Sustainable agriculture: Influence of macro - and micro - nutrient levels, mixture and humic acid on growth and quality parameters of kharif maize ( Zea mays L.)

A field experiment entitled “ Sustainable agriculture: Influence of macro - and micro - nutrient levels, mixture and humic acid on growth and quality parameters of kharif maize ( Zea mays L.) ” was conducted at Agricultural farm, Lovely Professional University, Punjab during the kharif season of 2020 - 2021. The experiment was carried out in a randomized block design with ten treatments and three replications each. The treatments were as follows - T0:Control (Recommended dose of fertilizer; RDF), T1: RDF+ soil application of MM 10 kg/ha at 30 DAS, T2: RDF+ foliar application of MM 1% at 30 DAS, T3: RDF+ seed priming with MM 1% before sowing, T4: 75% RDF+ soil application of MM 10 kg/ha at 30 DAS, T5: 75% RDF+ foliar application of MM 1% at 30 DAS, T6: 75% RDF+ seed priming with MM 1% before sowing, T7: T 4 + humic acid 1% at 30 DAS, T8: T 5 + humic acid 1% at 30 DAS, T9: T 6 + humic acid 1% at 30 DAS. Among the various treatments, T8 exhibited higher growth attributes after 30 DAS, including plant height (cm), number of leaves per plant, leaf area, leaf area index, dry matter accumulation, chlorophyll index, CGR, RGR and NAR. Additionally, maize treated with T 5 + humic acid 1% at 30 DAS showed improved quality in terms of protein contents in both grain and stover.


Introduction
Maize is the second most important crop in terms of production, following wheat, and it is grown in various regions, including the tropics, sub tropics and temperate zones.Maize is available in a range of cultivars, such as field corn, sweet corn, popcorn and baby corn (1).In India, it holds the third position in terms of production, following wheat and rice (2).India ranks 4 th in terms of area and 7 th in terms of production of maize among the various maize-growing countries.Due to its higher genetic production potential compared to other cereals, maize is renowned as the 'Queen of cereals' or the 'Miracle crop' worldwide (3).Maize is cultivated in over 166 countries, covering a total area of approximately 193.7 million hectares.These countries have collectively produced nearly 1147.7 million tonnes of maize, with an average productivity of 5.75 tonnes per ha.Among the total maize production, approximately 61% is utilized as animal feed, 17% is consumed by humans, and around 22% is used for industrial purposes (4).They have developed the Corn Belt, a large area dedicated exclusively to growing corn.Mexico is the largest importer of maize from the USA accounting for 99% of the imports.Mexico is also known for having the original maize plant called 'teosinte', which is valuable for further research and hybridization purposes (5).Due to the increasing demand and the need to combat hunger, the world requires higher maize production.It is crucial to address this need while ensuring sustainable practices and minimizing residual effects.Providing proper nourishment to the global population is a top priority for maize-growing countries.Among the current agro-management practices, fertilizer application is commonly followed to enhance the growth and yield of maize (6).Macro-nutrient fertilizers, such as nitrogen, phosphorous and potassium are of utmost importance as they play a major role in biochemical and physiological reactions, including photosynthesis and energy transfer metabolisms (7).During the time of the green revolution, the primary focus was on NPK nutrition to crops.However, it is important to note that plants require 17 essential elements for their growth and development.Among these elements, micro-nutrients which are needed in small quantities, also play a significant role in plant metabolic activities and overall improvement (8).While the content of the trace elements in in the soil may be adequate, their availability to plants is often insufficient.The presence of trace elements is crucial for proper metabolism of macro-nutrients like N, P, K, Mg, Ca and S.They act as a cofactor in various plant activities (9).Humic acids improve soil physical, chemical and biological conditions and they have both direct and indirect effects on plant growth (10).These compounds are widely used by many growers as an alternative to pesticides, as they have positive effect such as increased minerals uptake, promotion of root length, and increased fresh and dry weight of plants.Additionally, it also assists in seed germination, root and shoot development and the uptake of macro-and micro-nutrients (10).The aim of this research was to evaluate the impact of different macro-nutrient levels, a micronutrient mixture along with humic acid on growth and quality parameters of kharif maize.

Materials and Methods
A field experiment was conducted at agricultural research farm, Lovely Professional University in Phagwara, Punjab during kharif season 2021.The objective was to study the influence of macro-nutrient levels, micro-nutrient mixture (Zn, Cu, Fe, Mn and B) along with humic acid on kharif maize.The pH of the soil was near neutral due to the previous cropping systems involving like maize, wheat, rice and mustard.The farm is located at an elevation of 250 m, near the intersection of latitude 310° 22′ 31.81′′North and longitude 750° 23′ 03.02′′East and 20 Km away from Jalandhar city in Punjab, India.
The experiment used the RASI 3033 hybrid variety of maize, which was sown on July 2nd, 2021, with a spacing of 60 cm x 25 cm.For micro-nutrient mixture (MM) five salts of Zn, Fe, Cu, Mn and B was taken (Table 1).Proper cultural practices, disease and pest control precautions were taken throughout the crop period.Observations of growth parameters were taken at 30, 60 and 90 DAS including plant height (cm), leaf area index, Chlorophyll, dry matter accumulation along with weather parameters like, minimum and maximum temperature, relative humidity, rainfall, bright sunshine hrs and quality parameters like protein content in grains and Stover of kharif maize were monitored.Chlorophyll was measured with SPAD meter.The leaf area index was calculated with this following Equation .

Crop Growth rate: (gm -2 day -1 )
Crop growth rate (CGR) indicated the rate at which the crop was growing and it was represented as gm -2 day -1 .It was computed by formulae (11).

Relative growth rate (RGR) (gg -1 day -1 )
Relative growth rate is the rate of increase in dry weight per unit dry weight and is expressed in gram per gram per day.RGR was calculated by using the formula suggested by (12).

Weather and climatic conditions
The experiment location was situated in a subtropical zone characterized by mild winters and hot summers.The region experienced the heaviest rainfall during the summer months of July, August and September.The primary source of precipitation was the southwest monsoon, which typically started in the latter half of June and lasted until the end of September.The monsoon season brought the highest amount of rainfall, with June to September being the period of heaviest rainfall.The coldest weather occurred between January and December, although the specific temperatures are not provided.Total leaf area of plant (cm) LAI = Ground area (cm) data representing the precipitation levels during the month of July, which is a significant month for precipitation due to the monsoon.

Statistical data analysis
The data collected from the experiment were subjected to a randomized block design and analyzed using analysis of variance (ANOVA).The mean values along with their standard deviation (Mean ± SEm) were determined and presented in the Table 1.The data analysis was conducted using with SPSS software (California).Duncan multiple range test (DMRT) used to check the variation between the treatments.The relation between the growth parameters was found by Pearson's correlation at 1% and 5% level of significance (to check the significant and non-significant parameters).

Plant height (cm)
The plant height (cm) of maize exhibited significant variability across a wide range of macro-nutrient levels, micronutrient mixtures and humic acid concentrations at all intervals.The most substantial increases in plant height was observed up to 60 DAS.However, after reaching maturity, the growth rate of plant height significantly slowed down.

Leaf area index
The rise in the leaf area index (LAI) was observed until 60 days after sowing (DAS), indicating an increase in the leaf surface area.However, after reaching full maturity, the LAI started to decline.At 60 DAS, the treatment with 75% recommended dose of fertilizer (RDF) + foliar application of a 1% micro-nutrient mixture (MM) and 1% humic acid showed a significantly higher LAI (4.142) compared to the treatment with 75% RDF + foliar application of a 1% MM at 30 DAS (4.087) and the control group (RDF) (4.080).Conversely, seed priming with a 1% MM before planting resulted in the lowest LAI (2.829) among the treatments with 75% RDF.The high LAI observed at 30 DAS could be attributed to the application of the recommended dose of fertilizers.Meanwhile, at 60 DAS, the increase in LAI might be attributed to the application of humic acid and the micro-nutrient mixture, which could have influenced the uptake of both macro-and micro-nutrients.This, in turn, could enhance photosynthesis and other metabolic activities in the plant, leading to the generation and expansion of new leaves (14) (Table 4).

Chlorophyll Index (SPAD value)
During the vegetative stage of the maize plants, the chlorophyll content was observed to be high.As the plants progressed towards maturity, there was a gradual decrease in the chlorophyll content.At 60 DAS, chlorophyll content was significantly high in 75% RDF+ foliar application of MM 1%+ humic acid 1% at 30 DAS (55.53) which is statistically at par with RDF+ soil application of MM 10 kg ha -1 at 30 DAS (54.90).While low chlorophyll content (46.98) was noticed in: 75% RDF+ seed priming with MM 1% before sowing.The decrease of chlorophyll at later stages of crop growth might be due to aging of leaf as well as translocation of nitrogen (precursor of chlorophyll) more towards reproductive organs.And application of humic acid after 30 DAS resulted in higher chlorophyll content in 75% RDF+ foliar application of MM 1%+ humic acid 1% which might be due to enhances the uptake of macro-and micronutrient s which helped in the synthesis of chlorophyll.Similar findings were reported earlier (15) (Table 5).

Dry matter accumulation (g)
Dry matter accumulation (DMA) showed significant variations among different macro-nutrient levels, micronutrient mixture and humic acid.Maize plants continued to accumulate dry matter throughout the crop growth period.The dry matter accumulation was maximum between 60 to 90 DAS.While at 90 DAS treatment 75% RDF+ foliar application of MM 1%+ humic acid 1% at 30 DAS (219.80 g) showed significantly higher dry matter accumulation while the lowest dry matter accumulation was observed in 75% RDF+ seed priming with MM 1% before sowing (177.53 g).Similar findings have been reported in previous studies where the application of humic acid and foliar application of micro-nutrient mixture might result in higher uptake of both macro-and micronutrients (16).That might increase photosynthesizing area and activity i.e., production of more no. of leaves as well as expansion of leaves through influencing the growth of stem and node (Table 5).
Crop growth rate (gm -2 day -1 ) Among various macro-nutrient levels, micro-nutrient mixture along with humic acid treatments there exists a significant variation in crop growth rate (CGR).At 30-60 DAS interval CGR was significantly highest (21.27 gm -2day -1 ) in 75% RDF+ foliar application of MM 1%+ humic acid 1%.While the lowest (15.80 gm -2day -1 ).CGR was observed in 75% RDF+ seed priming with MM 1% before sowing.While at 60-90 DAS, 75% RDF+ foliar application of MM 1%+ humic acid 1% showed significantly higher (24.1316.68gm -2day -1 ) CGR which is statistically at par with 75% RDF+ soil application of MM 10 kg ha -1 + humic acid 1%.While the lowest (19.76 gm -2day -1 ) was recorded at 75% RDF+ seed priming with MM 1% before sowing.The crop growth rate is the direct reflection of dry matter accumulation.Similar findings were reported by (16) in which high CGR was noticed when a micro-nutrient mixture was applied by foliar method and mentioned high nitrogen resulted in chlorophyll synthesis, while phosphorus and potassium ensured root development and stomatal opening of capture of sunlight as well as CO2, resulting in high photosynthesis and thereby, high CGR (Table 6).

Relative growth rate (RGR; mgg -1 day -1 )
At 30-60 days after sowing, the RGR was substantially highest (28.62 mgg which ultimately results in a reduction in the activity of photosynthetic and RGR.These results are in corroborate with the findings of ( 17) (Table 3).

Net assimilation rate (gm -2 day -1 )
The net assimilation rate (NAR) of kharif maize under different levels of macro-nutrients and micro-nutrient mixture, along with humic acid, was measured at 2 specific time intervals: 30-60 days after sowing (DAS) and 60-90 DAS.NAR was significantly highest (0.0029 gm -2day -1 ) in 75% RDF+ foliar application of MM 1%+ humic acid 1% at 30-60 DAS while the lowest (0.0018 gm -2day -1 ) RGR was observed in 75% RDF+ seed priming with MM 1% before sowing.While at 60-90 DAS, 75% RDF+ foliar application of MM 1%+ humic acid 1% showed significantly highest (0.0022 gm -2day -1 ) NAR which is statistically at par with 75% RDF + soil application of MM 10 kg ha -1 + humic acid 1% having 0.0021 gm -2day -1 .And lowest (0.0014 gm -2day -1 ) NAR was recorded in 75% RDF+ seed priming with MM 1% before sowing.During later intervals, the NAR was reduced and we know that LAI is a contributing factor to NAR, so by an increase in leaf area index during growing season, more leaves are completely or partially are under shadow and this decrease NAR.While during early stages there was less competition between shrubs and canopy, higher NAR was observed.These results are in corroboration with the findings of (18) Table 6.

Correlation
A correlation coefficient of pooled values of 2 successive years for different plant growth contributing characters were studied with themselves and found positive.The value of correlation coefficient varied from 0.959 to 0.781, that indicated these parameters were highly significant and inter correlated with each other.The data revealed that CGR and RGR showed the greatest correlation coefficient (0.959), however least con-elation coefficient (0.728) was observed for plant height (cm) and dry matter accumulation (Table 3).

Protein content in maize grain and stover
Protein contents of maize grain under different levels of macro-nutrients and micro-nutrient mixture along with humic acid were analysed in laboratory and data as discussed below.75%RDF+ foliar application of MM 1%+ humic acid 1% at 30 DAS showed significantly higher protein (%) in grain of maize i.e., 8.21 % and is statistically at par with 75% RDF+ soil application of MM 10 kg ha -1 + humic acid 1% at 30 DAS (7.83%) and found to be better among the other macro-and micro-nutrient level treatments.While the lowest protein (%) in grain of maize was observed in treatment T6: 75% RDF+ seed priming with MM 1% before sowing i.e., 6.83%.According to data represented, 75% RDF+ foliar application of MM 1%+ humic acid 1% at 30 DAS (2.73%) showed significantly high protein content in stover which is found to be better among the other macro-and micro-nutrient level treatments.While 75% RDF+ seed priming with MM 1% before sowing showed low protein content in stover i.e., 1.38%.Humic acid helps in better translocation of macro-and micronutrients that were applied (19).Nitrogen is considered a precursor of protein.Increased uptake of nitrogen thereby, probably resulted in increased protein content of maize grain as well as stover (19)

Conclusion
Based on the research conducted on the application of micro-nutrient mixture, macro-nutrient levels and humic acid on kharif maize, it has been confirmed that the combined application of organic and inorganic sources of nutrients, using appropriate application methods such as soil or foliar, along with the inclusion of micro-nutrients alongside NPK, positively influences the growth and quality of kharif maize production.Considering the findings of the investigation, it is recommended that maize growers in the kharif season in Punjab, India, apply 75% recommended dose of fertilizer (RDF) along with foliar application of a 1% micro-nutrient mixture and 1% humic acid at 30 days after sowing (DAS).This recommended treatment has shown potential for achieving better growth and quality in kharif maize production.

Table 1 .
Ten treatments were taken in three replications using randomized block design *

Table 2 .
Monthly averages of meteorological data of Kapurthala, recorded during the period of crop growth

Table 5 .
Influence of micro-nutrient mixture, macro-nutrient levels and humic acid on chlorophyll content and dry matter accumulation of kharif maize (Z.mays L.) -1 day -1 and lowest (8.29 mgg -1 day -1 ) RGR was recorded in 75% RDF+ seed priming with MM 1% before sowing.RGR decreased as the plant aged as a result of an increase in the construction of developing tissues to replace older tissues.Additionally, the leaves of the plant progressively lose chlorophyll and become dried out, T0: Control (RDF), T1: RDF+ soil application of MM 10kg/ha at 30 DAS, T2: RDF+ foliar application of MM 1% at 30 DAS, T3: RDF+ seed priming with MM 1% before sowing, T4: 75% RDF+ soil application of MM 10kg/ha at 30 DAS, T5: 75% RDF+ foliar application of MM 1% at 30 DAS, T6: 75% RDF+ seed priming with MM 1% before sowing, T7: T4+ humic acid 1% at 30 DAS, T8: T5+ humic acid 1% at 30 DAS, T9: T6 + humic acid 1% at 30 DAS.Data is in the form of Mean ±SDM at p<0.05.The mean followed by different letters was significantly different at p<0.05, according to DMRT for separation of means.

Table 3 .
Simple correlation between different growth parameters (Averaged over one year)