Evaluation of Bacillus safensis-3F under semi-storage conditions for control of naturally occurring Penicillium digitatum and preservation of orange quality

In orange warehouses in northern Iran, hot-water treatment is used as part of standardized postharvest handling practices before subsequent cold storage. In this region, fruits disinfection is routinely performed by dipping in 42 °C hot water containing either Xedamix3 ( Syzygium aromaticum essential oil + ortho-phenylphenate) or CitroGuard (imazalil + pyrimethanil). In the current study, native bacterial epiphytes isolated from Thomson navel orange surface and screened for antagonism against native P. digitatum IR1037C, the causal agent of citrus green mold. Following in vitro inhibitory screenings, four isolates (3F, 2B, 2ND, and 2NF) were selected for further assessment. Among them, isolates 3F retained their inhibitory potential under low temperature, suppressed completely rotting symptoms on healthy fruits in wound inoculation method, and formed the highest rate of biofilm. Based on further assessments, 3F isolate characterized as Bacillus s afensis, harbored the lipopeptide-associated genes ( srf , spa , itu ) and was compatible with thiabendazole and Xedamix3. 3F metabolites contributed to fungal mycelium disruption, as presented by morphological abnormalities and electrolyte leakage. Moreover, its volatiles and cell free supernatant restricted the mycelium development of P. digitatum IR1037C during in vivo trials. During two independent in vivo challenges at low temperature, consisting of wound protection assay, and a semi-storage procedure with natural infection, strain 3F consistently prevented green mold. Its biocontrol efficiency was similar to or greater than thiabendazole and less than Xedamix3. In addition, 3F treatment did not modify total soluble solids, titratable acidity and pH, reduced fruit weight loss and enhanced vitamin C content compared with other treatments. These findings suggest that B. safensis 3F could be a promising approach for managing green mold during cold storage in citrus packinghouses. However, additional efforts are requested to evaluate bacterial population dynamics and its metabolic activity during storage, develop bacterial formulation and confirm its performance.