Improved group-specific primers based on the full SILVA 16S rRNA gene reference database.

Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics.

Microbial communities show parallels at sites with distinct litter and soil characteristics.

Plant and microbial community composition in connection with soil chemistry determines soil nutrient cycling. The study aimed at demonstrating links between plant and microbial communities and soil chemistry occurring among and within four sites: two pine forests with contrasting soil pH and two grasslands of dissimilar soil chemistry and vegetation. Soil was characterized by C and N content, particle size, and profiles of low-molecular-weight compounds determined by high-performance liquid chromatography (HPLC) of soil extracts. Bacterial and actinobacterial community composition was assessed by terminal restriction fragment length polymorphism (T-RFLP) and cloning followed by sequencing. Abundances of bacteria, fungi, and actinobacteria were determined by quantitative PCR. In addition, a pool of secondary metabolites was estimated by erm resistance genes coding for rRNA methyltransferases. The sites were characterized by a stable proportion of C/N within each site, while on a larger scale, the grasslands had a significantly lower C/N ratio than the forests. A Spearman's test showed that soil pH was correlated with bacterial community composition not only among sites but also within each site. Bacterial, actinobacterial, and fungal abundances were related to carbon sources while T-RFLP-assessed microbial community composition was correlated with the chemical environment represented by HPLC profiles. Actinobacteria community composition was the only studied microbial characteristic correlated to all measured factors. It was concluded that the microbial communities of our sites were influenced primarily not only by soil abiotic characteristics but also by dominant litter quality, particularly, by percentage of recalcitrant compounds.

Xylella fastidiosa in Olive: A Review of Control Attempts and Current Management.

Since 2013, Xylella fastidiosa Wells et al. has been reported to infect several hosts and to be present in different areas of Europe. The main damage has been inflicted on the olive orchards of southern Apulia (Italy), where a severe disease associated with X. fastidiosa subspecies pauca strain De Donno has led to the death of millions of trees. This dramatic and continuously evolving situation has led to European and national (Italian and Spanish) measures being implemented to reduce the spread of the pathogen and the associated olive quick decline syndrome (OQDS). Research has been also carried out to find solutions to better and directly fight the bacterium and its main insect vector, Philaenus spumarius L. In the course of this frantic effort, several treatments based on chemical or biological substances have been tested, in addition to plant breeding techniques and integrated pest management approaches. This review aims to summarize the attempts made so far and describe the prospects for better management of this serious threat, which poses alarming questions for the future of olive cultivation in the Mediterranean basin and beyond.

The role of plant-microbiome interactions in weed establishment and control.

The soil microbiome plays an important role in the establishment of weeds and invasive plants. They associate with microorganisms supporting their growth and health. Weed management strategies, like tillage and herbicide treatments, to control weeds generally alter soil structure going alongside with changes in the microbial community. Once a weed population establishes in the field, the plants build up a close relationship with the available microorganisms. Seeds or vegetative organs overwinter in soil and select early in the season their own microbiome before crop plants start to vegetate. Weed and crop plants compete for light, nutrition and water, but may differently interact with soil microorganisms. The development of new sequencing technologies for analyzing soil microbiomes has opened up the possibility for in depth analysis of the interaction between 'undesired' plants and crop plants under different management systems. These findings will help us to understand the functions of microorganisms involved in crop productivity and plant health, weed establishment and weed prevention. Exploitation of the knowledge offers the possibility to search for new biocontrol methods against weeds based on soil and plant-associated microorganisms. This review discusses the recent advances in understanding the functions of microbial communities for weed/invasive plant establishment and shows new ways to use plant-associated microorganisms to control weeds and invasive plants in different land management systems.

Rapid and dissimilar response of ammonia oxidizing archaea and bacteria to nitrogen and water amendment in two temperate forest soils.

Biochemical processes relevant to soil nitrogen (N) cycling are performed by soil microorganisms affiliated with diverse phylogenetic groups. For example, the oxidation of ammonia, representing the first step of nitrification, can be performed by ammonia oxidizing bacteria (AOB) and, as recently reported, also by ammonia oxidizing archaea (AOA). However, the contribution to ammonia oxidation of the phylogenetically separated AOA versus AOB and their respective responsiveness to environmental factors are still poorly understood. The present study aims at comparing the capacity of AOA and AOB to momentarily respond to N input and increased soil moisture in two contrasting forest soils. Soils from the pristine Rothwald forest and the managed Schottenwald forest were amended with either NH(4)(+)-N or NO(3)(-)-N and were incubated at 40% and 70% water-filled pore space (WFPS) for four days. Nitrification rates were measured and AOA and AOB abundance and community composition were determined via quantitative PCR (qPCR) and terminal restriction length fragment polymorphism (T-RFLP) analysis of bacterial and archaeal amoA genes. Our study reports rapid and distinct changes in AOA and AOB abundances in the two forest soils in response to N input and increased soil moisture but no significant effects on net nitrification rates. Functional microbial communities differed significantly in the two soils and responded specifically to the treatments during the short-term incubation. In the Rothwald soil the abundance and community composition of AOA were affected by the water content, whereas AOB communities responded to N amendment. In the Schottenwald soil, by contrast, AOA responded to N addition. These results suggest that AOA and AOB may be selectively influenced by soil and management factors.

Nitrifiers and denitrifiers respond rapidly to changed moisture and increasing temperature in a pristine forest soil.

Complete cycling of mineral nitrogen (N) in soil requires the interplay of microorganisms performing nitrification and denitrification, whose activity is increasingly affected by extreme rainfall or heat brought about by climate change. In a pristine forest soil, a gradual increase in soil temperature from 5 to 25 degrees C in a range of water contents stimulated N turnover rates, and N gas emissions were determined by the soil water-filled pore space (WFPS). NO and N(2)O emissions dominated at 30% WFPS and 55% WFPS, respectively, and the step-wise temperature increase resulted in a threefold increase in the NO(3)(-) concentrations and a decrease in the NH(4)(+) concentration. At 70% WFPS, NH(4)(+) accumulated while NO(3)(-) pools declined, indicating gaseous N loss. AmoA- and nirK-gene-based analysis revealed increasing abundance of bacterial ammonia oxidizers (AOB) with increasing soil temperature and a decrease in the abundance of archaeal ammonia oxidizers (AOA) in wet soil at 25 degrees C, suggesting the sensitivity of the latter to anaerobic conditions. Denitrifier (nirK) community structure was most affected by the water content and nirK gene abundance rapidly increased in response to wet conditions until the substrate (NO(3)(-)) became limiting. Shifts in the community structure were most pronounced for nirK and most rapid for AOA, indicating dynamic populations, whereas distinct adaptation of the AOB communities required 5 weeks, suggesting higher stability.

Dynamics of ammonia-oxidizing communities in barley-planted bulk soil and rhizosphere following nitrate and ammonium fertilizer amendment.

Oxidation of ammonia by nitrifying microorganisms is a major pathway that fertilizer nitrogen (N) may take upon application to agricultural soils, but the relative roles of bacterial (AOB) vs. archaeal (AOA) ammonia oxidizers are controversial. We explored the effects of various forms of mineral N fertilizer on the AOB and AOA community dynamics in two different soils planted with barley. Ammonia oxidizers were monitored via real-time PCR and terminal restriction fragment length polymorphism analysis of bacterial and archaeal amoA genes following the addition of either [NH4]2SO4, NH4NO3 or KNO3. AOB and AOA communities were also studied specifically in the rhizospheres of two different barley varieties upon [NH4]2SO4 vs. KNO3 addition. AOB changed in community composition and increased in abundance upon ammonium amendment in bulk soil and rhizosphere, with changes in bacterial amoA copy numbers lagging behind relative to changes in soil ammonium. In both soils, only T-RFs corresponding to phylotypes related to Nitrosospira clade 3a underwent significant community changes. Increases in AOB abundance were generally stronger in the bulk soil than in the rhizosphere, implying significant ammonia uptake by plant roots. AOA underwent shifts in the community composition over time and fluctuated in abundance in all treatments irrespective of ammonia availability. AOB were thus considered as the main agents responsible for fertilizer ammonium oxidation, while the functions of AOA in soil N cycling remain unresolved.

Phage-type specific markers identified by Diversity Arrays Technology (DArT) analysis of Salmonella enterica ssp. enterica serovars Enteritidis and Typhimurium.

Diversity Arrays Technology (DArT) was applied to differentiate between S. enterica serovar Enteritidis and Typhimurium strains, respectively. Ten and eleven, mainly phage and plasmid-related markers were identified for serovars Enteritidis and Typhimurium. In combination, these markers can be used for subtyping among and within phage types.

Epidemic multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport strains contain three phage regions and a MDR resistance plasmid.

Multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport has caused serious disease in animals and humans in North America, whereas in the UK S. enterica serovar Newport is not associated with severe disease and usually sensitive to antibiotics; MDR S. Newport (not AmpC) strains have only been isolated from poultry. We found that UK poultry strains belonged to MLST type ST166 and were distinct from cattle isolates for being able to utilize D-tagotose and when compared by pulsed-field gel electrophoresis (PFGE), comparative genomic hybridization (CGH) and diversity arrays technology (DArT). Cattle strains belonged to the ST45 complex differing from ST166 at all seven loci. PFGE showed that 19 out of 27 cattle isolates were more than 85% similar to each other and some UK and US strains were indistinguishable. Both CGH and DArT identified genes (including phage-related ones) that were uniquely present in the US isolates and two such genes identified by DArT showed sequence similarities with the pertussis-like (artAB) toxin. This work demonstrates that MDR-AmpC S. Newport from the USA are genetically closely related to pan-susceptible strains from the UK, but contained three extra phage regions and a MDR plasmid.

Comparison of diversities and compositions of bacterial populations inhabiting natural forest soils.

The diversity and composition of soil bacterial communities were compared among six Austrian natural forests, including oak-hornbeam, spruce-fir-beech, and Austrian pine forests, using terminal restriction fragment length polymorphism (T-RFLP, or TRF) analysis and sequence analysis of 16S rRNA genes. The forests studied differ greatly in soil chemical characteristics, microbial biomass, and nutrient turnover rates. The aim of this study was to relate these differences to the composition of the bacterial communities inhabiting the individual forest soils. Both TRF profiling and clone sequence analysis revealed that the bacterial communities in soils under Austrian pine forests, representing azonal forest types, were distinct from those in soils under zonal oak-hornbeam and spruce-fir-beech forests, which were more similar in community composition. Clones derived from an Austrian pine forest soil were mostly affiliated with high-G+C gram-positive bacteria (49%), followed by members of the alpha-Proteobacteria (20%) and the Holophaga/Acidobacterium group (12%). Clones in libraries from oak-hornbeam and spruce-fir-beech forest soils were mainly related to the Holophaga/Acidobacterium group (28 and 35%), followed by members of the Verrucomicrobia (24%) and the alpha-Proteobacteria (27%), respectively. The soil bacterial communities in forests with distinct vegetational and soil chemical properties appeared to be well differentiated based on 16S rRNA gene phylogeny. In particular, the outstanding position of the Austrian pine forests, which are determined by specific soil conditions, was reflected in the bacterial community composition.