Liberibacter crescens biofilm formation in vitro: establishment of a model system for pathogenic \u2018Candidatus Liberibacter spp.\u2019

Eber Naranjo,Mukesh Jain,Dean W Gabriel,Leonardo De La Fuente,Ofir Bahar,Michael J Davis,Marcus V Merfa,Virginia Ferreira

doi:10.1038/s41598-019-41495-5

Abstract

The Liberibacter genus comprises insect endosymbiont bacterial species that cause destructive plant diseases, including Huanglongbing in citrus and zebra chip in potato. To date, pathogenic ‘Candidatus Liberibacter spp.’ (CLs) remain uncultured, therefore the plant-associated Liberibacter crescens (Lcr), only cultured species of the genus, has been used as a biological model for in vitro studies. Biofilm formation by CLs has been observed on the outer midgut surface of insect vectors, but not in planta. However, the role of biofilm formation in the life cycle of these pathogens remains unclear. Here, a model system for studying CLs biofilms was developed using Lcr. By culture media modifications, bovine serum albumin (BSA) was identified as blocking initial cell-surface adhesion. Removal of BSA allowed for the first time observation of Lcr biofilms. After media optimization for biofilm formation, we demonstrated that Lcr attaches to surfaces, and form cell aggregates embedded in a polysaccharide matrix both in batch cultures and under flow conditions in microfluidic chambers. Biofilm structures may represent excellent adaptive advantages for CLs during insect vector colonization helping with host retention, immune system evasion, and transmission. Future studies using the Lcr model established here will help in the understanding of the biology of CLs.

Highlights

‘Candidatus Liberibacter’ species are the causal agents of devastating plant diseases worldwide that include Huanglongbing (HLB) or citrus greening, and zebra chip (ZC) of potato[1,2]
To facilitate comparison with the media nomenclature used throughout this manuscript (Table 1), BM7 medium is referred here as “bBM7 + Fetal bovine serum (FBS)” that corresponds to bBM7 containing FBS
For all experiments that were started from cultures on bBM7 + FBS, a higher growth rate of Liberibacter crescens (Lcr) was observed than in those that started from cultures on bBM7 + 1.0 mβc agar plates (Figs 1 and 2)

Summary

Introduction

‘Candidatus Liberibacter’ species are the causal agents of devastating plant diseases worldwide that include Huanglongbing (HLB) or citrus greening, and zebra chip (ZC) of potato[1,2]. For all experiments that were started from cultures on bBM7 + FBS, a higher growth rate of Lcr was observed than in those that started from cultures on bBM7 + 1.0 mβc agar plates (Figs 1 and 2).

Results

Conclusion