Endophytic diazotrophic communities from rice roots are diverse and weakly associated with soil diazotrophic community composition and soil properties.

AIM: Bacteria that promote plant growth, such as diazotrophs, are valuable tools for achieving a more sustainable production of important non-legume crops like rice. Different strategies have been used to discover new bacteria capable of promoting plant growth. This work evaluated the contribution of soil diazotrophs to the endophytic communities established in the roots of rice seedlings cultivated on seven representative soils from Uruguay. METHODS AND RESULTS: The soils were classified into two groups according to the C and clay content. qPCR, terminal restriction fragment length polymorphism (T-RFLP), and 454-pyrosequencing of the nifH gene were used for analyzing diazotrophs in soil and plantlets' roots grown from seeds of the same genotype for 25 days under controlled conditions. A similar nifH abundance was found among the seven soils, roots, or leaves. The distribution of diazotrophs was more uneven in roots than in soils, with dominance indices significantly higher than in soils (nifH T-RFLP). Dominant soils' diazotrophs were mainly affiliated to Alphaproteobacteria and Planctomycetota. Conversely, Alpha, Beta, Gammaproteobacteria, and Bacillota were predominant in different roots, though undetectable in soils. Almost no nifH sequences were shared between soils and roots. CONCLUSIONS: Root endophytic diazotrophs comprised a broader taxonomic range of microorganisms than diazotrophs found in soils from which the plantlets were grown and showed strong colonization patterns.

Methylogaea oryzae gen. nov., sp. nov., a mesophilic methanotroph isolated from a rice paddy field.

A novel methanotroph, designated strain E10(T), was isolated from a rice paddy field in Uruguay. Strain E10(T) grew on methane and methanol as sole carbon and energy sources. Cells were Gram-negative, non-motile, non-pigmented, slightly curved rods showing type I intracytoplasmic membranes arranged in stacks. The strain was neutrophilic and mesophilic; optimum growth occurred at 30-35 °C with no growth above 37 °C. The strain possessed only a particulate methane monooxygenase (pmoA). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was most closely related to the moderately thermophilic strains Methylocaldum szegediense OR2(T) (91.6 % sequence similarity) and Methylococcus capsulatus Bath (91.5 %). Comparative sequence analysis of pmoA genes also confirmed that strain E10(T) formed a new lineage among the genera Methylocaldum and Methylococcus with 89 and 84 % derived amino acid sequence identity to Methylococcus capsulatus Bath and Methylocaldum gracile VKM-14L(T), respectively. The DNA G+C content was 63.1 mol% and the major cellular fatty acid was C(16 :0) (62.05 %). Thus, strain E10(T) (=JCM 16910(T) = DSM 23452(T)) represents the type strain of a novel species within a new genus, for which the name Methylogaea oryzae gen. nov., sp. nov. is proposed.

Dynamics, diversity and function of endophytic siderophore-producing bacteria in rice.

Siderophore production confers to bacteria competitive advantages to colonize plant tissues and to exclude other microorganisms from the same ecological niche. This work shows that the community of endophytic siderophore-producing bacteria (SPB) associated to Oryza sativa cultivated in Uruguayan soils is dynamic and diverse. These bacteria were present in grains, roots, and leaves, and their density fluctuated between log(10) 3.44 and log(10) 5.52 cfu g(-1) fresh weight (fw) during the plant growth. Less than 10% of the heterotrophic bacteria produced siderophores in roots and leaves of young plants, but most of the heterotrophic bacteria were siderophore-producers in mature plants. According to their amplified restriction DNA ribosomal analysis (ARDRA) pattern, 54 of the 109 endophytic SPB isolated from different plant tissues or growth stages from replicate plots, were unique. Bacteria belonging to the genera Sphingomonas, Pseudomonas, Burkholderia, and Enterobacter alternated during plant growth, but the genus Pantoea was predominant in roots at tillering and in leaves at subsequent stages. Pantoea ananatis was the SPB permanently associated to any of the plant tissues, but the genetic diversity within this species-revealed by BOX-PCR fingerprinting- showed that different strains were randomly distributed along time and plant tissue, suggesting that a common trait of the species P. ananatis determined the interaction with the rice plant. Several isolates were stronger IAA producers than Azospirillum brasilense or Herbaspirillum seropedicae. In vitro inhibition assays showed that SPB of the genus Burkholderia were good antagonists of pathogenic fungi and that only one SPB isolate of the genus Pseudomonas was able to inhibit A. brasilense and H. seropedicae. These results denoted that SPB were selected into the rice plant. P. ananatis was the permanent and dominant associated species which was unable to inhibit two of the relevant plant growth-promoting bacteria.

Bacterial community analysis of the water surface layer from a rice-planted and an unplanted flooded field.

The bacterial communities in floodwater, from a rice-planted and an unplanted field were characterized at the beginning (flooding stage) and at the end (harvest stage) of the rice cropping cycle. Most probable number estimations and plate counts of aerobic and anaerobic heterotrophic bacteria and of several metabolic bacterial groups (methanogens, sulfate-reducers, anaerobic sulfur and nonsulfur phototrophs, denitrifiers and ammonifiers) were similar in rice and unplanted floodwater at both sampling times. The analysis of denitrifiers and methanogens by fluorescent in situ hybridization revealed a shift in the phylogenetic affiliation only of the former group in the rice-planted floodwater. Terminal restriction fragment length polymorphism of 16S rRNA gene amplicons indicated that the bacterial communities of the rice-planted and unplanted soils were consistently diverse and strongly influenced by the season.

Performance of a commercial inoculum for the aerobic biodegradation of a high fat content dairy wastewater.

The effectiveness of a commercial inoculum for degrading a dairy wastewater with high fat content was evaluated, and compared with an activated sludge inoculum from a dairy wastewater treatment pond. Both inocula reached similar chemical oxygen demand removal in batch experiments. The population dynamics was also studied by determining heterotrophic counts. Predominant microorganisms were differentiated by colony morphology and genomic fingerprinting (BOX-PCR) analysis. The higher population diversity and the wider range of CO2 production rate observed in batch reactors inoculated with activated-sludge, indicated that microorganisms from this inoculum were well adapted and may have had synergic activity for the degradation of the dairy effluent. When the bioreactor was operated with the commercial inoculum in continuous mode, according to its microbial growth kinetics, other microorganisms became predominant. These results showed that inoculated microorganisms did not persist in the open system and periodic addition of microorganisms may be needed to achieve a high performance treatment.

Strong shift in the diazotrophic endophytic bacterial community inhabiting rice (Oryza sativa) plants after flooding.

Flooding impacts soil microbial communities, but its effect on endophytic communities has rarely been explored. This work addresses the effect of flooding on the abundance and diversity of endophytic diazotrophic communities on rice plants established in a greenhouse experiment. The nifH gene was significantly more abundant in roots after flooding, whereas the nifH gene copy numbers in leaves were unaffected and remained low. The PCA (principal component analysis) of T-RFLP (terminal restriction fragment length polymorphism) profiles indicated that root communities of replicate plots were more similar and diverse after flooding than before flooding. The nifH libraries obtained by cloning and 454 pyrosequencing consistently showed a remarkable shift in the diazotrophic community composition after flooding. Gammaproteobacteria (66-98%), mainly of the genus Stenotrophomonas, prevailed in roots before flooding, whereas Betaproteobacteria was the dominant class (26-34%) after flooding. A wide variety of aerotolerant and anaerobic diazotrophic bacteria (e.g. Dechloromonas, Rhodopseudomonas, Desulfovibrio, Geobacter, Chlorobium, Spirochaeta, Selenomonas and Dehalobacter) with diverse metabolic traits were retrieved from flooded rice roots. These findings suggest that endophytic communities could be significantly impacted by changes in plant-soil conditions derived from flooding during rice cropping.

Molecular and culture-dependent analyses revealed similarities in the endophytic bacterial community composition of leaves from three rice (Oryza sativa) varieties.

The endophytic bacterial communities of the three most important rice varieties cultivated in Uruguay were compared by a multiphasic approach. Leaves of mature plants grown in field experiments for two consecutive crop seasons were studied. No significant differences were found in the heterotrophic bacterial density for the three varieties. Pantoea ananatis and Pseudomonas syringae constituted 51% of the total of the isolates. These species were always present regardless of the variety or the season. Molecular analysis based on the 16S rRNA gene was performed by terminal restriction fragment length polymorphism (T-RFLP) and cloning. T-RFLP analysis revealed that bacterial communities grouped according to the variety, although the three varieties presented communities that showed 74% or higher similarities. Brevundimonas, the dominant genus in the clone library (18% of the clones), which might be present in all varieties according to T-RFLP profiles, was not recovered by cultivation. Conversely, bacteria from the genus Pseudomonas were not detected in the clone library. These results indicate that communities established in leaves of physiologically different rice varieties were highly similar and composed by a reduced group of strongly associated and persistent bacteria that were partially recovered by cultivation.

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact.

Lonar Lake is a unique saline and alkaline ecosystem formed by meteor impact in the Deccan basalts in India around 52,000 years ago. To investigate the role of methylotrophy in the cycling of carbon in this unusual environment, stable-isotope probing (SIP) was carried out using the one-carbon compounds methane, methanol and methylamine. Denaturing gradient gel electrophoresis fingerprinting analyses performed with heavy (13)C-labelled DNA retrieved from sediment microcosms confirmed the enrichment and labelling of active methylotrophic communities. Clone libraries were constructed using PCR primers targeting 16S rRNA genes and functional genes. Methylomicrobium, Methylophaga and Bacillus spp. were identified as the predominant active methylotrophs in methane, methanol and methylamine SIP microcosms, respectively. Absence of mauA gene amplification in the methylamine SIP heavy fraction also indicated that methylamine metabolism in Lonar Lake sediments may not be mediated by the methylamine dehydrogenase enzyme pathway. Many gene sequences retrieved in this study were not affiliated with extant methanotrophs or methylotrophs. These sequences may represent hitherto uncharacterized novel methylotrophs or heterotrophic organisms that may have been cross-feeding on methylotrophic metabolites or biomass. This study represents an essential first step towards understanding the relevance of methylotrophy in the soda lake sediments of an unusual impact crater structure.

Bacterial community analysis of the water surface layer from a rice-planted and an unplanted flooded field

The bacterial communities in floodwater, from a rice-planted and an unplanted field were characterized at the beginning (flooding stage) and at the end (harvest stage) of the rice cropping cycle. Most probable number estimations and plate counts of aerobic and anaerobic heterotrophic bacteria and of several metabolic bacterial groups (methanogens, sulfate-reducers, anaerobic sulfur and nonsulfur phototrophs, denitrifiers and ammonifiers) were similar in rice and unplanted floodwater at both sampling times. The analysis of denitrifiers and methanogens by fluorescent in situ hybridization revealed a shift in the phylogenetic affiliation only of the former group in the rice-planted floodwater. Terminal restriction fragment length polymorphism of 16S rRNA gene amplicons indicated that the bacterial communities of the rice-planted and unplanted soils were consistently diverse and strongly influenced by the season.