Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage

Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant

Cultivation of hitherto-uncultured bacteria belonging to the Verrucomicrobia subdivision 1 from the potato (Solanum tuberosum L.) rhizosphere

The role of dominant bacterial groups in the plant rhizosphere, e.g., those belonging to the phyla Acidobacteria and Verrucomicrobia, has, so far, not been elucidated, and this is mainly due to the lack of culturable representatives. This study aimed to isolate hitherto-uncultured bacteria from the potato rhizosphere by a combination of cultivation approaches. An agar medium low in carbon availability (oligotrophic agar medium) and either amended with potato root exudates or catalase or left unamended was used with the aim to improve the culturability of bacteria from the potato rhizosphere. The colony forming unit numbers based on colonies and microcolonies were compared with microscopically determined fluorescence-stained cell numbers. Taxonomical diversity of the colonies was compared with that of library clones made from rhizosphere DNA, on the basis of 16S rRNA gene comparisons. The oligotrophic media amended or not with catalase or rhizosphere extract recovered up to 33.6% of the total bacterial numbers, at least seven times more than the recovery observed on R2A. Four hitherto-uncultured Verrucomicrobia subdivision 1 representatives were recovered on agar, but representatives of this group were not found in the clone library. The use of oligotrophic medium and its modifications enabled the growth of colony numbers, exceeding those on classical agar media. Also, it led to the isolation of hitherto-uncultured bacteria from the potato rhizosphere. Further improvement in cultivation will certainly result in the recovery of other as-yet-unexplored bacteria from the rhizosphere, making these groups accessible for further investigation, e.g., with respect to their possible interactions with plants

Soil microbial diversity affects the plant-root colonization by arbuscular mycorrhizal fungi

Terrestrial plants establish symbiosis with arbuscular mycorrhizal fungi (AMF) to exchange water and nutrients. However, the extent to which soil biodiversity influences such association remains still unclear. Here, we manipulated the soil microbial diversity using a "dilution-to-extinction" approach in a controlled pot microcosm system and quantified the root length colonization of maize plants by the AMF Rhizophagus clarus. The experiment was performed by manipulating the soil microbiome within a native and foreign soil having distinct physicochemical properties. Overall, our data revealed significant positive correlations between the soil microbial diversity and AMF colonization. Most importantly, this finding opposes the diversity-invasibility hypothesis and highlights for a potential overall helper effect of the soil biodiversity on plant-AMF symbiosis

Unravelling the interplay of ecological processes structuring the bacterial rare biosphere

Most ecological communities harbor many rare species (i.e., the rare biosphere), however, relatively little is known about how distinct ecological processes structure their existence. Here, we used spatiotemporal data on soil bacterial communities along a natural ecosystem gradient to model the relative influences of assembly processes structuring the rare and common biospheres. We found a greater influence of homogeneous selection (i.e., imposed by spatiotemporally constant variables) mediating the assembly of the rare biosphere, whereas the common biosphere was mostly governed by variable selection (i.e., imposed by spatial and/or temporal fluctuating variables). By partitioning the different types of rarity, we found homogeneous selection to explain the prevalence of permanently rare taxa, thus suggesting their persistence at low abundances to be restrained by physiological traits. Conversely, the dynamics of conditionally rare taxa were mostly structured by variable selection, which aligns with the ability of these taxa to switch between rarity and commonness as responses to environmental spatiotemporal variations. Taken together, our study contributes to the establishment of a link between conceptual and empirical developments in the ecology of the soil microbial rare biosphere. Besides, this study provides a framework to better understand, model, and predict the existence and dynamics of microbial rare biospheres across divergent systems and scales

Comparing the influence of assembly processes governing bacterial community succession based on DNA and RNA data

Quantifying which assembly processes structure microbiomes can assist prediction, manipulation, and engineering of community outcomes. However, the relative importance of these processes might depend on whether DNA or RNA are used, as they differ in stability. We hypothesized that. RNA-inferred community responses to (a)biotic fluctuations are faster than those inferred by DNA; the relative influence of variable selection is stronger in RNA-inferred communities (environmental factors are spatiotemporally heterogeneous), whereas homogeneous selection largely influences DNA-inferred communities (environmental filters are constant). To test these hypotheses, we characterized soil bacterial communities by sequencing both 16S rRNA amplicons from the extracted DNA and RNA transcripts across distinct stages of soil primary succession and quantified the relative influence of each assembly process using ecological null model analysis. Our results revealed that variations in α-diversity and temporal turnover were higher in RNA- than in DNA-inferred communities across successional stages, albeit there was a similar community composition; in line with our hypotheses, the assembly of RNA-inferred community was more closely associated with environmental variability (variable selection) than using the standard DNA-based approach, which was largely influenced by homogeneous selection. This study illustrates the need for benchmarking approaches to properly elucidate how community assembly processes structure microbial communities