Comparative analysis of endophytic bacterial localization and microbiome diversity in plant varieties under varied growth conditions through microscopic imaging and sequencing techniques

Abstract

Introduction: Intracellular colonization by endophytic bacteria (EB) is a relatively new and less explored aspect of plant microbiome research. In this study, we investigated the presence and localization of EB in Nicotiana tabacum var. Podali and Vigna radiata var. Pratap using SYTO9 (S9) and Propidium Iodide (PI) staining.

Methodology: Confocal Laser Scanning Microscopy (CLSM) was used to visualized bacterial localization, MitoTracker Deep Red (MDR) was used to confirm non- overlapping mitochondrial staining. Time-lapse imaging was employed to observe bacterial motility. For microbial community profiling next-generation sequencing (NGS) of the 16S rRNA gene was conducted to analyze bacterial diversity and composition.

Results: Confocal laser scanning microscopy (CLSM) revealed S9-labelled live bacteria located close to the nucleus in Podali tissues and suspension cultures, while PI selectively stained dead cells. MitoTracker Deep Red (MDR) confirmed that there was no overlap with mitochondrial staining. Interestingly, time-lapse imaging captured the movement of bacteria within the cells, indicating possible bacterial motility. EB were observed in both in vitro and field-grown Podali plants, whereas they were detected only in field-grown Pratap plants. Next-generation sequencing revealed that Podali harbored a much higher bacterial diversity, with 37 bacterial families identified mainly from Burkholderiaceae and Enterobacteriaceae. In contrast, Pratap plants showed lower diversity, with only 10 bacterial families, dominated by Rhizobiaceae.

Conclusion: This study is among the first to report intracellular EB localization in these plant varieties and demonstrates how environmental conditions and growth methods can influence the composition of plant-associated microbiomes.

Keywords: endophytic bacteria; intracellular colonization; microbiome diversity; next-generation sequencing; plant-microbe interactions.

Figure 1

Treatment of plant cell suspension with different antibiotics. (A) Control plant cell suspension stained with Syto9 shows endophyte around the nucleus and cell boundary shown by blue arrow. (B–F) Plant cell suspension treated with different antibiotics (100 μg) showed elimination of endophyte. In (B) rifampicin treated cell suspension display few bacteria cells. In each cell, nucleus was stained with Syto9 shown with red arrow. Image size 25 μm.

Figure 2

Cell viability in plant cell suspension and intracellular association of EB. (A) Yellow arrow showed staining of EB by S9 in single cell suspensions, around the nucleus and plasma membrane. (B) White arrow indicates staining of nucleus with PI of non-viable cells. (C) Merge image of (A,B). Image size 25 μm.

Figure 3

Distinction between intracellular bacteria and mitochondria in plant cell suspension culture. Cells were stained with MDR for mitochondria and S9 for bacteria and nucleus. Confocal micrograph show: (A) intracellular bacteria stained with S9, indicated by yellow arrow. (B) Mitochondria stained with MDR, indicated by white arrow. Size of the mitochondria ranges from 1.45–2.27 μm. Presence of bacteria and mitochondria together indicates the cytosolic nature of EB. Image size 10 μm.

Figure 4

Intracellular bacteria and chloroplast in the tobacco leaves tissue. (A) Syto9 staining of tobacco leaves tissue section showed the presence of intracellular bacteria (green color) around the nucleus and the cell membrane, indicated by yellow arrow. (B) Auto-fluorescence of chloroplasts (magenta color) from leaves tissue indicated by red arrow. (C) Merged image of (A,B). Image size 25 μm.

Figure 5

Observation of endophytic bacteria in Pratap Plants grown under aseptic condition and in open field using LCM. (A) S9 stains only the nucleus (indicated by magenta arrows) of leaf tissue of Pratap plants grown under aseptic condition in MS medium. (B) Auto-fluorescence of chloroplast in aseptically grown Pratap plants (indicated by white arrow). (C) Merge image of (A,B). (D) S9 stains both the nucleus (indicated by magenta arrow) and endophytic bacteria (indicated by white arrow) in leaf tissue sections of Pratap plants grown in open field. (E) Auto-fluorescence of chloroplast of Pratap grown in open field (indicated by white arrow). (F) Merge image of (D,E). Image size (A–C) 10 μm and (D–F) 25 μm.

Figure 6

Diversity of bacterial endophyte In Tobacco Plant (TS) and Green Gram plants (PS). (A) Relative abundance at Phylum level. (B) Represent the absolute abundance of 3 replicates of tobacco var. Podali and Green Gram var. Pratap. (C) Relative abundance at genus level. (D) Relative abundance at family level.