Cytobacts: Abundant and Diverse Vertically Seed-Transmitted Cultivation-Recalcitrant Intracellular Bacteria Ubiquitous to Vascular Plants

Pious Thomas,Thekepat P Rajendran,Christopher M M Franco

doi:10.3389/fmicb.2022.806222

Abstract

We have recently described ‘Cytobacts’ as abundant intracellular endophytic bacteria inhabiting live plant cells based on the observations with callus and cell suspension cultures of grapevine and other plant species with the origin ascribable to field explants. In this study, we investigated the prevalence of such cytoplasmic bacterial associations in field plants across different taxa, their cultivability, and the extent of taxonomic diversity and explored the possibility of their embryo-mediated vertical transmission. Over 100 genera of field plants were surveyed for ‘Cytobacts’ through bright-field live-cell imaging as per our previous experience using fresh tissue sections from surface-sterilized shoot-tissues with parallel cultivation-based assessments. This revealed widespread cellular bacterial associations visualized as copious motile micro-particles in the cytoplasm with no or sparse colony forming units (CFU) from the tissue-homogenates indicating their general non-cultivability. Based on the ease of detection and the abundance of ‘Cytobacts’ in fresh tissue sections, the surveyed plants were empirically classified into three groups: (i) motile bacteria detected instantly in most cells; (ii) motility not so widely observed, but seen in some cells; and (iii) only occasional motile units observed, but abundant non-motile bacterial cells present. Microscopy versus 16S-rRNA V3–V4 amplicon profiling on shoot-tip tissues of four representative plants—tomato, watermelon, periwinkle, and maize—showed high bacterial abundance and taxonomic diversity (11–15 phyla) with the dominance of Proteobacteria followed by Firmicutes/Actinobacteria, and several other phyla in minor shares. The low CFU/absence of bacterial CFU from the tissue homogenates on standard bacteriological media endorsed their cultivation-recalcitrance. Intracellular bacterial colonization implied that the associated organisms are able to transmit vertically to the next generation through the seed-embryos. Microscopy and 16S-rRNA V3–V4 amplicon/metagenome profiling of mature embryos excised from fresh watermelon seeds revealed heavy embryo colonization by diverse bacteria with sparse or no CFU. Observations with grapevine fresh fruit-derived seeds and seed-embryos endorsed the vertical transmission by diverse cultivation-recalcitrant endophytic bacteria (CREB). By and large, Proteobacteria formed the major phylum in fresh seed-embryos with varying shares of diverse phyla. Thus, we document ‘Cytobacts’ comprising diverse and vertically transmissible CREBs as a ubiquitous phenomenon in vascular plants.

Highlights

All plants and plant organs are known to harbor endophytic bacteria, the prokaryotes that inhabit plants internally without apparent adverse effects on the host (Hallmann et al, 1997; Kandel et al, 2017)
Pure cultures of different bacteria isolated as endophytes were observed under bright-field microscopy, and the captured videos for some representative organisms—Klebsiella pneumoniae, Microbacterium esteraromaticum, Micrococcus terrus—have been provided to serve as a reference to assess the similarity of the motile micro-particles in the tissue sections to the bacterial cells in terms of size, shape, and motility patterns (Supplementary Movies 01–03) along with Bacillus pumilus (Supplementary Movie 04) isolated as external contaminant
Three distinct plant categories were observed: (i) motile bacteria detected instantly in most cells, as documented with tomato, sweet pepper, hot pepper, egg-plant, garden peas, cowpea, French bean, coriander, amaranth, chrysanthemum, marigold, gerbera, crossandra, centella, money plant, and pineapple (Supplementary Movies 05–09); (ii) motility not so widely observed, but seen in some cells with non-motile bacteria obvious in most cells; and (iii) no or occasional motile bacteria observed but abundant non-motile microparticles corresponding to bacteria present

Summary

Introduction

All plants and plant organs are known to harbor endophytic bacteria, the prokaryotes that inhabit plants internally without apparent adverse effects on the host (Hallmann et al, 1997; Kandel et al, 2017). Lower bacterial diversity commonly observed in root tissues relative to the rhizosphere has contributed to the assumption that plants selectively recruit a subset of their choice organisms from soil as endophytes (Compant et al, 2010, 2021; Hardoim et al, 2015). One major and initial route of plant entry of endophytic bacteria is through root hairs (Prieto et al, 2011; Compant et al, 2021). This is applicable for seed-associated bacteria making their entry at seed germination from the spermosphere (Nelson, 2018). As per the general understanding, the organisms move further upward through the vascular system and reach various plant parts displaying intercellular colonization (Compant et al, 2005, 2008, 2011; Kandel et al, 2017; Liu et al, 2017). Plant acquisition of endophytes from the aerial parts through natural openings such as stomata, lenticels, and injuries incited by insects and other pests is proposed, but in such cases too, the colonization is generally intercellular (Frank et al, 2017; Kandel et al, 2017)

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Results

Conclusion