Scale-dependent variation among destructive and non-destructive chlorophyll estimation methods across crop species

Chlorophyll estimation is fundamental in plant physiology, crop management, and ecological studies; however, destructive and non-destructive methods are often interpreted interchangeably despite differing measurement principles. The present study compared four chlorophyll estimation approaches—two non-destructive (SPAD meter and GreenSeeker) and two destructive (80% acetone and DMSO extraction)—across eight crop species under uniform field conditions. Significant interspecific variation was observed for all methods. Correlation and regression analyses revealed generally weak relationships among methods, particularly between leaf-level (SPAD, solvent extraction) and canopy-level (GreenSeeker) measurements, reflecting scale-dependent behavior and methodological differences. Moderate associations were observed between SPAD and acetone-extracted chlorophyll for certain traits, whereas GreenSeeker showed poor agreement with solvent-based estimates. Differences between DMSO and acetone extraction further highlighted solvent-specific extraction efficiency. The results demonstrate that chlorophyll estimation methods are not directly interchangeable and should be selected based on study objectives, biological scale, and leaf anatomical characteristics. Species-specific calibration and integration of canopy structural parameters are required to improve cross-method interpretability.