Vertical Transmission of Diverse Cultivation-Recalcitrant Endophytic Bacteria Elucidated Using Watermelon Seed Embryos

Abstract

Seed transmission of endophytic microorganisms is a growing research area in plant biology and microbiology. We employed cultivation versus cultivation-independent approaches on excised embryos from watermelon seeds (6–12 months in storage) and on embryo-derived in vitro seedlings (EIVS) to assess the vertical transmission of endophytic bacteria. Surface-disinfected watermelon seeds bore abundant residual bacteria in the testa and perisperm tissues, predominantly Bacillus spp. propounding the essentiality of excluding all non-embryonic tissues for vertical transmission studies. Tissue homogenates from re-disinfected seed embryos displayed no cultivable bacteria during the 1-week monitoring. Bright-field live microscopy revealed abundant bacteria in tissue homogenates and in embryo sections as intracellular motile particles. Confocal imaging on embryo sections after SYTO-9 staining and eubacterial fluorescent in situ hybridization (FISH) endorsed enormous bacterial colonization. Quantitative Insights Into Microbial Ecology (QIIME)-based 16S rRNA V3–V4 taxonomic profiling excluding the preponderant chloroplast and mitochondrial sequences revealed a high bacterial diversity in watermelon seed embryos mainly Firmicutes barring spore formers followed by Proteobacteria, Bacteroidetes, and Actinobacteria, and other minor phyla. Embryo-base (comprising the radicle plus plumule parts) and embryo-cotyledon parts differed in bacterial profiles with the abundance of Firmicutes in the former and Proteobacteria dominance in the latter. EIVS displayed a higher bacterial diversity over seed embryos indicating the activation from the dormant stage of more organisms in seedlings or their better amenability to DNA techniques. It also indicated embryo-to-seedling bacterial transmission, varying taxonomic abundances for seed embryos and seedlings, and differing phylogenic profiles for root, hypocotyl, and cotyledon/shoot-tip tissues. Investigations on different watermelon cultivars confirmed the embryo transmission of diverse cultivation recalcitrant endophytic bacteria. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes formed the core phyla across different cultivars with 80–90% similarity at genus to phylum levels. Conversely, freshly harvested seeds displayed a dominance of Proteobacteria. The findings revealed that dicot seeds such as in different watermelon cultivars come packaged with abundant and diverse vertical and seedling-transmissible cultivation recalcitrant endophytic bacteria with significant implications for plant biology.

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

“…Recent reports on endophytic bacteria in pollen grains ( Maniranjan et al, 2017 ), excised seed-embryos in wheat ( Kuźniar et al, 2020a , b ), and in the in vitro raised tomato seedlings after seed-coat removal post-seed germination illustrate this significant result ( Shaik and Thomas, 2019 ). Very recent studies employing excised seed-embryos, different parts of embryos, and embryo-derived in vitro seedlings of watermelon have clearly established vertical transmission of abundant and diverse bacteria prevailing as CREBs ( Thomas and Sahu, 2021 ). Intracellular colonization facilitates the diverse organisms to reach the embryo and in turn all the cells of the zygote-derived new plant allowing a concoction of the organisms from either parent.…”

“…Tomato, watermelon, and maize are common annual crops accessible the world over, the former two representing dicots and the latter, a monocot. The first two also formed the candidates for seed and embryo bacterial transmission studies ( Thomas and Shaik, 2020 ; Thomas and Sahu, 2021 ). Periwinkle, a semi-perennial shrub, formed the source of cell cultures employed for the elucidation of intracellular bacteria ( Thomas and Franco, 2021 ) along with grapevine which was covered in detail earlier ( Thomas et al, 2017 ).…”

“…One major question with respect to the intracellular bacterial colonization pertained to how the organisms gain cytoplasmic entry throughout the plant system without damaging the cells, concurring with the widely accepted concept of plant recruitment of endophytic bacteria from soil or atmosphere. This becomes feasible in the case of embryo-mediated vertical transmission ( Thomas and Sahu, 2021 ). Intracellular colonization implies that the organisms are able to move to the daughter cells/gametes through mitosis/meiosis and then to the embryo, which allows their distribution to all parts of the new plant similar to cell organelles.…”

“…In such studies, there is a possibility of carry-over of externally associated bacteria despite extensive surface-sterilization treatments. It is essential that the embryo be studied distinctly excluding the seed coat tissues to confirm the vertical transmission ( Thomas and Sahu, 2021 ). The report on ‘Cytobacts’ as a widespread association in different plant tissues and as a general phenomenon in cell and callus cultures across plant species substantiated the possibility of their movement to the gametes, possible integration in the new embryos during fertilization, and the continued perpetuation in the new plant.…”

“…Thus, the present study was taken up as a continuation of the observations on the prevalence of abundant and diverse endophytic bacteria in cell cultures of different plant species with their origin traced to the field explants ( Thomas and Franco, 2021 ) and their possible vertical transmission ( Thomas and Sahu, 2021 ). Further, we found bright-field live-tissue imaging as an easy and simple way to observe the intracellular bacteria ( Thomas and Sekhar, 2014 ; Thomas and Franco, 2021 ), whereas the deep-sequencing or omics-based tools help in studying the diversity of conventionally uncultivable plant microbiome ( Kaul et al, 2016 ; Matsumoto et al, 2021 ).…”