Ecology of Root Colonizing Massilia (Oxalobacteraceae)

Citation: Ofek M, Hadar Y, Minz D ( Ecology of Root Colonizing Massilia (Oxalobacteraceae) Maya Ofek 0 Yitzhak Hadar 0 Dror Minz 0 Boris Alexander Vinatzer, Virginia Tech, United States of America 0 1 Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, Bet Dagan, Israel, 2 The Robert H. Smith Faculty of Agriculture , Food and Environment , The Hebrew University of Jerusalem , Rehovot , Israel Background: Ecologically meaningful classification of bacterial populations is essential for understanding the structure and function of bacterial communities. As in soils, the ecological strategy of the majority of root-colonizing bacteria is mostly unknown. Among those are Massilia (Oxalobacteraceae), a major group of rhizosphere and root colonizing bacteria of many plant species. Methodology/Principal Findings: The ecology of Massilia was explored in cucumber root and seed, and compared to that of Agrobacterium population, using culture-independent tools, including DNA-based pyrosequencing, fluorescence in situ hybridization and quantitative real-time PCR. Seed- and root-colonizing Massilia were primarily affiliated with other members of the genus described in soil and rhizosphere. Massilia colonized and proliferated on the seed coat, radicle, roots, and also on hyphae of phytopathogenic Pythium aphanidermatum infecting seeds. High variation in Massilia abundance was found in relation to plant developmental stage, along with sensitivity to plant growth medium modification (amendment with organic matter) and potential competitors. Massilia absolute abundance and relative abundance (dominance) were positively related, and peaked (up to 85%) at early stages of succession of the root microbiome. In comparison, variation in abundance of Agrobacterium was moderate and their dominance increased at later stages of succession. Conclusions: In accordance with contemporary models for microbial ecology classification, copiotrophic and competitionsensitive root colonization by Massilia is suggested. These bacteria exploit, in a transient way, a window of opportunity within the succession of communities within this niche. - Funding: This research was supported by research grant no. US-4264-09 from the US-Israel Binational Agricultural Research and Development Fund (BARD) and research grant no. 1298/09 from the Israel Science Foundation (ISF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Within the soil, the plant rhizosphere is nothing short of a gold mine in terms of carbon availability for its microbiota. Soil bacteria maintain a large number of adaptive traits which enable them to prosper in this highly competitive niche [1]. Those traits have been studied extensively for some bacterial species, including pathogens [2,3], mutual symbionts [4] and a few commensal, plant-growth-promoting species [1,5]. Technical advances in recent years have enabled comprehensive cultivation-independent studies of the composition of root and rhizosphere bacterial communities [6]. As a result of such studies, the number of bacterial species and genera classified as root-associated continues to rise [6,7]. For most of these novel root-associates, their ecology and life strategies are unknown. Among those are Massilia (Oxalobacteraceae), a group of bacteria of an emerging interest in recent years, described in a broad range of niches. Members of the genus Massilia were first isolated from clinical samples [8,9] and were characterized as aerobic, flagellated, nonspore forming rods [8,10]. Massilia were thereafter isolated and detected in environmental samples of many sources, including air, aerosols and dust samples [1113], freshwater [14], soils [15,16] and phyllosphere [17,18]. In recent years, Massilia had been detected in- and isolated from the rhizosphere and roots of many plant species [1929]. Although this group of bacteria appears to be an important component of the rhizosphere, Massilia and Oxalobacteraceae in general were seldom specifically examined in rhizosphere studies [19,20]. Therefore, association of Massilia with roots and different root compartments as well as population dynamics in the root niche is little known. In previous studies, we have reported that indigenous Massilia (Oxalobacteraceae) populations can achieve high dominance in the cucumber rhizosphere and spermosphere, but are also sensitive to conditions in the growth medium [30,31]. Here, a qualitative and quantitative investigation of Massilia colonization of cucumber seeds and roots was performed in order to gain further understanding of their association with different rhizosphere and spermosphere components, and their dynamics in this environment. Composition of Seed and Root Colonizing Massilia and Other Oxalobacteraceae The composition of bacteria colonizing cucumber seeds, seedlings and roots was assessed by high-throughput sequencing of 16S rRNA gene fragments, amplified directly from the extracted total DNA. Samples were collected at distinct growth stages: radicle emergence (24 hours), primary root extension (48 hours), first true leaf (7 days) and vine-tip-over (45 true leaves, 21 days). Table S1 lists the number of sequences obtained from each of the different treatment and plant ages. Across all samples, a total of 181,163 bacterial sequences were obtained following elimination of suspected chimeras, low-quality sequences and short sequences (,300 bp). Of these, 73,067 sequences (40.3%) were affiliated with the Oxalobacteraceae (Fig. 1a). Four different genera among Oxalobacteraceae were substantially (.1%) represented in the Oxalobacteraceae sequence pool (Fig. 1b). Among these, Massilia were by far the most predominant, followed by the genus Naxibacter. The eleven most abundant OTUs (,97% sequence similarity between the sequences within each OTU) comprised 58% of Oxalobacteraceae sequences, and are presented along with their closest published relatives in Figure 2. The most abundant Massilia sequences were identical or highly similar (99% and above) to sequences previously retrieved from roots and rhizosphere of different plants and soils. Massilia Colonization of Seeds and Roots FISH-CLSM was used to examine seed and root surface colonization by Massilia, using a specifically designed probe (Fig. 3). Massilia were undetected on the coat or radicle of seed germinated in perlite for 24 h. In contrast, clusters of few Massilia cells, as well as dense Massilia clusters were found on the coat of seeds germinated for 24 h in compost-amended perlite (Fig. 3a). Those were found in mixed colonies with other bacteria, but also in exclusive ones, over the surface of the coat cells as well as in grooves between adjacent cells. The dense clusters morphology indicated multiplication taking place (Fig. 3b). By then, Mas (...truncated)