Host signature effect on plant root-associated microbiomes revealed through analyses of resident vs. active communities.

Plant roots create specific microbial habitat in the soil - the rhizosphere. In this study, we characterized the rhizosphere microbiome of four host plant species to get insight into the impact of the host (host signature effect) on resident vs. active communities. Results show a distinct plant host specific signature found among wheat, maize, tomato and cucumber, based on the following three parameters: (i) each plant promoted the activity of a unique suite of soil bacterial populations; (ii) significant variations were observed in the number and the degree of dominance of active populations; and (iii) the level of contribution of active (rRNA-based) populations to the resident (DNA-based) community profiles. In the rhizoplane of all four plants, a significant reduction of diversity was observed, relative to the bulk soil. Moreover, an increase in DNA-RNA correspondence indicated higher representation of active bacterial populations in the residing rhizoplane community. This study demonstrates that the host plant determines the bacterial community composition in its immediate vicinity, especially with respect to the active populations.

Characterization of fluoroquinolone resistance and qnr diversity in Enterobacteriaceae from municipal biosolids.

Municipal biosolids produced during activated sludge treatment applied in wastewater treatment plants, are significant reservoirs of antibiotic resistance, since they assemble both natural and fecal microbiota, as well as residual concentrations of antibiotic compounds. This raises major concerns regarding the environmental and epidemiological consequences of using them as fertilizers for crops. The second generation fluoroquinolone ciprofloxacin is probably the most abundant antibiotic compound detected in municipal biosolids due to its widespread use and sorption properties. Although fluoroquinolone resistance was originally thought to result from mutations in bacterial gyrase and topoisomerase IV genes, it is becoming apparent that it is also attributed to plasmid-associated resistance factors, which may propagate environmental antibiotic resistance. The objective of this study was to assess the impact of the activated sludge process on fluoroquinolone resistance. The scope of resistances and mobile genetic mechanisms associated with fluoroquinolone resistance were evaluated by screening large collections of ciprofloxacin-resistant Enterobacteriaceae strains from sludge (n = 112) and from raw sewage (n = 89). Plasmid-mediated quinolone resistance determinants (qnrA, B, and S) were readily detected in isolates from both environments, the most dominant being qnrS. Interestingly, all qnr variants were significantly more abundant in sludge isolates than in the isolates from raw sewage. Almost all ciprofloxacin-resistant isolates were resistant to multiple antibiotic compounds. The sludge isolates were on the whole resistant to a broader range of antibiotic compounds than the raw sewage isolates; however, this difference was not statistically significant. Collectively, this study indicates that the activated sludge harbors multi-resistant bacterial strains, and that mobile quinolone-resistance elements may have a selective advantage in the activated sludge.

The role of the bacterial community in the nutritional ecology of the bulb mite Rhizoglyphus robini (Acari: Astigmata: Acaridae).

The biology of many arthropods can only be understood when their associated microbiome is considered. The nutritional requirements of the bulb mite Rhizoglyphus robini Claparede (Acari: Astigmata: Acaridae) in the laboratory have been shown to be very easily satisfied, and in the field the mites prefer fungus-infected over uninfected plants. To test whether symbiotic bacteria facilitate the survival of R. robini on a temporarily nutritionally unbalanced diet, we investigated the composition of its microbiome. Using 454 pyrosequencing of 16S rRNA gene fragments, 3 genera were found to dominate the bacterial community: Myroides (41.4%), Serratia (11.4%), and Alcaligenes (4.5%); the latter 2 are known to include chitinase-producing species. Laboratory experiments demonstrated that mite fecundity is significantly higher (2 times) on fungus than on controls (sterilized potato dextrose agar and filter paper). Also, when mite homogenate was applied to a chitin layer, the halo produced through degradation was clearly visible, while the saline control did not produce a halo. We thus concluded that R. robini utilizes fungal chitin, at least to a certain extent, as a food source with the help of its associated bacteria. This information supports the general concept of multigenome organisms and the involvement of bacteria in the mite's nutritional ecology.

Soil suppressiveness to fusarium disease: shifts in root microbiome associated with reduction of pathogen root colonization.

Soil suppressiveness to Fusarium disease was induced by incubating sandy soil with debris of wild rocket (WR; Diplotaxis tenuifolia) under field conditions. We studied microbial dynamics in the roots of cucumber seedlings following transplantation into WR-amended or nonamended soil, as influenced by inoculation with Fusarium oxysporum f. sp. radicis-cucumerinum. Disease symptoms initiated in nonamended soil 6 days after inoculation, compared with 14 days in WR-amended soil. Root infection by F. oxysporum f. sp. radicis-cucumerinum was quantified using real-time polymerase chain reaction (PCR). Target numbers were similar 3 days after inoculation for both WR-amended and nonamended soils, and were significantly lower (66%) 6 days after inoculation and transplanting into the suppressive (WR-amended) soil. This decrease in root colonization was correlated with a reduction in disease (60%) 21 days after inoculation and transplanting into the suppressive soil. Fungal community composition on cucumber roots was assessed using mass sequencing of fungal internal transcribed spacer gene fragments. Sequences related to F. oxysporum, Fusarium sp. 14005, Chaetomium sp. 15003, and an unclassified Ascomycota composed 96% of the total fungal sequences in all samples. The relative abundances of these major groups were highly affected by root inoculation with F. oxysporum f. sp. radicis-cucumerinum, with a 10-fold increase in F. oxysporum sequences, but were not affected by the WR amendment. Quantitative analysis and mass-sequencing methods indicated a qualitative shift in the root's bacterial community composition in suppressive soil, rather than a change in bacterial numbers. A sharp reduction in the size and root dominance of the Massilia population in suppressive soil was accompanied by a significant increase in the relative abundance of specific populations; namely, Rhizobium, Bacillus, Paenibacillus, and Streptomyces spp. Composition of the Streptomyces community shifted significantly, as determined by PCR denaturing gradient gel electrophoresis, resulting in an increase in the dominance of a specific population in suppressive soils after only 3 days. This shift was related mainly to the increase in Streptomyces humidus, a group previously described as antagonistic to phytopathogenic fungi. Thus, suitable soil amendment resulted in a shift in the root's bacterial communities, and infection by a virulent pathogen was contained by the root microbiome, leading to a reduced disease rate.

Ecology of root colonizing Massilia (Oxalobacteraceae).

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 competition-sensitive 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.

Colonization of cucumber seeds by bacteria during germination.

Detailed analysis revealed fundamental differences between bacterial association with cucumber (Cucumis sativus) seeds and seedlings roots. Seed colonization by bacteria seems to result from passive encounter between bacteria, conveyed by imbibed soil solution, and the germinating seed. In accordance, the seed-associated bacterial community composition directly reflected that of the germination medium and was characterized by low dominance. Transition from seed to root was marked by a shift in bacterial community composition and in an increase in dominance values. Furthermore, settlement of bacteria on roots was tightly controlled by the specific properties of each root segment. Size and richness of the seed-associated bacterial community were clearly determinate by the community in the germination medium. In contrast, for fully developed and active roots, the medium effect on these parameters was negligible. Perturbation of the seed environment by a pathogen (Pythium aphanidermatum) had major consequences on the seed bacterial community. However, those were mostly related to direct pathogen-bacteria rather than seed-bacteria interactions. In conclusion, simple, even passive processes may determine the initial stage of plant-microbe association during seed germination, prior to extension of the primary root. Therefore, seed germination is a unique phase in the plant life cycle, with respect to its interaction with the below-ground microbiome.

Microbial aspects of accelerated degradation of metam sodium in soil.

Preplant soil fumigation with metam sodium is used worldwide to control soilborne diseases. The development of accelerated degradation of pesticides in soil, including metam sodium, results in reduced pesticide efficacy. Therefore, we studied microbial involvement in accelerated degradation of methyl isothiocyanate (MITC) following repeated soil applications of the parent compound, metam sodium. MITC degradation was reduced in soil with a history of metam sodium applications following sterilization, indicating the key role of microorganisms in accelerated degradation. Accelerated degradation of MITC was induced by inoculation of soil with no previous application of metam sodium with soil with a history of metam sodium applications. We developed a method to extract the active microbial fraction responsible for MITC degradation from soil with a history of metam sodium applications. This concentrated soil extract induced accelerated degradation of MITC when added to two different soils with no previous application of metam sodium. An extensive shift in total bacterial community composition in concentrated soil extracts occurred after a single metam sodium application. Two Oxalobacteraceae strains, MDB3 and MDB10, isolated from Rehovot soil following triple application of metam sodium rapidly degraded MITC in soil with no previous application of metam sodium. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community composition showed relative enrichment of MDB3 following metam sodium application, suggesting its potential in situ involvement in accelerated degradation development in Rehovot soil. Responses of resident Oxalobacteraceae community members to metam sodium applications differed between Rehovot and En Tamar soils. Isolate MDB10 did not induce accelerated degradation of MITC in En Tamar soil and, with the slow dissipation of MITC, soil suppressiveness of accelerated degradation is suggested. The isolation and identification of MITC-degrading bacteria might be helpful in developing tools for managing accelerated degradation.

Comparison of effects of compost amendment and of single-strain inoculation on root bacterial communities of young cucumber seedlings.

Compost amendment and inoculations with specific microorganisms are fundamentally different soil treatment methods, commonly used in agriculture for the improvement of plant growth and health. Although distinct, both methods affect the rhizosphere and the plant roots. In the present study we used a 16S rRNA gene approach to achieve an overview of early consequences of these treatments on the assemblage of plant root bacterial communities. For this purpose, cucumber seedlings were grown, under controlled conditions, in perlite potting mix amended with biosolid compost or straw compost, or inoculated with Streptomyces spp. A uniform trend of response of root bacterial communities for all treatments was observed. Root bacterial density, measured as bacterial targets per plant tef gene by real-time PCR, was reduced in 31 to 67%. In addition, increased taxonomic diversity accompanied shifts in composition (alpha-diversity). The magnitude of change in these parameters relative to the perlite control varied between the different treatments but not in relation to the treatment method (compost amendments versus inoculations). Similarity between the compositions of root and of potting mix bacterial communities (beta-diversity) was relatively unchanged. The abundance of Oxalobacteraceae was >50% of the total root bacterial community in the untreated perlite. Root domination by this group subsided >10-fold (straw compost) to >600-fold (Streptomyces sp. strain S1) after treatment. Thus, loss of dominance appears to be the major phenomenon underlining the response trend of the root bacterial communities.