Molecular characterization of epiphytic ice nucleation-active bacteria on deciduous fruit trees in northwest Iran

Late-spring frosts mediated by epiphytic ice nucleation-active (INA) bacteria pose significant risks to temperate fruit production. We systematically isolated epiphytic bacteria from the phyllosphere and caulosphere of eight deciduous fruit species across nine sites in East Azerbaijan, northwest Iran, during autumn 2023 and spring 2024. Of 94 purified isolates, 35 (37.2%) were INA-positive in the droplet-freezing assay. Among these, 22 strains with the highest freezing efficiency were selected for polyphasic characterization. BOX-PCR fingerprinting resolved these into four genomic clusters at 45% Jaccard similarity, revealing notable inter-cluster heterogeneity. One representative strain per cluster was selected for detailed analysis. Phenotypic profiling, 16 S rRNA sequencing with EzBioCloud-based identity scoring, and Bayesian phylogenetic analysis confirmed their taxonomic identities as Pantoea agglomerans Ab445 (GenBank accession PV889035.1), Erwinia billingiae Ab432 (PV916234.1), and Bacillus safensis Ab399 (PV916233.1), while strain Ab431 was assigned to Bacillus wiedmannii based on 98.0% sequence identity. Temperature–time kinetic assays showed that P. agglomerans and B. safensis were strong ice nucleators, initiating crystallization at − 2 °C within 20–30 min, whereas B. wiedmannii and E. billingiae strains functioned as weak nucleators requiring lower temperatures and extended exposure. A pronounced geographic pattern emerged: isolates from high-altitude regions such as Tabastanagh and Uzbak were exclusively strong nucleators, while those from lower-elevation areas like Marand and Ashan were uniformly weak. To our knowledge, this represents the first systematic report of Bacillus strains exhibiting strong ice nucleation activity in Iranian deciduous orchards, and among few such accounts globally. Further protein-level characterization and targeted gene analyses are warranted to elucidate the molecular mechanisms underlying Bacillus -mediated ice nucleation.