	21.0 ±7.07	22.0 ±5.65	17.0 ±1.41	20.0 ±2.64	17.0 ±3.89*	8.0 ±1.10*	10.0 ±0.47*	17.0 ±9.85*
T11	24.50 ±16.26*	44.0 ±11.31*	32.50 ±27.57*	33.66 ±9.80*	13.66 ±9.60*	13.0 ±1.87*	4.0 ±1.12*	13.66 ±10.11*

Values expressed in µg/g; *Significant @ P<0.05 (t-test)

Table 8: Effect of micronutrients on the nitrogen content of mulberry leaf

Treatment	45th day of pruning				60th day of pruning
	Top	Middle	Bottom	Mean	Top	Middle	Bottom	Mean
T0	3.99 ±0.55	3.21 ±0.06	3.12 ±0.22	3.44 ±0.47	3.11 ±0.43	3.10 ±0.64	3.10 ±0.27	3.10 ±0.05
T1	4.32 ±0.76	3.24 ±0.15	3.46 ±0.15*	3.67 ±0.57	3.19 ±0.75	3.12 ±0.47	3.17 ±0.84	3.16 ±0.03
T2	4.29 ±0.48	3.61 ±0.21*	3.31 ±0.30	3.73 ±0.50	3.18 ±0.83	3.16 ±0.32	3.24 ±0.69	3.19 ±0.04*
T3	4.23 ±0.48	3.54 ±0.19*	3.27 ±0.28	3.68 ±0.49	3.28 ±0.90	3.25 ±0.18	3.27 ±0.91	3.24 ±0.03*
T4	4.53 ±0.72*	3.50 ±0.01	3.48 ±0.25*	3.83 ±0.60*	3.14 ±0.83	3.16 ±0.42	3.12 ±0.01	3.14 ±0.02
T5	4.63 ±0.73*	3.59 ±0.02*	3.62 ±0.23*	3.94 ±0.59*	3.40 ±0.53	3.31 ±0.13	3.29 ±0.98	3.33 ±0.05*
T6	4.01 ±0.01	3.99 ±0.28*	3.59 ±0.19*	3.86 ±0.23*	3.28 ±0.42	3.08 ±0.43	3.05 ±0.76	3.13 ±0.12
T7	4.45 ±0.09*	4.32 ±0.28*	3.91 ±0.22*	4.22 ±0.28*	3.32 ±0.19	3.20 ±0.14	3.17 ±0.91	3.23 ±0.07
T8	4.76 ±0.52*	4.02 ±0.16*	3.78 ±0.28*	4.18 ±0.51*	3.49 ±0.25*	3.18 ±0.06	3.16 ±0.57	3.27 ±0.18
T9	5.20 ±0.67*	4.24 ±0.12*	4.06 ±0.31*	4.50 ±0.61*	3.51 ±0.23*	3.28 ±0.79	3.25 ±0.12	3.34 ±0.14
T10	3.87 ±0.24	4.22 ±0.62*	3.34 ±0.33	3.81 ±0.44*	3.53 ±0.47*	3.46 ±0.54*	3.41 ±0.32*	3.46 ±0.06*
T11	4.08 ±0.26	3.71 ±0.14*	3.50 ±0.18*	3.76 ±0.29	3.45 ±0.63*	3.38 ±0.93	3.32 ±0.25	3.38 ±0.06*

Values expressed in %; *Significant @ P<0.05 (t-test)

Figure 1: Effect of micronutrients on biochemical contents of mulberry leaves Click here to view Figure

Discussion

Micronutrients have an impact on the mulberry plant in terms of quality, growth and yield,²⁶ and contribute to the foremost part in numerous metabolic actions accountable for synthesis of proteins, sugars and enzymes, which leads to superior production of quality mulberry leaf.²⁷. Machii and Katagiri²⁸ and Suryanarayan and Shivashankar²⁹ reported that increased contents of nitrogen and amino acids in mulberry leaves are nutritionally greater and are positively linked to silkworm development and growth. The present results opined that soil application of micronutrients supplemented the betterment of yield and quality in terms of proteins and carbohydrates, as these two biochemical constituents determines the feeding value of silkworm which in turn reflects upon the silk production. 

Leaf moisture

Moisture content in mulberry leaves is a very important factor which influences silkworm growth, as it has an intimate relation to an easy ignition of leaves to silkworm, as high moisture make it more palatable to them. Micronutrients treated mulberry leaves revealed significant variations in moisture and moisture retention capacity. Yokoyama³⁰ reported 64-83% moisture content in mulberry leaves. On the 45^th and 60^th day of pruning, the moisture capacity in the present study increased to 10.30% – 12.39% as the maximum in T₈ treatment (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) when compared to control. Higher moisture content in T₈ treatment may be due to genetic character, and influence of balanced fertilizers combination which helped in uptake of available nutrients. During the study period, the tender leaves had more moisture content than middle and bottom leaves. Leaves possessing higher leaf moisture content are recognised as good quality leaves,^31,32 and are considerably related to the silkworm growth and nutritional parameters³³ The moisture content in mulberry leaves plays an important part in uplifting the nutritional levels, which in turn improves the scrumptiousness and digestibility of leaves by silkworms, feeding efficiency of silkworm larvae, increased growth rate, as well as quality development of cocoon.^34,35 An optimum level of leaf moisture content is directly related to the growth of silkworm, since low leaf moisture content influences the assimilation and conversion efficiency of food (energetic parameters) to decrease. Sinha et al.³⁶ reported that corresponding increased leaf growth and maturity can lead to a gradual decreased in the moisture content of mulberry leaves. The present findings correlated with the above facts. The enhancement of leaf moisture due to application of micronutrients might be due to enhancement of organic matter and water holding capacity in the soil, thereby, increasing the water absorption by plant.³⁷ The current observations are related to the results of Babu et al.³⁸ too. Increased leaf moisture content, and fresh leaf weight are due to the water retention capacity which steadily supplies moisture to the leaf. Further, microbial inoculants in the soil rhizosphere may also facilitate in the moisture accessibility, towards water uptake and added metabolic activities for upholding normal plant growth.

Leaf moisture retention

Moisture retention capacity also play a vital role, because silkworms prefer fresh leaves with high moisture for longer time.³⁹ Micronutrients influences the moisture retention capacity of mulberry leaves due to water retention capacity of organic manures which steadily supplied the moisture.^40-42 Increased moisture retention capacity in T₈ treatment (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) was 11.02% – 29.32% higher when compared to control on the 45^th and 60^th day of pruning. This may be due to increased stomata size due to uptake of water, and balanced mixture of zinc, copper and iron. 

Chlorophyll

Chlorophyll content plays a vital role in computing the photosynthetic productivity of plant, and is a critical component in evaluating leaf quality. Total chlorophyll content was recorded high in T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) and T₉ (CuSO₄ 15Kg/ha + ZnSO₄ 15Kg/ha + FeSO₄ 30Kg/ha) treated mulberry leaves on the 45^th and 60^th day of pruning, which increased to 21.87% and 26.25% when compared with control. Micronutrients significantly increased the levels of chlorophyll reflecting its impact on the increased qualitative parameters of leaf. The amplified volume of chlorophyll content in leaves specifies the photosynthetic effectiveness, the primary criterion for measuring photosynthetic proportion in mulberry.³² The positive effect of micronutrients on chlorophyll content of mulberry leaves might be attributed to the fact that nitrogen, an essential constituent of chlorophyll helps in harnessing more solar energy, and plays a greater role in improving the chlorophyll synthesis, an essential constituent of photosynthesis.⁴³ Increased chlorophyll content of mulberry leaves be due to foliar nutrition.^37,44 In the present study, increased chlorophyll content was obviously due to zinc which acted as a structural and catalytic components of protein, enzymes and as cofactors for normal development of pigment biosynthesis.⁴⁵

Carbohydrate

Leaf quality determined by the presence of sugar, as the main source of energy, induces the silkworms to bite the leaves (biting factor) and to cherish it well, which in turn influences the healthy growth and development of silkworms. Carbohydrates, in mulberry leaves are present abundantly, and are stated to be the silkworm’s primary source of energy.^46,47 High content of total sugar in mulberry leaf was accredited to the enhanced mineralization resulting in superior production of plant growth substances and enzyme activity due to soil application of micronutrients. The photosynthetic action aids in synthesis of carbohydrates in mulberry leaves, and principally the sugar content are in close relation with the silkworm health. The carbohydrate content significantly varied among the treatments and the highest carbohydrate content was found in the T₁₀ (CuSO₄ 20Kg/ha + ZnSO₄ 20Kg/ha + FeSO₄ 40Kg/ha) and T₅ (CuSO₄ 5Kg/ha + FeSO₄ 10Kg/ha) treated mulberry leaves which increased by 22.25% and 24.99% when compared to control on the 45^th and 60^th day of pruning respectively. This might be due to rapid conversion of starch to sugar. Further, optimum combination of micronutrients amplified nutrient uptake from soil, and caused the plant to drive additional sugars and extra exudates from its roots. 

Protein

Leaf protein is a key element for silkworm nutrition, and approximately 70% of protein content of raw silk are biosynthesized directly from leaf protein, and 30% from the body tissue of silkworm.^48,49 Protein content in mulberry leaves have a direct bearing on silkworm, mostly on larval growth with special reference to development of silk gland and cocoon characters.³¹ In the present study, the protein content was maximum in T₉ (CuSO₄ 15Kg/ha + ZnSO₄ 15Kg/ha + FeSO₄ 30Kg/ha) and T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) treated mulberry leaves which increased by 60.56% and 70.69%, when compared with control. This was due to zinc in combination with copper and iron. Zinc and copper are taken up in the form of their respective ions, by the plants, and iron in the form of ferrous or ferric ions, which are needed for protein synthesis, and are involved in the activation of several enzyme systems. Further, the protein content of middle leaves was more when compared with top and bottom leaves, which was contradictory to the results of Murthy et al.⁵⁰ but correlates with the findings of Singhvi et al.⁵¹ Protein, the core constituent of mulberry leaf, contributes to silk synthesis,⁵² and increase in the protein content is directly revealed on the leaf yield. Increased protein content might be due to the accessibility of adequate amount of nitrogen to the plants. Increase in crude protein was largely accredited to the soil application of micronutrients which were rapidly absorbed by the leaves, thereby activating plant metabolism, and thus leading to production of healthy green foliage, and the same was stated by Singhvi et al.⁵¹ too. Increased protein content in mulberry leaves due to micronutrient application was strongly supported by Sterling,⁵³ through microorganisms, as it signifies increased nutritional status of mulberry leaves with regard to biochemical aspects.⁵ 

Free amino acid

Amino acids determine the leaf quality.⁵⁴ Mulberry leaves, the significant constituent for silkworm nutrition are rich in amino acids.^8,55 Amino acids help silkworm in food selection, and are utilised for the formation of haemolymph, development of silk glands and cocoon production. In the present study, the highest amino acid content was recorded in T₈ (CuSO₄ 10Kg/ha + ZnSO₄ 10Kg/ha + FeSO₄ 20Kg/ha) and T₄ (CuSO₄ 5Kg/ha + ZnSO₄ 5Kg/ha) treated apical mulberry leaves which may be due to the application of required amount and combined effect of zinc, copper and iron, and also by the age of the plant, growth stage and maturity.

Nitrogen

Nitrogen also determines leaf quality,⁵⁴ and is the vital component of protein, nucleic acids, chlorophyll and growth hormones.⁵⁶ The present study recorded maximum nitrogen content in T₉ (CuSO₄ 15Kg/ha + ZnSO₄ 15Kg/ha + FeSO₄ 30Kg/ha) and T₁₀ (CuSO₄ 20Kg/ha + ZnSO₄ 20Kg/ha + FeSO₄ 40Kg/ha) treated mulberry leaves, which corroborates the results of Rafiq et al.⁵⁷. Nitrogen uptake from soil, assimilation and consumption via proliferated mulberry roots directly improve numerous strategic roles, like, transfer of energy, photosynthesis level, conversion of sugars, movement of nutrients within the plant cell, metabolic constituents, several physiological process, chlorophyll, and protein matters. Consequently, nitrogen is involved in production of leaf area, and net assimilation rate with regard to growth parameters like, leaf yield and leaf quality viz., moisture, chlorophyll ‘a’ and ‘b’, total sugar, soluble carbohydrate, reducing sugar and crude protein. Besides these, the nitrogen fixing microorganisms which increases the nitrogen accessibility might have increased the protein content in the leaves too in the present study.

Conclusion

The present investigation showed that the micronutrients improved the biochemical parameters of mulberry leaves, and indicated variations amongst the treatments due to the effect of micronutrients. Further, more research with more varied and different combination of treatments should be carried out to support the current findings in regard to the biochemical aspects of mulberry leaves so as to yield mulberry leaves of more quality and quantity.

Acknowledgment

The authors are thankful to the Department of Zoology, Scott Christian College, Nagercoil, Tamil Nadu, India for the help and support rendered. 

Funding Sources

The authors received no financial support for this research. 

Conflict of Interest

The authors declare no conflict of interest.

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