A reproducible workflow for isolating and characterizing bacterial endophytes, pathogens, and saprophytic colonizers...

A reproducible workflow is presented that integrates fruit‑health stratification, stringent surface sterilization, culture‑based isolation, molecular identification, enzyme phenotyping, and a low‑injury needle‑transfer pathogenicity assay to isolate, classify and functionally characterize bacterial endophytes, saprophytic colonizers and pathogens associated with postharvest tomato (Solanum lycopersicum) fruits. The method is designed to distinguish ecological guilds (endophytes vs soft‑rot pathogens vs saprophytes) rather than simply list "bacteria present", and can be implemented in standard microbiology laboratories without specialized equipment. Tomato fruits were stratified by fruit-health status and surface‑sterilized to distinguish internal endophytes from epiphytic and saprophytic surface‑associated communities. All bacterial isolates were cultured on tryptone soya agar, purified, assigned to an ecological niche (healthy or spoiled/diseased fruits), and tested for in planta pathogenicity on tomato fruits. All the isolates were identified using biochemical and 16S rRNA gene sequencing, while preliminary phenotypic screening was used to quantify cell wall‑degrading activities relevant to soft‑rot. The workflow yielded 14 characterized bacterial isolates spanning three ecological groups (non‑pathogenic endophytic Bacillus species, soft‑rot‑inducing Enterobacterales, and saprophytic colonizers), with ecological niche separation statistically supported by Fisher's exact test (p < 0.001). The method can be adapted to other fruit or vegetable systems to link bacterial community composition with plant health outcomes.•Provides a low-injury and contamination-reduced approach for fruit pathogenicity assays based on a needle‑transfer inoculation technique adapted for routine microbiology laboratories.•Enables the functional differentiation of endophytic, saprophytic, and pathogenic bacterial isolates relevant to fruits and vegetables through combined ecological sources, in planta pathogenicity, and enzyme phenotyping.•The approach is adaptable to multiple fruit and vegetable host crops in a resource-limited and efficient laboratory setting.