Multivariate physiological indices explain greengram yield response under conservation agriculture and nitrogen management practices in wheat-greengram-maize system

Introduction

Greengram (Vigna radiata L. Wilczek) is a key short-duration pulse crop in cereal-based cropping systems of the Indo-Gangetic Plains, contributing to dietary protein security, biological nitrogen fixation, and system diversification during the summer season (Giller 2001; Thiyagarajan et al. 2025) [9,22] . Its inclusion between two nitrogen-intensive cereals such as wheat and maize improves system resilience, soil biological activity, and nutrient cycling; however, greengram productivity remains highly unstable because of its shallow root system, short growth duration, and strong dependence on residual soil fertility (Berg 1997; Kadam et al. 2022) [2,14] . In intensive cereal-pulse-cereal rotations, inefficient nitrogen management in preceding crops frequently results in sub-optimal residual nitrogen availability, which constrains early canopy development, physiological vigour, and subsequent yield formation of greengram (Govaerts et al. 2009; Rashid et al. 2019) [10,19] . Conservation agriculture (CA), founded on minimal soil disturbance, residue retention, and diversified crop rotations, has been widely promoted to improve soil structure, moisture conservation, and nutrient cycling in cereal-based systems (Hobbs et al. 2008; Kassam et al. 2019) [11,15] . Residue-retained CA systems enhance soil organic matter turnover, microbial activity, and nitrogen mineralisation while reducing nitrogen losses through erosion and volatilisation, thereby improving nitrogen availability to succeeding crops (Bhattacharyya et al. 2021; Ladha et al. 2020) [3,18] . Nevertheless, CA alone may not fully compensate for inadequate nitrogen supply in preceding cereals, particularly in short-duration pulses such as greengram, where early nitrogen availability governs rapid canopy establishment before effective nodulation and biological nitrogen fixation become functional (Berg 1997; Kadam et al. 2022) [2,14] . Recent advances in nitrogen management practices, including reduced basal nitrogen rates combined with synchronised foliar applications of prilled urea or nano urea, have demonstrated significant improvements in nitrogen-use efficiency and nitrogen synchrony in cereals (Upadhyay et al. 2023a; Kumar et al. 2024) [16, 23] . Such strategies alter residual nitrogen dynamics by improving nitrogen uptake efficiency, reducing losses, and enhancing internal nitrogen recycling, thereby creating favourable physiological conditions for succeeding crops without direct fertiliser application (Devi et al. 2025) [5] . Greengram grown solely on residual fertility thus serves as an effective bio-indicator for assessing upstream nitrogen synchronisation within cereal-pulse-cereal cropping systems. Conventional univariate approaches often fail to capture the complex interactions among physiological, biochemical, and reproductive traits governing yield formation under residual fertility conditions. Multivariate analytical tools such as principal component analysis (PCA) and hierarchical clustering on principal components (HCPC) enable integration of multiple traits and identification of dominant physiological drivers regulating productivity and nitrogen responsiveness (Sadras and Lemaire 2014; Kumar et al. 2025) [17,20] . However, information remains limited on how multivariate physiological indices explain greengram yield response under combined CA and nitrogen management strategies in cereal-pulse-cereal systems. The present study was therefore undertaken to evaluate the multivariate physiological and agronomic responses of summer greengram grown under conservation agriculture and nitrogen management practices applied to preceding wheat and maize. Specifically, the study aimed to determine whether nitrogenregulated physiological synchrony established during cereal phases governs greengram performance and whether multivariate discrimination can identify nitrogen-efficient treatment combinations capable of sustaining greengram productivity without direct nitrogen inputs.

Materials and Methods

A two-year field experiment was conducted during Rabi 2021-22 to Kharif 2023 at the Research Farm of ICAR-Indian Agricultural Research Institute, New Delhi (28°38′N, 77°09′E; 228 m amsl). The region experiences a semi-arid subtropical climate with hot summers, cool winters, and mean annual rainfall of about 1,015 mm, predominantly received during July-August, typical of the western Indo-Gangetic Plains (Gathala et al. 2019) [8] . Pre-sowing soil analysis (0-15 cm) indicated sandy loam texture, alkaline reaction (pH 7.9), low organic carbon (0.43%), low available nitrogen (224.5 kg ha⁻¹), and medium available phosphorus (19.0 kg ha⁻¹) and potassium (246.2 kg ha⁻¹), determined following standard soil analytical procedures (Jackson 1973) [13] .

The experiment was laid out in a split-plot design with three replications. Crop establishment methods-conventional tillage (CT), zero tillage with residue retention (ZT+R), and permanent raised beds with residue retention (PB+R)-were assigned to main plots, while nitrogen management practices were allotted to sub-plots. Crop residues (~3 t ha⁻¹) were retained in ZT+R and PB+R but removed in CT, consistent with the principles of conservation agriculture emphasizing minimal soil disturbance and residue retention (FAO 2016; Kassam et al. 2019) [15] . Crops were grown sequentially as wheat (Rabi) → greengram (summer) → maize (Kharif) in both years to assess residual nitrogen effects within a cereal-pulse-cereal rotation (Govaerts et al. 2009) [10] . Seven nitrogen management treatments were imposed only on wheat and maize: N₁ (0 N), N₂ (100% recommended dose of nitrogen, RDN), N₃ (75% RDN), N₄ (75% RDN + one foliar spray of 2% prilled urea), N₅ (75% RDN + one foliar spray of nano urea), N₆ (50% RDN + two foliar sprays of 2% prilled urea), and N₇ (50% RDN + two foliar sprays of nano urea). The RDN was 150 kg N ha⁻¹ for wheat and 180 kg N ha⁻¹ for maize, applied as per regional recommendations. Foliar nitrogen sprays were scheduled at critical growth stages to enhance nitrogen synchrony and improve nitrogen-use efficiency, as reported in earlier nitrogen management studies (Adhikari et al. 2020; Dabhi et al. 2025) [1,4] . No nitrogen fertiliser was applied to greengram to enable evaluation of residual soil nitrogen availability generated through preceding cereal management (Berg 1997) [2] . Wheat variety HD 3226, greengram PUSA 1431, and maize hybrid PJHM-1 were sown using recommended agronomic practices. Greengram observations included leaf area index measured using a canopy analyser (LAI-2200C), chlorophyll index using a SPAD-502 Plus meter, canopy vigour using a GreenSeeker NDVI sensor, dry matter accumulation after oven drying, leaf nitrogen concentration determined by the Kjeldahl method, nitrogen uptake computed from biomass and nitrogen concentration, yield attributes, and seed yield, following standard physiological and analytical procedures (Jackson 1973; Evans 1989) [6,13] . Data were analysed using analysis of variance appropriate for a split-plot design, with mean separation at p<0.05. Multivariate analyses, including principal component analysis (PCA) and hierarchical clustering on principal components (HCPC), were performed using R software with FactoMineR and factoextra packages to integrate physiological, nitrogen, and yield-related traits (Husson et al. 2010) [12] .

Results and Discussion

Nitrogen management practices imposed on the preceding wheat and maize crops exerted a pronounced influence on the physiological expression, nitrogen assimilation, and yield formation of residual summer greengram grown under conservation agriculture (CA). Despite the absence of direct nitrogen fertilisation to greengram, substantial variability was observed in canopy greenness, dry matter accumulation, and reproductive traits across nitrogen management practices, indicating strong carry-over effects of fertiliser nitrogen synchronisation and residue-mediated mineralisation under CAbased cropping systems. These responses confirm that greengram performance in cereal-pulse-cereal rotations are governed largely by upstream nitrogen availability and its temporal synchrony rather than by in-season nitrogen supply alone (Berg 1997; Kadam et al. 2022) [2,14] . Principal component analysis revealed that the first two principal components together accounted for more than 97% of the total variation among measured physiological, nitrogen, and yield attributes (Table 1; Fig. 1). The dominance of PC1, explaining 92.03% of the total variance, reflects a highly consolidated physiological axis regulated primarily by nitrogen availability. Traits with strong positive on PC1 included SPAD values, chlorophyll concentration, NDVI, leaf nitrogen concentration, total nitrogen uptake, and dry matter accumulation, demonstrating a tight coupling between nitrogen status, photosynthetic capacity, and biomass production. This indicates that residual nitrogen availability governs canopy establishment and physiological efficiency in greengram, particularly during early vegetative stages when nodulation and biological nitrogen fixation are still developing (Giller 2001; Sadras and Lemaire 2014) [9,20] . In contrast, PC2 explained only 4.99% of the total variation and was primarily associated with reproductive traits such as pods per plant, seeds per pod, pod length, and test weight, suggesting that yield differentiation along this axis was influenced by assimilate partitioning and sink strength during later growth stages rather than by nitrogen acquisition percentage. The correlation structure among traits further clarified these relationships (Fig. 2). Strong positive associations were observed among canopy greenness indices (SPAD and NDVI), leaf nitrogen concentration, nitrogen uptake, and dry matter accumulation, confirming that nitrogen-driven chlorophyll stability and photosynthetic efficiency underpin greengram growth under residual fertility conditions. These findings align with earlier reports that nitrogen availability stabilises chloroplast structure, prolongs leaf functional duration, and enhances carbon assimilation in short-duration legumes (Evans 1989; Tegeder 2014) [6,21] . Pod-related traits exhibited moderate correlations with physiological attributes, indicating that while nitrogen availability establishes the physiological ceiling for yield formation, reproductive efficiency and assimilate remobilisation ultimately determine the extent to which this potential is realised. Such decoupling between vegetative vigour and reproductive output is common in greengram under heatprone summer environments, where sink limitation and flower shedding can constrain yield despite adequate early growth (Kumar et al. 2025) [17] . Clear discrimination among nitrogen management treatments was evident in the PCA biplot (Fig. 3). Nitrogen-adequate and synchronised treatments, namely N₂ (100% RDN), N₅ (75% RDN + nano urea), N₄ (75% RDN + prilled urea), and N₃ (75% RDN), clustered along the positive PC1 axis, corresponding to higher SPAD values, NDVI, leaf nitrogen concentration, nitrogen uptake, dry matter accumulation, and yield-attributing traits. These treatments benefitted from improved residual nitrogen availability and sustained nitrogen release through crop residue decomposition, which collectively enhanced canopy vigour and photosynthetic efficiency during the critical early growth stages of greengram. In contrast, treatments lacking adequate nitrogen supply or effective synchronisation (N₁, N₆, and N₇) were positioned along the negative PC1 axis, reflecting poor chlorophyll stability, reduced biomass accumulation, and inferior reproductive performance. These results confirm that synchronised nitrogen management in cereals exerts strong downstream effects on greengram physiology and yield formation, even in the absence of direct fertilisation (Govaerts et al. 2009; Rashid et al. 2019) [10,19] . Hierarchical clustering on principal components grouped the treatments into two statistically distinct clusters (Table 2; Fig. 4). Cluster I, comprising N₂, N₅, N₄, and N₃, recorded significantly higher cluster means for chlorophyll concentration, NDVI, nitrogen uptake, dry matter accumulation, pods per plant, and grain yield. This cluster represents nitrogen-efficient treatment combinations where moderate reductions in basal nitrogen application to cereals were effectively compensated through synchronised foliar nitrogen interventions, resulting in favourable residual fertility conditions for greengram. Notably, the inclusion of reduced basal nitrogen treatments within this cluster demonstrates the potential for nitrogen savings in cereal phases without compromising pulse productivity, provided nitrogen synchrony is maintained (Upadhyay et al. 2023a; Nayak et al. 2025) [23] . In contrast, Cluster II, consisting of N₁, N₆, and N₇, consistently exhibited inferior physiological and yield performance, highlighting that CA alone cannot overcome nitrogen deficiency in short-duration greengram when residual nitrogen availability is inadequate. [3,18] . Physiological traits such as SPAD, NDVI, and leaf nitrogen concentration emerged as reliable indicators of residual nitrogen sufficiency and were closely linked to biomass accumulation and yield formation, supporting their use as diagnostic tools for evaluating nitrogen synchrony in CA systems. Residual greengram productivity in wheat-greengram-maize rotations is governed primarily by nitrogen-regulated physiological synchrony established during preceding cereal crops, while conservation agriculture provides complementary moderation of soil and microclimatic conditions. Multivariate analysis confirmed that synchronised nitrogen management in cereals sustains canopy greenness, nitrogen assimilation, biomass accumulation, and pod development in greengram under residual fertility. These findings indicate that combining residue-retained conservation agriculture with synchronised nitrogen strategies in cereals offers a practical pathway to reduce fertiliser inputs without compromising greengram productivity, thereby improving nitrogen-use efficiency and sustainability of cereal-pulse systems in semi-arid environments.

Acknowledgement

AcknowledgementThe authors sincerely acknowledge the facilities and support provided by the ICAR-Indian Agricultural Research Institute, New Delhi, for conducting the field and laboratory investigations. The assistance of the technical staff and farm personnel of the Division of Agronomy during field experimentation and data collection is gratefully acknowledged.

References

1. Adhikari T, Kundu S, Biswas AK, Tarafdar JC. Nano fertilizers: A review on synthesis and application. Indian J Agric Sci. 2020;90(1):3-17.
2. Berg A. Plant nutrition and nitrogen fixation in grain legumes. Plant Soil. 1997;190:1-7.
3. Bhattacharyya R, Dhaliwal JK, Ghosh A, Mondal S, Meena BP, Sharma C. Conservation agriculture increases nitrogenuse efficiency via soil biological processes in cereal systems. Soil Tillage Res. 2021;209:104926.
4. Dabhi MS, Patel AS, Vihol KJ, Patel VM, Patel MD. Effect of nano urea on increasing nitrogen-use efficiency and productivity of wheat under irrigated conditions. Int J Adv Biochem Res. 2025;9(Spec Iss 1):874-877.
5. Devi S, Singh R, Meena RS, Gathala MK. Nitrogen synchrony and productivity of cereal-pulse systems under conservation agriculture. Field Crops Res. 2025;298:108931.
6. Evans JR. Photosynthesis and nitrogen relationships in leaves of C₃ plants. Oecologia. 1989;78:9-19.
7. Food and Agriculture Organization of the United Nations. Conservation Agriculture. Rome: FAO; 2016.
8. Gathala MK, Timsina J, Islam S, Rahman M, Hossain MI, Govaerts B. Conservation agriculture-based practices improve soil organic carbon, water productivity and yield in cereal systems of South Asia. Field Crops Res. 2019;238:95-108.
9. Giller KE. Nitrogen fixation in tropical cropping systems. 2nd ed. Wallingford (UK): CABI Publishing; 2001.
10. Govaerts B, Verhulst N, Castellanos-Navarrete A, Sayre KD, Dixon J, Dendooven L. Conservation agriculture and soil carbon sequestration: Between myth and farmer reality. Crit Rev Plant Sci. 2009;28:97-122.
11. Hobbs PR, Sayre K, Gupta R. The role of conservation agriculture in sustainable agriculture. Philos Trans R Soc B. 2008;363:543-555.
12. Husson F, Josse J, Le S, Mazet J. FactoMineR: Multivariate exploratory data analysis and data mining. R package version 1.15; 2010.
13. Jackson ML. Soil chemical analysis. New Delhi: Prentice Hall of India Pvt Ltd; 1973.
14. Kadam SR, Gokhale DN, Jadhav MG, Deshmukh AV. Residual nitrogen effects on growth and yield of summer greengram in cereal-based cropping systems. Legume Res. 2022;45(6):759-765.
15. Kassam A, Friedrich T, Derpsch R, Kienzle J. Overview of the worldwide spread of conservation agriculture. Field Actions Sci Rep. 2019;12:1-8.
16. Kumar A, Singh R, Meena RS, Yadav GS. Nano-nitrogen formulations improve nitrogen-use efficiency and crop productivity: A meta-analysis. Agron J. 2024;116(1):120134.
17. Kumar S, Singh G, Meena BL, Choudhary R. Physiological determinants of yield formation in summer greengram under heat stress environments. J Plant Physiol. 2025;282:153973.
18. Ladha JK, Pathak H, Krupnik TJ, Six J, van Kessel C. Efficiency of fertilizer nitrogen in cereal production: Retrospects and prospects. Adv Agron. 2020;163:85-156.
19. Rashid MA, Iqbal MA, Akbar N, Ahmad R. Residual soil fertility effects on mungbean performance following cereal crops. Pak J Agric Sci. 2019;56(2):375-382.
20. Sadras VO, Lemaire G. Quantifying crop nitrogen status for comparisons of agronomic practices and genotypes. Field Crops Res. 2014;164:54-64.
21. Tegeder M. Transporters for amino acids in plant cells: Some functions and many unknowns. Curr Opin Plant Biol. 2014;22:1-8.
22. Thiyagarajan TM, Somasundaram E, Singh R. Role of pulses in sustainable intensification of cereal-based cropping systems. Indian J Agron. 2025;70(1):1-10.
23. Upadhyay PK, Singh R, Parihar CM, Shivay YS. Synchronised nitrogen management improves nitrogen-use efficiency and yield of wheat under conservation agriculture. Indian J Agric Sci. 2023;93(8):801-807.