Dynamics of Seed-Borne Rice Endophytes on Early Plant Growth Stages

Bacterial endophytes are ubiquitous to virtually all terrestrial plants. With the increasing appreciation of studies that unravel the mutualistic interactions between plant and microbes, we increasingly value the beneficial functions of endophytes that improve plant growth and development. However, still little is known on the source of established endophytes as well as on how plants select specific microbial communities to establish associations. Here, we used cultivation-dependent and -independent approaches to assess the endophytic bacterrial community of surface-sterilized rice seeds, encompassing two consecutive rice generations. We isolated members of nine bacterial genera. In particular, organisms affiliated with Stenotrophomonas maltophilia and Ochrobactrum spp. were isolated from both seed generations. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) of seed-extracted DNA revealed that approximately 45% of the bacterial community from the first seed generation was found in the second generation as well. In addition, we set up a greenhouse experiment to investigate abiotic and biotic factors influencing the endophytic bacterial community structure. PCR-DGGE profiles performed with DNA extracted from different plant parts showed that soil type is a major effector of the bacterial endophytes. Rice plants cultivated in neutral-pH soil favoured the growth of seed-borne Pseudomonas oryzihabitans and Rhizobium radiobacter, whereas Enterobacter-like and Dyella ginsengisoli were dominant in plants cultivated in low-pH soil. The seed-borne Stenotrophomonas maltophilia was the only conspicuous bacterial endophyte found in plants cultivated in both soils. Several members of the endophytic community originating from seeds were observed in the rhizosphere and surrounding soils. Their impact on the soil community is further discussed

Exploration of Hitherto-Uncultured Bacteria from the Rhizosphere

Well-studied microorganisms commonly found in the rhizosphere are considered to play important roles in plant growth support. These microorganisms grow well in culture media as single-species cultures. However, the vast majority of the species present in soil and in the rhizosphere remain uncultured so far. Virtually nothing is known about their eventual interactions with plants and microorganisms indigenous to the rhizosphere and endosphere. Eventual occurrences of plant and microbe interactions may be studied with culture-independent technologies, like metagenomics. However, for experimentation in the rhizosphere, culturable representatives of hitherto uncultured are favored. This chapter focuses on Acidobacteria and Verrucomicrobia, two deep-rooting lineages in bacterial taxonomy because representatives of both phyla are fastidious in their growth. A few culturable representatives have been found so far in different natural ecosystems, including soils. Recently, culturable representatives of the two phyla were found in the rhizospheres of leek (Allium porrum) and potato (Solanum tuberosum), and their identities were different from the ones found in soils. Two representatives of Acidobacteria subdivision 8 (class Holophagae) were found in the leek rhizosphere, whereas in soil, representatives of subdivisions 1, 2, 3, 4, and 6 were found. Further, three taxonomically (based on 16S rRNA gene comparisons with public database sequences) distinguishable groups of Verrucomicrobia subdivision 1 were found in the rhizospheres of leek and potato, whereas in soil, culturable representatives of Verrucomicrobia subdivisions 2 and 3 (Spartobacteria) were found. Acidobacteria subdivision 8 and one particular subgroup of Verrucomicrobia subdivision 1 (whose genus was proposed as Candidatus genus Rhizophora) were shown to be present in the leek rhizosphere. Demonstrating rhizosphere competence will be the first step in the exploration of the roles of these groups in rhizospheres and endospheres of different plant species.</p

Impact of bacterial-fungal interactions on the colonization of the endosphere

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Biplot ordination diagrams of rice shoot and root bacterial endophytes.

<p>RDA diagrams generated from PCR-DGGE profiles of endophytic bacterial community sampled from shoot (A and B) and root (C and D) tissues of plants cultivated on K (A and C) and V (B and D) soils are shown. Squares and circle represent PCR-DGGE patterns of bacterial communities from plants submitted to, respectively, flooded and unflooded regimes and exposed to low- (empty symbol) and high- (full symbol) BID. Triangles (control treatment) represent PCR-DGGE patterns of bacterial communities from plants submitted to unflooded regime and cultivated in uninoculated soils. Six replicates of each treatment are shown. Stars represent nominal environmental variables. Arrows represent PCR-DGGE bands in which only the most descriptive communities are shown.</p

Biplot ordination diagrams of rice rhizosphere and bulk soil bacterial communities.

<p>RDA diagrams generated from PCR-DGGE profiles of bacterial community sampled from rhizosphere (A and B) and bulk (C and D) soil of plants cultivated in K (A and C) and V (B and D) soils are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone-0030438-g003" target="_blank">Fig. 3</a> for symbol description.</p

Heat map composition of selected bacterial communities.

<p>Distribution of select endophytic bacterial communities (rows) from two soil types (K and V) and four different habitats (root-free and rhizosphere soil, root and shoot endosphere) is shown. Cells are coloured in spectrum of grey that correlates with percentage of observed bacterium in a given habitat. Habitat from which the assessed bacterium was most likely to be originated from ‘artificial’ soil community is labelled with “inoculated”. Unlabelled cells are most likely represented by assessed bacterium originated from rice seeds.</p

Identification of isolated seed-borne strains.

a<p>Rice strains isolated from first (R1-R4) and second (R5-R16) generation of seeds.</p><p>*The 16S rRNA gene sequences of strains R6 and R8 were identical to PCR-DGGE products of the bands 12 and 9, respectively.</p>b<p>Source of the closest rice associated bacteria, LE – Leaf Endophyte <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a>; LS – Leaf surface <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a>; PF – Paddy Field (Islam et al., unpublished); PS – Paddy Soil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Shrestha1" target="_blank">[28]</a>; R - Rhizosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Steindler1" target="_blank">[25]</a>; RE1 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Hardoim2" target="_blank">[20]</a>; RE2 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a> and SE – Seed endophyte <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano1" target="_blank">[5]</a>.</p

Identification of excised PCR-DGGE bands.

a<p>Source of the closest rice associated bacteria: PF – Paddy Field <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Cuong1" target="_blank">[65]</a>; PS – Paddy Soil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Shrestha1" target="_blank">[28]</a>; R - Rhizosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Steindler1" target="_blank">[25]</a>; RE1 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Hardoim2" target="_blank">[20]</a> and RE2 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Sun1" target="_blank">[64]</a>.</p