Hexachlorocyclohexane (HCH) is a highly recalcitrant pesticide that persists in soils. Three novel HCHdegrading strains (DS2, DS2-2 and DS3-1) were isolated after enrichment from HCH-contaminated soil from Germany. These strains efficiently degraded the a a a a -, g g g g -and d d d d -isomers of HCH, while strain DS3-1 also degraded b b b b -HCH. Based on 16S rDNA analysis, strain DS3-1 was closely related to Sphingomonas taejonensis , while strains DS2 and DS2-2 were closely related to Sphingomonas flava and seven HCHdegrading strains recently isolated from HCH-contaminated Spanish soil. Hence, geographic origin of the strains was not reflected in their phylogenetic affiliation. Subsequently, lin genes involved in HCH degradation, virtually identical to those from Sphingomonas paucimobilis strains UT26 from Japan and B90A from India, were identified in strains DS3-1, DS2, DS2-2 and five of the strains from Spain. The conserved lin gene sequences and structural organization, as well as the close association with IS 6100 , suggest a shared lin gene origin and recent horizontal gene transfer among phylogenetically diverged Sphingomonas strains in remote geographic locations. The loss of the ability to degrade g g g g -HCH was associated with the deletion of the linA gene, probably due to recombination involving IS 6100 elements, of which several copies are located in the lin cluster region.

SummaryWe used a two‐step enrichment approach to isolate root‐colonizing hexachlorocyclohexane (HCH)‐degrading microorganisms. The first step consists of the use of classical liquid enrichment to isolate γ‐HCH degraders. The γ‐HCH‐degrading microbes were attached in mass to corn seeds sown in soil with γ‐HCH, and after plant development we rescued bacteria growing on root tips. Bacteria were then subjected to a second enrichment round in which growth on liquid medium with γ‐HCH and inoculation of corn seeds were repeated. We then isolated bacteria on M9 minimal medium with γ‐HCH from root tips. We were able to isolate four Sphingomonas strains, all of which degraded α‐, β‐, γ‐ and δ‐HCH. Two of the strains were particularly good colonizers of corn roots, reaching high cell density in vegetated soil and partly removing γ‐HCH. In contrast, these bacteria performed poorly in unplanted soils. This study supports the hypothesis that the removal of persistent toxic chemicals can be accelerated by combinations of plants and bacteria, a process generally known as rhizoremediation.

Symbiotic associations with arbuscular mycorrhizal fungi (AMF) offer an effective indirect mechanism to reduce heavy metal (HM) stress; however, it is still not clear which AMF species are more efficient as bioremediating agents. We selected different species of AMF: Rhizoglomus custos (Custos); Rhizoglomus sp. (Aznalcollar); and Rhizophagus irregularis (Intraradices), in order to study their inoculation in wheat grown in two soils contaminated with two levels of HMs; we tested the phytoprotection potential of the different AMF symbioses, as well as the physiological responses of the plants to HM stress. Plants inoculated with indigenous Aznalcollar fungus exhibited higher levels of accumulation, mainly in the shoots of most of the HM analyzed in heavily contaminated soil. However, the plants inoculated with the non-indigenous Custos and Intraradices showed depletion of some of the HM. In the less-contaminated soil, the Custos and Intraradices fungi exhibited the greatest bioaccumulation capacity. Interestingly, soil enzymatic activity and the enzymatic antioxidant systems of the plant increased in all AMF treatments tested in the soils with both degrees of contamination. Our results highlight the different AMF strategies with similar effectiveness, whereby Aznalcollar improves phytoremediation, while both Custos and Intraradices enhance the bioprotection of wheat in HM-contaminated environments.