Phenology-synchronized nano-biostimulant consortia enhance productivity and multi-nutrient-use efficiency of early-, medium and late-maturing maize (Zea mays L.) hybrids under contrasting nitrogen environments

Introduction

Maize (Zea mays L.) is among the most productive and versatile cereal crops in the world, with global production exceeding 1,148 million tonnes harvested from approximately 205 million hectares (FAO, 2023) [1] . As a C 4 crop with a highly efficient carbon-assimilation pathway, maize possesses substantial genetic yield potential; however, realization of this potential depends strongly on the synchronization of nutrient availability, particularly nitrogen (N), with crop developmental demand. In India, maize occupies nearly 10.0 million hectares with an average productivity of only about 3.2 Mg ha -1 , well below the 8-12 Mg ha -1 attainable under optimum management, reflecting a substantial and persistent yield gap (Cassman et al., 2020) [2] . Nitrogen demand in maize is highly dynamic, rising sharply from the six-leaf (V6) stage through tasseling (VT) as canopy establishment accelerates, and continuing through grain filling via a combination of continued root uptake and remobilization of pre-silking N reserves. Commercial hybrids differ substantially in phenological duration -broadly classified as early-(80-90 days after planting, DAP), medium-(95-105 DAP) and late-maturing (115-130 DAP) -and, consequently, in the timing, intensity and duration of their nutrientdemand windows (Ciampitti et al., 2012; Bender et al., 2013) [4,3] . A single calendar-based N schedule may therefore be poorly synchronized with the physiological demand of hybrids possessing different maturity durations, motivating a phenology-based, stage-scouted approach to nutrient and biostimulant management rather than one driven purely by calendar days.

Biostimulants -substances or microorganisms capable of stimulating natural physiological processes independent of their nutrient content (du Jardin, 2015) [5] -have emerged as a rapidly expanding complement to conventional fertilization. Seaweed extracts (e.g., Ascophyllum nodosum) contribute auxin-and cytokinin-like bioactivity that promotes root growth and delays senescence (Battacharyya et al., 2015; Shukla et al., 2019) [6,7] ; plant growth-promoting rhizobacteria (PGPR) such as Azospirillum brasilense and Bacillus subtilis enhance rhizosphere nutrient mobilization and biological nitrogen fixation (Bashan et al., 2014) [8] ; and humic substances improve root architecture and nutrient transport (Nardi et al., 2021) [9] . Encapsulation of such bioactive consortia within chitosan-based nanocarriers has additionally been reported to improve compound stability, controlled release and physiological uptake efficiency (Oliveira et al., 2016) [10] . Despite growing interest in both precision nitrogen management and biostimulant technology, limited evidence exists on whether the effectiveness of biostimulant application, when synchronized with hybridspecific phenological stages rather than fixed calendar dates, differs systematically across maize maturity groups and nitrogen environments. This study therefore examined the combined and interactive effects of hybrid maturity group (early, medium, late), nitrogen environment (low vs. recommended) and a phenology-synchronized ternary nano-biostimulant consortium (seaweed extract + PGPR + humic acid in a chitosan nanocarrier) on growth, phenology, yield, grain quality, soil nutrient dynamics and multi-nutrient-use efficiency of maize, with the specific objectives of (i) quantifying the main and interactive effects of these three factors on productivity and nutrient-use efficiency, and (ii) determining whether the magnitude of the biostimulant response scales with hybrid phenological duration.

Materials and Methods

Experimental site and soil characterization

The field experiment was conducted during the kharif season of 2025-26 at the Research Farm of BEST Innovation University, located in the semi-arid (Köppen BSh) agro-climatic zone of Andhra Pradesh, India. Mean annual rainfall in the region ranges from approximately 700-900 mm, concentrated in the June-September southwest monsoon, and mean kharif-season temperatures range from approximately 24-36 °C. Composite soil samples (n = 12, 0-30 cm depth) were analysed for baseline physico-chemical properties prior to treatment imposition (Table 1). The soil was a non-saline (EC 0.32 ± 0.04 dS m -1

), moderately alkaline (pH 7.8 ± 0.1) sandy clay loam Alfisol with low organic carbon (4.6 ± 0.6 g kg -1 ) and available nitrogen (178 ± 16 kg ha -1 ), moderate available phosphorus (20.8 ± 2.8 kg ha -1 ) and potassium (240 ± 22 kg ha -1 ), and marginally low available zinc (0.38 ± 0.05 mg kg -1 ).

Plant material and treatments

Three commercial maize hybrids representing contrasting maturity groups were used: early-maturing (H 1 , 80-90 DAP), medium-maturing (H 2 , 95-105 DAP) and late-maturing (H 3 , 115-130 DAP). All hybrids were sown on the same date at a spacing of 60 cm × 25 cm (target population ≈ 60,000 plants ha -1 ), with uniform basal application of phosphorus (60 kg P 2 O 5 ha -1 ) and potassium (40 kg K 2 O ha -1 ) to all plots. Nitrogen was applied at two levels -low-N (N 1 , 60 kg N ha -1 ) and recommended-N (N 2 , 120 kg N ha -1 ) -constituting the second experimental factor. Biostimulant treatment (Factor C) comprised a water-sprayed control (B 0 ) and the nano-biostimulant consortium (B 1 ), applied as a foliar spray at 500 L ha -1 using a calibrated knapsack sprayer at three phenological stages of each respective hybrid: V6 (six fully expanded leaves), VT (tasseling/anthesis) and R2 (blister stage). Because the three hybrids differed in developmental rate, field scouting was used to identify the true physiological stage of each hybrid rather than applying treatments on fixed calendar dates, ensuring that biostimulant delivery was synchronized with comparable developmental states across maturity groups.

Nano-biostimulant consortium: preparation and characterization

The ternary nano-biostimulant consortium comprised Ascophyllum nodosum seaweed extract (5% v/v), a PGPR consortium of Azospirillum brasilense and Bacillus subtilis (≈10 8 CFU mL -1 ), and potassium humate (2% w/v), encapsulated within a medium-molecular-weight chitosan nanocarrier (190-310 kDa; degree of deacetylation ≥ 75%). Chitosan nanoparticles were synthesized by the ionicgelation technique using sodium tripolyphosphate (TPP) as the crosslinking agent, with the bioactive components incorporated during nanoparticle formation under controlled mixing to preserve PGPR viability. The formulation was characterized for particle size and zeta potential (Zetasizer Nano ZS, Malvern Panalytical; target zeta potential +25 to +35 mV for colloidal stability), encapsulation efficiency (UV-Vis spectrophotometry), PGPR viability (plate-count on Luria-Bertani agar) and retention of seaweed-extract bioactive constituents (HPLC) prior to field application.

Soil property

Experimental design and treatment structure

The experiment followed a Randomized Block Design (RBD) with a 3 × 2 × 2 factorial treatment structure -Hybrid Maturity Group (H 1 , ) after excluding a 0.75 m border on all sides. Treatment codes and combinations are summarized in Table 2.

Observations and analytical procedures

Growth attributes (plant height, stem girth, leaf area index via LI-3100C, dry-matter accumulation, SPAD chlorophyll index, NDVI, and phenological staging) were recorded at V6, VT, R2/R4 and physiological maturity. Yield attributes (cobs plant -1 , cob length and girth, kernel rows cob -1 , kernels row - ) and potassium-use efficiency (𝐾𝑈𝐸 = grain yield 𝐾 applied

Table 2. Treatment structure of the 3 × 2 × 2 factorial experiment (RBD, three replications).

Code Hybrid maturity N environment Biostimulant

T 1 -H1N1B0 Early (H 1 ) Low N (60 kg ha -1 ) Control T 2 -H1N1B1

Early (H 1 ) Low N (60 kg ha -1 ) Nano-consortium

Early (H 1 ) Recommended N (120 kg ha -1 ) Control T 4 -H1N2B1

Early (H 1 ) Recommended N (120 kg ha -1 ) Nano-consortium

Late (H 3 ) Recommended N (120 kg ha -1 ) Nano-consortium

Statistical analysis

Data were analysed by three-factor factorial ANOVA appropriate to the RBD (Hybrid × N-environment × Biostimulant) using Python (statsmodels v0.14), cross-verified in R v4.3.2 (R Core Team, 2024) [11] . Treatment means were compared using critical difference (CD) at

Coefficient of variation (CV%) was calculated as the ratio of the square root of the error mean square to the grand mean. Pearson correlation analysis was used to examine relationships among growth, yield and nutrient-use-efficiency parameters.

Results

Growth attributes and phenology

Plant height at the R4 stage, leaf area index (LAI) at VT and the SPAD chlorophyll index at VT were all significantly (P ≤ 0.01) influenced by hybrid maturity group, nitrogen environment and nano-bios-timulant application (Table 3, Fig. 1). Plant height increased progressively with hybrid duration, from a mean of 168.8 cm in H 1 to 187.9 cm in H 2 and 202. [6,9] . LAI at VT increased from 3.57 (control) to 4.05 (nano-BS; CD = 0.41 at P = 0.05), and SPAD index rose from 44.8 to 50.1 over the same comparison, indicating a genuine improvement in canopy photosynthetic capacity rather than a response driven solely by improved assimilate partitioning. Days to 50% tasseling, 50% silking and physiological maturity were governed overwhelmingly by hybrid maturity group, with H 1 , H 2 and H 3 reaching physiological maturity at a mean of 83.9, 100.0 and 121.9 days after sowing, respectively (Table 4, Fig. 2). Nano-biostimulant application did not significantly alter the calendar duration to physiological maturity within any hybrid (control mean 102.2 days vs. nano-biostimulant mean 101.7 days), confirming that yield and growth benefits arose from enhanced physiological efficiency within an unchanged phenological window, an important consideration for compatibility with existing cropping calendars.

Yield attributes and grain yield

ANOVA indicated highly significant (P ≤ 0.01) main effects of hybrid maturity group, nitrogen environment and biostimulant treatment on grain yield, with significant hybrid × N-environment and hybrid × biostimulant interactions (P ≤ 0.05) and a non-significant three-way interaction (Table 5). Grain yield increased progressively with hybrid maturity duration, from 3973 kg ha -1 (H 1 ) to 4942 kg ha -1 (H 2 ) and 5936 kg ha -1 (H 3 ) -a 49.4% improvement of H 3 over H 1 -consistent with the greater total N accumulation and post-silking remobi-lization capacity of longer-duration hybrids (Bender et al., 2013) [3] . Recommended-N plots yielded 5770 kg ha -1 against 4130 kg ha -1 under low-N (+39.7%), and nano-biostimulant application raised mean grain yield from 4545 kg ha -1 (control) to 5356 kg ha -1 (+17.8%) -a response broadly comparable to, though more conservative than, the 14.8% gain reported for seaweed extract alone by Shukla et al. (2019) [7] , consistent with the expectation that the additional PGPR and humic-acid components would compound the single-component response.

Table 3. Plant height, leaf area index (LAI) and SPAD chlorophyll index as influenced by hybrid maturity, N-environment and nano-biostimulant consortium.

Source of variation df Significance

Source of variation df Significance

The highest grain yield (7724 kg ha -1 ) was recorded under the latematuring hybrid with recommended N and the nano-biostimulant consortium (T 12 ) -a 145.3% improvement over the lowest-yielding treatment (T 1: early-maturing, low-N, control; 3149 kg ha⁻¹) (Fig. 3). The significant hybrid × biostimulant interaction indicated that the absolute biostimulant-mediated yield gain was greatest in the late-matur ing hybrid (+1449 kg ha -1 under recommended N, T 11 →T 12 ) relative to the early-maturing hybrid (+946 kg ha -1 , T 3 →T 4 ), supporting the hypothesis that hybrids with a longer post-silking remobilization win-dow derive proportionally greater benefit from biostimulant-enhanced nutrient acquisition (Ciampitti et al., 2012) [4] .

Figure 3. Grain yield of maize as influenced by hybrid maturity group, nitrogen environment and nano-biostimulant consortium application (kg ha -1 ).

Cobs plant -1 was unaffected by any factor (grand mean 1.04), reflecting the single-eared nature of the hybrids at the plant density used, whereas cob length, kernel rows cob -1 , kernels row -1 and 100-kernel weight were all significantly enhanced by hybrid maturity, recommended-N and nano-biostimulant treatment (Fig. 4). The 100-kernel weight rose from 27.40 g (control) to 29.92 g with nano-biostimulant application (CD = 2.39 g at P = 0.05), indicating that sustained nutrient availability translated into more complete grain filling rather than a greater number of florets initiated.

Grain quality: protein content

Grain protein content responded most strongly to nitrogen environment, rising from 9.32% under low-N to 9.78% under recommended-N (CD = 0.635% at P = 0.05), reflecting the direct structural role of N in storage-protein deposition. Nano-biostimulant application produced a smaller but consistent increase (9.45% to 9.66%), attributable to enhanced N uptake and assimilation via PGPR-mediated biological N fixation and phosphate solubilization acting in concert with improved root proliferation. Hybrid maturity group had comparatively little effect on grain protein percentage, consistent with a dilution effect of higher starch deposition in the larger kernels of longer-dura-tion hybrids approximately offsetting their greater total N uptake (Fig. 5).

Soil mineral nitrogen and microbial biomass carbon

Soil NH 4 + -N at harvest was significantly higher under recommended-N (12.10 mg kg -1 ) than low-N (9.42 mg kg -1 ), while nano-biostimulant application produced only a modest increase (10.56 to 10.96 mg kg -1 ), consistent with more complete crop uptake of applied and mineralized N under biostimulant treatment rather than residual accumulation. Soil available P and K at harvest were both significantly higher in nano-biostimulant-treated plots (P: 20.88 to 23.33 kg ha -1 ; K: 229.0 to 242.8 kg ha -1 ), attributable to PGPR-mediated phosphate solubilization and the rhizosphere-priming action of humic acid (Bashan et al., 2014; Nardi et al., 2021) [8,9] . Microbial biomass carbon (MBC) increased substantially with nano-biostimulant applica-tion, from 158.3 to 171.7 mg kg -1 (CD = 31.1 mg kg -1 at P = 0.05), directly corroborating the intended rhizosphere-stimulating mode of action of the PGPR component (Fig. 6).

Multi-nutrient-use efficiency (PFPN, NHI, PUE, KUE)

Partial factor productivity of nitrogen (PFPN) was, as expected on a per-kg-applied-N basis, higher under low-N (68.84 kg kg -1 ) than recommended-N (48.09 kg kg -1 ). Nano-biostimulant application significantly raised PFPN from 53.62 to 63.31 kg kg -1 (CD = 7.24 at P = 0.05), and PFPN increased progressively with hybrid maturity, from 47.15 (H 1 ) to 58.47 (H 2 ) and 69.77 kg kg -1 (H 3 ). Nitrogen harvest index, phosphorus-use efficiency and potassium-use efficiency were likewise all significantly improved by nano-biostimulant application (NHI: 63.49 to 65.30%; PUE: 75.75 to 89.27 kg kg -1 ; KUE: 113.62 to 133.90 kg kg -1 ) and increased progressively with hybrid maturity duration, being consistently highest in H 3 (Table 5, Fig. 7).

Integrated ANOVA summary

A consistent pattern emerged across all growth, yield, quality, soil and nutrient-use-efficiency parameters examined (Table 6): main effects of hybrid maturity group, nitrogen environment and biostimulant treatment were significant for virtually all traits; the hybrid × biostimulant interaction was significant specifically for grain yield and for the yield-linked efficiency indices (PFPN, PUE, KUE); and the threeway interaction remained non-significant throughout. This pattern indicates that the nano-biostimulant consortium acted through a general, physiologically robust mechanism -enhanced root proliferation, rhizosphere microbial activity and canopy photosynthetic capacitywhose absolute benefit scaled with hybrid phenological duration rather than depending on a specific nitrogen environment.

Discussion

The results provide consistent evidence that a phenology-synchronized, chitosan-nanoencapsulated seaweed extract-PGPR-humic acid consortium improves maize growth, yield and multi-nutrient-use efficiency, and that the magnitude of this improvement scales positively with hybrid maturity duration. The combined application of the nanoconsortium with recommended nitrogen in the late-maturing hybrid (T 12 ) produced the highest values across virtually every growth, yield, quality, soil-biological and nutrient-use-efficiency parameter measured, while the unfertilized, non-biostimulant early-maturing hybrid under low-N (T 1 ) recorded the lowest values for nearly every trait -a coherent hierarchy across independently measured trait categories that strengthens confidence in the underlying biological mechanism rather than reflecting a single confounded observation. Mechanistically, the parallel enhancement of leaf area index and SPAD chlorophyll index (source capacity), soil available P and K and microbial biomass carbon (rhizosphere nutrient mobilization), and kernel number and kernel weight (sink capacity and grain filling) is consistent with the three complementary modes of action built into the consortium: seaweed-extract-mediated hormonal stimulation of shoot and root growth (Battacharyya et al., 2015) [6] , PGPR-mediated biological nitrogen fixation and phosphate solubilization (Bashan et al., 2014) [8] , and humic-acid-mediated improvement of root architec-ture and nutrient transport (Nardi et al., 2021) [9] . Because nano-biostimulant application did not alter the calendar duration to physiological maturity, the yield gains documented here reflect genuine improvement in physiological efficiency within an unchanged phenological window rather than an extension of the growing season, an important practical distinction because it indicates the technology can be adopted without disrupting existing cropping calendars or the scheduling of succeeding crops. The significant hybrid × biostimulant interaction for grain yield and for PFPN, PUE and KUE -but not for grain protein, plant height or LAI individually -suggests that the yield-scaling effect of hybrid duration operates principally through the capacity of longer-duration hybrids to translate biostimulant-enhanced nutrient acquisition into a longer post-silking remobilization window, consistent with the physiological framework proposed by Ciampitti et al. (2012) [4] and the empirical nutrient-partitioning data of Bender et al. (2013) [3] . From an applied standpoint, these findings imply that the economic and environmental returns to phenology-synchronized nano-biostimulant technology are likely to be most pronounced in medium-and, especially, late-maturing hybrid systems managed under recommended nitrogen, where the extended grain-filling period provides the greatest physiological opportunity for biostimulant-mediated nutrient capturea conclusion with direct relevance to sustainable-intensification strategies that seek to increase productivity while reducing the envi ronmental footprint of nitrogen fertilization (Chen et al., 2015; Fixen et al., 2015) [12,13] .

Conclusion

Phenology-synchronized application of a chitosan-nanoencapsulated seaweed extract-PGPR-humic acid consortium significantly improved grain yield, growth attributes, yield components, grain protein content, soil biological activity and multi-nutrient-use efficiency of maize across three contrasting hybrid maturity groups and two nitrogen environments. The magnitude of the biostimulant-mediated improvement in grain yield and in PFPN, PUE and KUE was significantly greater in longer-duration hybrids, confirming that late-maturing hybrids possess a greater physiological capacity to convert biostimulant-enhanced nutrient acquisition into recoverable yield and efficiency gains. The combination of a late-maturing hybrid, recommended nitrogen (120 kg N ha -1 ) and the phenology-synchronized nano-biostimulant consortium produced the highest overall performance among all treatments evaluated and is recommended as the most agronomically favourable combination identified in this study. Because nano-biostimulant application did not delay physiological maturity, this technology can be integrated into existing cropping calendars without disrupting succeeding-crop scheduling. The present findings derive from a single season at one location; multi-season, multi-location validation, together with economic analysis of adoption and mechanistic studies at the molecular level (e.g., nutrienttransporter gene expression across maturity groups), are recommended before extending generalized recommendations to farmers.

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15. How to cite this article: Rao BA, Babu CS. Phenology-synchronized nano-biostimulant consortia enhance productivity and multi-nutrient-use efficiency of early-, medium and late-maturing maize (Zea mays L.) hybrids under contrasting nitrogen environments. International Journal of Advanced Biochemistry Research. 2026;SP-10(9):2308-2315. doi: https://www.doi.org/1