Comparison of specific endophytic bacterial communities in different developmental stages of Passiflora incarnata using culture-dependent and culture-independent analysis

Abstract

Plants and endophytic microorganisms have coevolved unique relationships over many generations. Plants show a specific physiological status in each developmental stage, which may determine the occurrence and dominance of specific endophytic populations with a predetermined ecological role. This study aimed to compare and determine the structure and composition of cultivable and uncultivable bacterial endophytic communities in vegetative and reproductive stages (RS) of Passiflora incarnata. To that end, the endophytic communities were assessed by plating and Illumina-based 16S rRNA gene amplicon sequencing. Two hundred and four cultivable bacterial strains were successfully isolated. From the plant's RS, the isolated strains were identified mainly as belonging to the genera Sphingomonas, Curtobacterium, and Methylobacterium, whereas Bacillus was the dominant genus isolated from the vegetative stage (VS). From a total of 133,399 sequences obtained from Illumina-based sequencing, a subset of 25,092 was classified in operational taxonomy units (OTUs). Four hundred and sixteen OTUs were obtained from the VS and 66 from the RS. In the VS, the most abundant families were Pseudoalteromonadaceae and Alicyclobacillaceae, while in the RS, Enterobacteriaceae and Bacillaceae were the most abundant families. The exclusive abundance of specific bacterial populations for each developmental stage suggests that plants may modulate bacterial endophytic community structure in response to different physiological statuses occurring at the different plant developmental stages.

Keywords: 16S rRNA gene sequencing; diversity; endophytic microbiome; microbial ecology; plant development.

Figure 1

Graphical representation of culturable community recovered in each culture medium. Number of species and isolates from (a) vegetative stage and (b) reproductive stage of Passiflora incarnata. The values shown above of each pair bars are the richness index of Margalef (D_MG)

Figure 2

Rarefaction curves of partial sequences of 16S rRNA gene. Rarefaction analysis of 16S rRNA gene sequence data to estimate microbial diversity based on a cutoff <97% sequence identity. Abbreviation: OTUs, operational taxonomy units

Figure 3

Krona plots on 16S rRNA sequences of the bacterial communities associated with Passiflora incarnata leaves. The data represent taxonomic hierarchies of bacterial communities in the (a) vegetative stage and in the (b) reproductive stage in a multilevel diagram

Figure 4

Heat map of the bacterial community composition of each stage based on Bray–Curtis distance. Taxonomic distribution of the core endophytic bacterial community at family level, based on an analysis of the first 16 most abundant families. Clustering of samples based on Bray–Curtis distance indices calculated by operational taxonomy units at a distance of 3%

Figure A1

Comparison of taxonomic profiles between bacterial communities of vegetative and reproductive stages. (a) Scatter plot graph showing differences at the family level, (b) extended error bar percentage representation plot is showing differences in abundance between central bacterial populations at family level.