Physiological Mechanisms Underlying Chemical Fertilizer Reduction: Multiyear Field Evaluation of Microbial Biofertilizers in ‘Gala’ Apple Trees

This study is Part II of a five-year (2018-2022) field trial in western Portugal evaluating the effects of three microbial biofertilizers-Mycoshell^® (<i>Glomus</i> spp. + humic/fulvic acids), Kiplant iNmass^® (<i>Azospirillum brasilense</i>, <i>Bacillus megaterium</i>, <i>Saccharomyces</i> cerevisiae), and Kiplant All-Grip^® (<i>Bacillus megaterium</i>, <i>Pseudomonas</i> spp.)-applied at different dosages alongside two mineral fertilizer regimes, T100 (full dose) and T70 (70% of T100, alone or combined with biofertilizers), on the physiological performance of 'Gala Redlum' apple trees. Part I had shown that Myc4 (Mycoshell^®, 4 tablets/tree), iNM6, and iNM12 (Kiplant iNmass^®, 6 and L ha^-1, respectively) consistently enhanced fruit growth, yield, and selected quality traits. While Part I showed clear agronomic gains, Part II demonstrates that these improvements occurred without significant alterations in seasonal photosynthetic performance, canopy reflectance, or chlorophyll fluorescence parameters over five years, highlighting the contrast between observed yield improvements and physiological stability. Seasonal monitoring of physiological traits-including specific leaf area (SLA), chlorophyll content index (CCI), gas exchange (<i>A_n</i>, <i>g_s</i>, <i>E</i>, <i>C_i</i>), spectral indices (NDVI, OSAVI, SIPI, GM2), and chlorophyll fluorescence (OJIP). It is clear that physiological values remained largely stable across biofertilizer treatments and years. Importantly, this stability was maintained even under a 30% reduction in mineral fertilizer (T70), indicating that specific microbial biofertilizers can sustain physiological resilience under reduced nutrient inputs, thereby providing a physiological basis for the yield-enhancing effects observed and supporting their integration into fertilizer reduction strategies in Mediterranean orchards.