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.
Highlights
Endophytic bacteria colonize plants internally without any apparent adverse effects on the host and are normally considered beneficial to the host (Hallmann et al, 1997; Afzal et al, 2019)
Monitoring the seed external and internal bacterial load after the three treatments, namely, repeated filtered distilled water postautoclaving (FDW) washing followed by 1 min ethanol soaking (T1), T1 followed by 5 min NaOCl treatment (T2), and T1 succeeded by 5 min 0.1% HgCl2 (T3), indicated a large amount of seed external bacteria (1.0 × 104 colony-forming unit (CFU)/seed), a part of which (4.4 × 103 CFU/seed) was released with mere water-rinsing which comprised largely spore-forming Bacillus spp. (Table 1 and Supplementary Figure 1)
The bacterial population that emerged from the seed testa, embryo washes, or the embryo homogenate varied with the disinfectant employed: slower-growing non-sporulating Gram-positive colony types dominated the NaOCl (4% chlorine) treated set, the HgCl2 (0.1%) treated set showed a high population of fast growing Bacillus spp., while the mere ethanol-treated seeds showed an intermediate population; the reason for this differential outcome was not understood
Summary
Endophytic bacteria colonize plants internally without any apparent adverse effects on the host and are normally considered beneficial to the host (Hallmann et al, 1997; Afzal et al, 2019). Application of cultivation-independent molecular tools to study the endophytic bacterial diversity in the shoot tissues and in particular to the in vitro plant cultures which could be guarded against the external organisms revealed a huge bacterial diversity similar to the root system prevailing a cultivation recalcitrant form (Thomas and Sekhar, 2017; Thomas et al, 2017). This raised a question about the prospects of endophyte entry over and above the generally described routes
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