Cadmium tolerance and taxonomic diversity of cultivable bacteria isolated from Inga sp. nodules, Quararibea sp. roots and rhizosphere soil in cocoa agroforestry systems of the Peruvian Amazon

Abstract

Cadmium (Cd) contamination in the Peruvian Amazon cocoa soils threatens production sustainability and international market access. This study characterized the taxonomic and genomic diversity of bacteria isolated from Inga sp. nodules, Quararibea sp. roots, and rhizosphere soil in cocoa agroforestry systems in Bagua, Amazonas, Peru (1.02–3.54 mg Cd kg−1), and evaluated the Cd tolerance phenotypes in vitro. From 93 initial isolates, 53 grew under cadmium screening; 23 were selected for Sanger sequencing, yielding 12 high-quality 16S rRNA sequences suitable for downstream phylogenetic analysis (GenBank: PZ012327–PZ012338). These strains were identified via 16S rRNA gene sequencing and BOX-PCR fingerprinting, revealing distribution across three bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria) and five classes. Notably, no rhizobia sensu stricto were recovered from Inga sp. nodules; all nodule-derived isolates were non-rhizobial endophytes (NREs) belonging to Achromobacter, Pseudomonas, Klebsiella, Priestia, and Glutamicibacter. BOX-PCR identified 12 unique genotypes (Shannon H′ = 2.485) distributed in four genomic clusters unrelated to phylogenetic affiliation. The Cd tolerance (25–200 ppm) was assessed via OD₆₀₀ in TY medium. Tolerance indices (0.190–0.643) classified strains into quartiles: highly tolerant (n = 3), tolerant (n = 3), sensitive (n = 3), and highly sensitive (n = 3). JSRA_S05 (K. variicola) showed clear hormesis (TI = 1.235 at 25 ppm), whereas JSRA_S08 (Glutamicibacter sp.) exhibited a possible hormetic tendency (TI = 1.012 at 50 ppm). IC₅₀ values (0.18–719.7 ppm) showed low correlation with TI (ρ < 0.1), indicating complementary dimensions of tolerance. Tolerance was strain-specific, as evidenced by opposite responses of two K. variicola strains (highly tolerant JSRA_S05 versus highly sensitive JSRA_N14). Strains associated with Quararibea sp. were consistently among the most tolerant. Three strains (JSRA_S05, JSRA_S08, and JSRA_R15), combining high physiological tolerance with taxonomic and source diversity, are proposed as priority candidates for functional evaluation in biosorption, bioaccumulation, and plant-inoculation assays. These culture-dependent findings indicate that, under the cultivation conditions employed, the cultivable bacterial fraction recovered from Cd-impacted Inga sp. nodules in Amazonian cocoa agroecosystems is dominated by non-rhizobial endophytes, representing a promising biological resource for further bioremediation studies. Whether classical rhizobia are truly absent or simply not retrieved under these conditions requires culture-independent confirmation.