Ampullimonas aquatilis gen. nov., sp. nov. isolated from bottled mineral water.

Two isolates, designated B15.09-116T and B15.09-124, were recovered from bottled mineral water in Portugal. Based on 16S rRNA gene sequence analysis, these strains were related most closely to species of the genus Derxia (belonging to the family Alcaligenaceae) with pairwise sequence similarities of 93.0-93.6 %. The isolates were not pigmented and formed Gram-stain-negative, short, motile rod-shaped cells. The organisms were strictly aerobic, oxidase-positive and catalase-negative. These organisms also fixed N2. The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. Ubiquinone 8 was the major respiratory quinone. The DNA G+C content of strain B15.09-116T was 49.8 mol%. Based on phylogenetic, physiological and biochemical characteristics the two strains are considered to represent a novel species of a new genus, for which the name Ampullimonas aquatilis gen. nov., sp. nov. is proposed. The type strain of Ampullimonas aquatilis is B15.09-116T ( = CECT 8581T = LMG 28208T).

Agricultural value of Black Soldier Fly larvae frass as organic fertilizer on ryegrass.

Hermetia illucens L., known as Black Soldier Fly (BSF) appear as an opportunity to reuse vegetable by-products, as it is easy to reproduce and can be reared in agricultural side streams, allowing the production of both, animal feed (the larvae, after recycling of the vegetal debris) and soil organic fertilizer (insect frass). Although several organic fertilizers, from long ago, have been used in agriculture, there is yet a paucity of experimental data on the evaluation of the fertilization potential of BSF larvae frass (BSFF). The present study is a contribution to access the agronomic and environmental potential of the BSFF as an organic fertilizer. Within this aim, it was conducted a greenhouse experiment with ryegrass, using seven treatments of BSFF. Under the experimental conditions, the results showed a significant effect of BSFF on the overall ryegrass production, with a steady increase (significant at p ≤ 0.05, as accessed through the Tukey test) up to the treatment with a greater rate of application. In what concerns sustainability of soil productivity, at the end of the experiment, there was also positive indications, namely, a significant increase of OM, P and K, for treatments with higher N endowments, together with a constant increase of dehydrogenase activity, from the control to the higher treatment, which was significant for treatments receiving the higher dose of BSFF.

The Nexus between Fire and Soil Bacterial Diversity in the African Miombo Woodlands of Niassa Special Reserve, Mozambique.

(1) Background: the Miombo woodlands comprise the most important vegetation from southern Africa and are dominated by tree legumes with an ecology highly driven by fires. Here, we report on the characterization of bacterial communities from the rhizosphere of Brachystegia boehmii in different soil types from areas subjected to different regimes. (2) Methods: bacterial communities were identified through Illumina MiSeq sequencing (16S rRNA). Vigna unguiculata was used as a trap to capture nitrogen-fixing bacteria and culture-dependent methods in selective media were used to isolate plant growth promoting bacteria (PGPB). PGP traits were analysed and molecular taxonomy of the purified isolates was performed. (3) Results: Bacterial communities in the Miombo rhizosphere are highly diverse and driven by soil type and fire regime. Independent of the soil or fire regime, the functional diversity was high, and the different consortia maintained the general functions. A diverse pool of diazotrophs was isolated, and included symbiotic (e.g., Mesorhizobium sp., Neorhizobium galegae, Rhizobium sp., and Ensifer adhaerens), and non-symbiotic (e.g., Agrobacterium sp., Burkholderia sp., Cohnella sp., Microvirga sp., Pseudomonas sp., and Stenotrophomonas sp.) bacteria. Several isolates presented cumulative PGP traits. (4) Conclusions: Although the dynamics of bacterial communities from the Miombo rhizosphere is driven by fire, the maintenance of high levels of diversity and functions remain unchanged, constituting a source of promising bacteria in terms of plant-beneficial activities such as mobilization and acquisition of nutrients, mitigation of abiotic stress, and modulation of plant hormone levels.

Mining the Microbiome of Key Species from African Savanna Woodlands: Potential for Soil Health Improvement and Plant Growth Promotion.

(1) Aims: Assessing bacterial diversity and plant-growth-promoting functions in the rhizosphere of the native African trees Colophospermum mopane and Combretum apiculatum in three landscapes of the Limpopo National Park (Mozambique), subjected to two fire regimes. (2) Methods: Bacterial communities were identified through Illumina Miseq sequencing of the 16S rRNA gene amplicons, followed by culture dependent methods to isolate plant growth-promoting bacteria (PGPB). Plant growth-promoting traits of the cultivable bacterial fraction were further analyzed. To screen for the presence of nitrogen-fixing bacteria, the promiscuous tropical legume Vigna unguiculata was used as a trap host. The taxonomy of all purified isolates was genetically verified by 16S rRNA gene Sanger sequencing. (3) Results: Bacterial community results indicated that fire did not drive major changes in bacterial abundance. However, culture-dependent methods allowed the differentiation of bacterial communities between the sampled sites, which were particularly enriched in Proteobacteria with a wide range of plant-beneficial traits, such as plant protection, plant nutrition, and plant growth. Bradyrhizobium was the most frequent symbiotic bacteria trapped in cowpea nodules coexisting with other endophytic bacteria. (4) Conclusion: Although the global analysis did not show significant differences between landscapes or sites with different fire regimes, probably due to the fast recovery of bacterial communities, the isolation of PGPB suggests that the rhizosphere bacteria are driven by the plant species, soil type, and fire regime, and are potentially associated with a wide range of agricultural, environmental, and industrial applications. Thus, the rhizosphere of African savannah ecosystems seems to be an untapped source of bacterial species and strains that should be further exploited for bio-based solutions.

Identification of Sinorhizobium (Ensifer) medicae based on a specific genomic sequence unveiled by M13-PCR fingerprinting.

A collection of nodule isolates from Medicago polymorpha obtained from southern and central Portugal was evaluated by M13-PCR fingerprinting and hierarchical cluster analysis. Several genomic clusters were obtained which, by 16S rRNA gene sequencing of selected representatives, were shown to be associated with particular taxonomic groups of rhizobia and other soil bacteria. The method provided a clear separation between rhizobia and co-isolated non-symbiotic soil contaminants. Ten M13-PCR groups were assigned to Sinorhizobium (Ensifer) medicae and included all isolates responsible for the formation of nitrogen-fixing nodules upon re-inoculation of M. polymorpha test-plants. In addition, enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting indicated a high genomic heterogeneity within the major M13- PCR clusters of S. medicae isolates. Based on nucleotide sequence data of an M13-PCR amplicon of ca. 1500 bp, observed only in S. medicae isolates and spanning locus Smed_3707 to Smed_3709 from the pSMED01 plasmid sequence of S. medicae WSM419 genome's sequence, a pair of PCR primers was designed and used for direct PCR amplification of a 1399-bp sequence within this fragment. Additional in silico and in vitro experiments, as well as phylogenetic analysis, confirmed the specificity of this primer combination and therefore the reliability of this approach in the prompt identification of S. medicae isolates and their distinction from other soil bacteria.

Colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria.

Multi-strain inoculants have increased potential to accomplish a diversity of plant needs, mainly attributed to its multi-functionality. This work evaluated the ability of a mixture of three bacteria to colonize and induce a beneficial response on the pasture crop annual ryegrass. Pseudomonas G1Dc10 and Paenibacillus G3Ac9 were previously isolated from annual ryegrass and were selected for their ability to perform multiple functions related to plant growth promotion. Sphingomonas azotifigens DSMZ 18530T was included due to nitrogen fixing ability. The effects of the bacterial mixture were assessed in gnotobiotic plant inoculation assays and compared with single and dual inoculation treatments. Triple inoculation with 3×108 bacteria significantly increased plant dry weight and leaf pigments, indicating improved photosynthetic performance. Plant lipid biosynthesis was enhanced by 65%, mainly due to the rise of linolenic acid, an omega-3 fatty acid with high dietary value. Electrolyte leakage, an indicator of plant membrane stability under stress, was decreased pointing to a beneficial effect by inoculation. Plants physiological condition was more favoured by triple inoculation than by single, although benefits on biomass were only evident relative to non-inoculated plants. The colonization behaviour and coexistence in plant tissues were assessed using FISH and GFP-labelling, combined with confocal microscopy and a cultivation-based approach for quantification. The three strains occupied the same sites, localizing preferentially along root hairs and in stem epidermis. Endophytic colonization was observed as bacteria entered root and stem inner tissues. This study reveals the potential of this mixture of strains for biofertilization, contributing to improve crop productivity and nutritional value.

Annual ryegrass-associated bacteria with potential for plant growth promotion.

Annual ryegrass is a fast-growing cool-season grass broadly present in the Portuguese "montado", a typically Mediterranean agro-forestry-pastoral ecosystem. A culture-dependent approach was used to investigate natural associations of this crop with potentially beneficial bacteria, aiming to identify strains suitable for biofertilization purposes. Annual ryegrass seedlings were used to trap bacteria from three different soils in laboratory conditions. Using a nitrogen-free microaerophilic medium, 147 isolates were recovered from the rhizosphere, rhizoplane, and surface-sterilized plant tissues, which were assigned to 12 genera in classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria. All isolates were able to grow in the absence of nitrogen and several of them were able to perform in vitro activities related to plant growth promotion. Isolates of the genera Sphingomonas and Achromobacter were found to be the most effective stimulators of annual ryegrass growth under nitrogen limitation (47-92% biomass increases). Major enhancements were obtained with isolates G3Dc4 (Achromobacter sp.) and G2Ac10 (Sphingomonas sp.). The latest isolate was also able to increment plant growth in nitrogen-supplemented medium, as well as the phosphate solubilizer and siderophore producer, G1Dc10 (Pseudomonas sp.), and the cellulose/pectin hydrolyser, G3Ac9 (Paenibacillus sp.). This study represents the first survey of annual ryegrass-associated bacteria in the "montado" ecosystem and unveiled a set of strains with potential for use as inoculants.

Salinisphaera shabanensis gen. nov., sp. nov., a novel, moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep, Red Sea.

A novel, moderately halophilic bacterium was isolated from the brine-seawater interface of the Shaban Deep, northern Red Sea. A polyphasic approach was used for the taxonomic characterization of this isolate, with the phenotypic and phylogenetic data clearly showing the distinctiveness of this bacterium. Cells of isolate E1L3A were Gram-negative, monotrichous cocci that showed a remarkable physiological flexibility, as could be seen by the quite broad growth ranges for oxygen, temperature, NaCl, and, to a smaller degree, pH. In addition, it was able to grow from atmospheric pressure up to 15 MPa, making it a piezotolerant bacterium. Phylogenetically, strain E1L3A represents a new, deeply branching lineage within the gamma-Proteobacteria, as determined by 16S rRNA gene sequence analysis. No close relatives are known so far, with sequence similarity to other cultivated members of the gamma-Proteobacteria being lower than 88%. The creation of the new genus Salinisphaera and the new species Salinisphaera shabanensis (DSM 14853; JCM 11575) for this new and highly versatile microorganism is therefore proposed.

Response of a strict anaerobe to oxygen: survival strategies in Desulfovibrio gigas.

The biochemical response to oxygen of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio gigas was studied with the goal of elucidating survival strategies in oxic environments. Cultures of D. gigas on medium containing lactate and sulfate were exposed to oxygen (concentration 5-120 micro M). Growth was fully inhibited by oxygen, but the cultures resumed growth as soon as they were shifted back to anoxic conditions. Following 24 h exposure to oxygen the growth rate was as high as 70 % of the growth rates observed before oxygenation. Catalase levels and activity were enhanced by exposure to oxygen whereas superoxide-scavenging and glutathione reductase activities were not affected. The general pattern of cellular proteins as analysed by two-dimensional electrophoresis was altered in the presence of oxygen, the levels of approximately 12 % of the detected proteins being markedly increased. Among the induced proteins, a homologue of a 60 kDa eukaryotic heat-shock protein (Hsp60) was identified by immunoassay analysis. In the absence of external substrates, the steady-state levels of nucleoside triphosphates detected by in vivo (31)P-NMR under saturating concentrations of oxygen were 20 % higher than under anoxic conditions. The higher energy levels developed under oxygen correlated with a lower rate of substrate (glycogen) mobilization, but no experimental evidence for a contribution from oxidative phosphorylation was found. The hypothesis that oxygen interferes with ATP dissipation processes is discussed.