On-Site Detection of Nitrogen Content in Maize using Optical Sensor

Nutrients are essential for the growth of plants, and they also increase the yield. Among the major nutrients, nitrogen is the most important nutrient for crop growth. The current study aims to estimatechlorophyll content vis-a-vis nitrogen content using different techniquesand attempts to establish comparative benefits among available techniques. Maize crop,being highly known for fertilizer responsiveness, was chosen to carry out experiments with different levels of nitrogenous fertilizers. A preliminary field survey was taken in the farmer's field in Coimbatore and Salem districts. Two field experiments and one pot culture experiment at different doses of nitrogen input were conductedin 2019, 2020, and 2021 in the Department of Nano science and Technology at Tamil Nadu Agricultural University. In this study conventional nitrogen was applied with various dosage viz.,0kgha(T1), 50kgha(T2), 100kgha(T3), 150kgha(T4), 200kgha(T5), 250kgha(T6), 300kgha(T7). In the pot culture experiment, IFFCO Nano nitrogen was sprayed on the maize leaves with seven different doses, which were control (no spray) (T1),conventional nitrogen (T2), 0.5X RDF(Recommended Dose of Fertilizer) (T3), 1.0X RDF (T4), 1.5X RDF (T5), 2.0X RDF (T6), 2.5X RDF (T7). The chlorophyll contentin all the trials wasestimated using Dimethylsulfoxide(DMSO), a portable in-house developed nutrient meter, and Spectroradio meter method.Chlorophyll content was measured at two different growth stages of maize viz., 60 & 90 days after sowing.The total nitrogen was analyzed using Kjeldahl method in the maize leaf.Among the treatments, with different doses of nitrogen levels, 300kgha-1 RDN & 2.5X RDF (T7) recorded higher SPAD (Soil Plant Analysis Development) values, chlorophyll “a” and chlorophyll “b” content and total nitrogen content at 60 DAS (Days After Sowing) in all tested methods.Chlorophyll and total nitrogen content weresignificantly decreased in later days. In a stressed environment withno nitrogen (T1)applied condition,the chlorophyll and nitrogen content showed significantly lower levels.

J. Curr. Crop Sci. Technol., 2022;https://doi.org/10.29321/MAJ.10.000624 (online first draft) balanced nutrients from the soil minerals, inorganic fertilizers, and soil organic compounds. Alternatively higher levels of nutrient application also cause toxicity in the plant system, therefore,the application of a required amount of nutrients is crucial, whichresults in better growth and yield production.
Nitrogenis referred to as the kingpin among all other essential nutrients in crop growth. In chlorophyll,a major portion is made up ofnitrogen. Chlorophyll is the most important component for photosynthesis. In plants all enzymatic reaction requires nitrogen. Nitrogen deficiency leads to earlier maturity which causes reduced yield production and grain quality level. Severe lower nitrogen condition causes death in plants.
Pale green to light yellow color occurs on the older leaves due to chlorosis, a classical symptom of nitrogentranslocating from older leaves to younger leaves.Hence, it is highly crucial to monitor the nitrogen levels vis-a-vis chlorophyll content in the plant system to enable improved yield.
Currently, SPAD chlorophyll meter is used for detecting N in plants. In this method, it is assumed that a major part of N gets accumulated in chlorophyll and the estimation of chlorophyll is an indirect measure of the N content of leaves(Simko and Veres, 2019; Yang et al., 2018). Despite it beingdemonstrated well for many crops, the technique is highly sensitive to cloud cover. SPAD meter readings are reliable only for brighter sunny days.Traditional laboratory-based methods are laborious, time-consuming, and highly variable due to the timelag between sample collection and laboratory analysis. The study aims to compare the nitrogen vis-a-vis chlorophyll content in different treatments using various methods viz., chlorophyll estimation by DMSO method, total nitrogen Kjeldahl method, leaf nitrogen content by SPAD meter, and spectral reflectance using Spectral Radiometer signatures (Naik et al., 2020). Therefore on-site detection is quite relevant.A spot detection of nutrient deficiencies in crops will enable the farmers to undertake an immediate corrective measure of proper fertilization and ensure the productivity of crops (Gabriel et al.,2019).Diphylamine, phosphomolybdic acid, and cobalt nitrate are used for quick diagnosis of N, P, and K.

Survey and data collection
A field survey was conducted to ascertain the chlorophyll content prevailing in maize crops at farmers' fields located in Coimbatore and Salem districts. The field survey is representative of two geographical locations. The data on chlorophyll content was recorded on 60 DAS at farmer's field using SPAD meter. The geo-coordinates of the farmers' field were plotted in ArcGIS software.

Establishing field crop with gradient nitrogen fertilizer application
To assess the nitrogen content vis-a-vis chlorophyll content in plant systems using different methods viz., DMSO method (Total nitrogen), Chlorophyll estimation (SPAD meter), and Hyper Spectral Imaging. Two batches of maize crops were raised in the research plots of TNAU and operational details are tabulated (Table1). The fieldwas prepared using a mould board plough and raised furrows were formed as per the stipulated spacing. Prior to land preparation, the land was broadcasted with 12.5tha -1 of farm yard manure J. Curr. Crop Sci. Technol., 2022;https://doi.org/10.29321/MAJ.10.000624 (online first draft) (FYM) as per the TNAU agriculture crop guidelines. The field was divided into 21sub-plots, and each plot consistedof seven ridges.

Nutrient application
The nutrients application and the package of practices were carried out as per TNAU crop production guidelines. For the treatments, one-quarter dose of nitrogen, full dose of phosphorus, and potassium wasapplied as the basal before sowing the seeds. The remaining splits of nutrients wereapplied as top dressing on 60 DAS and 75 DAS (Table2). In addition, phosphorus and potassium at a ratio of75:75 kgha -1 ; zinc sulphate at 37.5 kgha -1 was applied on 60 DAS as per the TNAU crop production guidelines.

Establishing pot culture experiment with gradient doses of IFFCO nano urea
In addition to ascertaining the chlorophyll vis-à-vis nitrogen content in field conditions imposed by treatments of gradient nitrogen levels, attempts were made to study the nitrogen content in maize plants grown in pot cultures in controlled environmental conditions. IFFCO Nano urea is used as a sole source of nitrogen and it has been applied as a foliar spray. The parameters like chlorophyll content (a, b and total), total nitrogen content, SPAD values, and hyperspectral data were recorded in green house conditions and data was correlated to filed data. The 21 pots were prepared with a mixture of soil and vermin compost. CRD (completely randomized designs) was adopted to impose treatments using IFFCO Nano Urea Sprays. Chlorophyll estimation by DMSO method 0.1g of fresh maize leaf was placed in the 10mLof DMSO in a 100mLof volumetric flask for overnight incubation. After chlorophyll 'a', chlorophyll 'b' and total chlorophyll were quantified by reading the optical density at 663m and 645m. The chlorophyll content was calculated using the formula describedin (1), (2), (3) (Yoshida et al., 1971).
Where, W -Weight of the leaf sample (g) V -Volume of supernatant solution made-up OD -Optical Density.

Total nitrogen estimation
In maize leaf, total nitrogen was estimated by Kjeldahl method (Jackson,1883). It is a destructive method.
Collected leaves were taken to the laboratory and samples were placed in to the diacid for digestion. After Physiology, Tamil Nadu Agricultural University, was used for recording SPAD data (Bonneville and Fyles, 2006;Simet al., 2015). It is a non-destructive method. In each plot, five datasets were taken, and it was averaged to get mean values. The readings were recorded from the fully expanded leaves from the top of the plant at 60 and 90 DAS.

Spectroradiometer
Spectral reflectance data at different wavebands of green, blue,and red region and NIR region ranging from 350 -1050nm were recordedusing GER 1500Spectroradiometer (Geophysical and Environmental Research, UK) (Figure1). Data was recorded at 60, and 90 DAS in addition to the SPAD readings, and the datasets were correlated (Alchanatiset al., 2005;Fu et al., 2021) Results and discussion: Field survey J. Curr. Crop Sci. Technol., 2022;

Chlorophyll estimation by DMSO method
The DAS. Whereas, In the pot culture experiment, higher chlorophyll "a" content of 1.68 and 1.51 mg g -1 fl in T7 respectively 60 &90 DAS followed by T6. Lower chlorophyll "a" content was observed in T 1 (control) which was 0.67 and 0.61 mg g -1 fl. A high chlorophyll "b" content of 0.54& 0.43 mg g -1 fl was observed in T7 with 2.5X RDF at 60 and 90 DAS, respectively. This was followed by T6 with 2.0 X RDF. Lower chlorophyll "b" content of 0.24 & 0.20 mg g -1 fl was recorded in T1 control with no nitrogen application at 60 and 90 DAS.

Total Nitrogen by Kjeldahl method
In addition to the indirect measure of chlorophyll content by SPAD meter, the actual total nitrogen present in the plant samples wasanalysed using Kjeldahl method. In field experiment I, The highest total nitrogen level of 1.466% and 1.576% wererecorded in T7 (300kgha -1 ) followed by1.

Chlorophyll estimation using spectral reflectance
In field experiment I, the spectral reflectance data represents, in plots, subjected to lower levels of nitrogen applicationresulted from spectral reflectance in the visible spectrum region that had a higher percentage of reflectance. A higher dose of nitrogen applied conditions reflectance in the visible spectrum region has lower reflectance and higher reflectance in the near-infrared region.

Conclusion
Total Nitrogen content vis-à-vis chlorophyll content is an important factor that determines the growth and development of the plant system. Gaining insights in developing appropriate tools or yardsticks J. Curr. Crop Sci. Technol., 2022;https://doi.org/10.29321/MAJ.10.000624 (online first draft) to capture the nitrogen content with high precession will empower farmers to carry out remedial measures or supplement nutrients and enable them to prevent drastic yield loss (Fomariet al., 2020). In the current study, attempts were made to study the different methodologies in vogue to quantify the nitrogen content in plant system viz., chlorophyll estimation (DMSO method), Total Nitrogen estimation (Khjeldhal method), SPAD meter, and spectral reflectance method. Estimation by DMSO method and Khjeldhal method are destructive and involve relatively cumbersome laboratory analysis, whereas the reflectance-based methods (SPAD and spectral imaging) are nondestructive methods and data can be obtained instantaneously (Habibullahet al., 2020;Rongtinget al., 2020). From the current study, a correlation has been established among the four methods. It is evident that SPAD and spectral signature based methods are highly versatile and with sufficient reference datasets, the method can be effectively employed in chlorophyll vis-à-vis nitrogen estimation in fields with larger areas in a relatively short time span (Fenget al., 2021). Further, these imaging/spectral scanners are conducive to be in integrated withAI and drone based technology pipelines (Cheema et al., 2018) thereby providing increased efficiency and phenotyping applications.