Guo-Chun Ding

PhyloChip hybridization uncovered an enormous bacterial diversity in the rhizosphere of different potato cultivars: many common and few cultivar-dependent taxa

The phylogenetic composition of bacterial communities in the rhizosphere of three potato cultivars grown at two distant field sites was analysed. Ribosomal gene fragments amplified from total community DNA were hybridized to PhyloChips. A total of 2432 operational taxonomic units (OTUs) were detected by the PhyloChips, of which 65% were found in the rhizosphere of all cultivars at both field sites. From all detected OTUs, 9% revealed a cultivar-dependent abundance at the one or the other field site and 4% at both sites. Differential abundance on the three cultivars was mainly observed for OTUs belonging to the Pseudomonadales, Actinomycetales and Enterobacteriales. More than 40% of OTUs belonging to Bradyrhizobiales, Sphingomonadales, Burkholderiales, Rhodocyclales, Xanthomonadales and Actinomycetales differed significantly in their abundance between the sites. A sequence analysis of six 16S rRNA gene clone libraries corresponded well with the taxonomic community structure evidenced by the PhyloChip hybridization. Most ribotypes matched OTUs detected by the PhyloChip. Those OTUs that responded to the potato cultivar at both field sites might be of interest in view of cultivar-specific effects on bacterial biocontrol strains and pathogens.

Increased Abundance and Transferability of Resistance Genes after Field Application of Manure from Sulfadiazine-Treated Pigs

ABSTRACT Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes sul1 and sul2 in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both sul genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of sul genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the sul gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into Escherichia coli. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance.

Effect of the soil type on the microbiome in the rhizosphere of field-grown lettuce

The complex and enormous diversity of microorganisms associated with plant roots is important for plant health and growth and is shaped by numerous factors. This study aimed to unravel the effects of the soil type on bacterial communities in the rhizosphere of field-grown lettuce. We used an experimental plot system with three different soil types that were stored at the same site for 10 years under the same agricultural management to reveal differences directly linked to the soil type and not influenced by other factors such as climate or cropping history. Bulk soil and rhizosphere samples were collected 3 and 7 weeks after planting. The analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and pyrosequencing revealed soil type dependent differences in the bacterial community structure of the bulk soils and the corresponding rhizospheres. The rhizosphere effect differed depending on the soil type and the plant growth developmental stage. Despite the soil type dependent differences in the bacterial community composition several genera such as Sphingomonas, Rhizobium, Pseudomonas, and Variovorax were significantly increased in the rhizosphere of lettuce grown in all three soils. The number of rhizosphere responders was highest 3 weeks after planting. Interestingly, in the soil with the highest numbers of responders the highest shoot dry weights were observed. Heatmap analysis revealed that many dominant operational taxonomic units were shared among rhizosphere samples of lettuce grown in diluvial sand, alluvial loam, and loess loam and that only a subset was increased in relative abundance in the rhizosphere compared to the corresponding bulk soil. The findings of the study provide insights into the effect of soil types on the rhizosphere microbiome of lettuce.

Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

ABSTRACT A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHDα) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHDα gene amplicons from soil DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHDα genes of Gram-positive and Gram-negative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHDα genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHDα gene-carrying soil bacteria to phenanthrene spiking.

Dynamics of Soil Bacterial Communities in Response to Repeated Application of Manure Containing Sulfadiazine

Large amounts of manure have been applied to arable soils as fertilizer worldwide. Manure is often contaminated with veterinary antibiotics which enter the soil together with antibiotic resistant bacteria. However, little information is available regarding the main responders of bacterial communities in soil affected by repeated inputs of antibiotics via manure. In this study, a microcosm experiment was performed with two concentrations of the antibiotic sulfadiazine (SDZ) which were applied together with manure at three different time points over a period of 133 days. Samples were taken 3 and 60 days after each manure application. The effects of SDZ on soil bacterial communities were explored by barcoded pyrosequencing of 16S rRNA gene fragments amplified from total community DNA. Samples with high concentration of SDZ were analyzed on day 193 only. Repeated inputs of SDZ, especially at a high concentration, caused pronounced changes in bacterial community compositions. By comparison with the initial soil, we could observe an increase of the disturbance and a decrease of the stability of soil bacterial communities as a result of SDZ manure application compared to the manure treatment without SDZ. The number of taxa significantly affected by the presence of SDZ increased with the times of manure application and was highest during the treatment with high SDZ-concentration. Numerous taxa, known to harbor also human pathogens, such as Devosia, Shinella, Stenotrophomonas, Clostridium, Peptostreptococcus, Leifsonia, Gemmatimonas, were enriched in the soil when SDZ was present while the abundance of bacteria which typically contribute to high soil quality belonging to the genera Pseudomonas and Lysobacter, Hydrogenophaga, and Adhaeribacter decreased in response to the repeated application of manure and SDZ.

Changes of Soil Bacterial Diversity as a Consequence of Agricultural Land Use in a Semi-Arid Ecosystem

Natural scrublands in semi-arid deserts are increasingly being converted into fields. This results in losses of characteristic flora and fauna, and may also affect microbial diversity. In the present study, the long-term effect (50 years) of such a transition on soil bacterial communities was explored at two sites typical of semi-arid deserts. Comparisons were made between soil samples from alfalfa fields and the adjacent scrublands by two complementary methods based on 16S rRNA gene fragments amplified from total community DNA. Denaturing gradient gel electrophoresis (DGGE) analyses revealed significant effects of the transition on community composition of Bacteria, Actinobacteria, Alpha- and Betaproteobacteria at both sites. PhyloChip hybridization analysis uncovered that the transition negatively affected taxa such as Acidobacteria, Chloroflexi, Acidimicrobiales, Rubrobacterales, Deltaproteobacteria and Clostridia, while Alpha-, Beta- and Gammaproteobacteria, Bacteroidetes and Actinobacteria increased in abundance. Redundancy analysis suggested that the community composition of phyla responding to agricultural use (except for Spirochaetes) correlated with soil parameters that were significantly different between the agricultural and scrubland soil. The arable soils were lower in organic matter and phosphate concentration, and higher in salinity. The variation in the bacterial community composition was higher in soils from scrubland than from agriculture, as revealed by DGGE and PhyloChip analyses, suggesting reduced beta diversity due to agricultural practices. The long-term use for agriculture resulted in profound changes in the bacterial community and physicochemical characteristics of former scrublands, which may irreversibly affect the natural soil ecosystem.

Dynamics of Bacterial Communities in Two Unpolluted Soils after Spiking with Phenanthrene: Soil Type Specific and Common Responders

Considering their key role for ecosystem processes, it is important to understand the response of microbial communities in unpolluted soils to pollution with polycyclic aromatic hydrocarbons (PAH). Phenanthrene, a model compound for PAH, was spiked to a Cambisol and a Luvisol soil. Total community DNA from phenanthrene-spiked and control soils collected on days 0, 21, and 63 were analyzed based on PCR-amplified 16S rRNA gene fragments. Denaturing gradient gel electrophoresis (DGGE) fingerprints of bacterial communities increasingly deviated with time between spiked and control soils. In taxon specific DGGE, significant responses of Alphaproteobacteria and Actinobacteria became only detectable after 63 days, while significant effects on Betaproteobacteria were detectable in both soils after 21 days. Comparison of the taxonomic distribution of bacteria in spiked and control soils on day 63 as revealed by pyrosequencing indicated soil type specific negative effects of phenanthrene on several taxa, many of them belonging to the Gamma-, Beta-, or Deltaproteobacteria. Bacterial richness and evenness decreased in spiked soils. Despite the significant differences in the bacterial community structure between both soils on day 0, similar genera increased in relative abundance after PAH spiking, especially Sphingomonas and Polaromonas. However, this did not result in an increased overall similarity of the bacterial communities in both soils.

Plant age and genotype affect the bacterial community composition in the tuber rhizosphere of field-grown sweet potato plants.

The hypothesis that sweet potato genotypes containing different starch yields in their tuberous roots can affect the bacterial communities present in the rhizosphere (soil adhering to tubers) was tested in this study. Tuberous roots of field-grown sweet potato of genotypes IPB-149 (commercial genotype), IPB-052, and IPB-137 were sampled three and six months after planting and analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analysis of 16S rRNA genes PCR-amplified from total community DNA. The statistical analysis of the DGGE fingerprints showed that both plant age and genotypes influenced the bacterial community structure in the tuber rhizosphere. Pyrosequencing analysis showed that the IPB-149 and IPB-052 (both with high starch content) displayed similar bacterial composition in the tuber rhizosphere, while IPB-137 with the lowest starch content was distinct. In comparison with bulk soil, higher 16S rRNA gene copy numbers (qPCR) and numerous genera with significantly increased abundance in the tuber rhizosphere of IPB-137 (Sphingobium, Pseudomonas, Acinetobacter, Stenotrophomonas, Chryseobacterium) indicated a stronger rhizosphere effect. The genus Bacillus was strongly enriched in the tuber rhizosphere samples of all sweet potato genotypes studied, while other genera showed a plant genotype-dependent abundance. This is the first report on the molecular identification of bacteria being associated with the tuber rhizosphere of different sweet potato genotypes.

Shifts in abundance and diversity of mobile genetic elements after the introduction of diverse pesticides into an on-farm biopurification system over the course of a year.

ABSTRACT Biopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1, intI2) and genes encoding resistance to sulfonamides (sul1, sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1 trfA gene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1β and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

Truffle Brûlés Have an Impact on the Diversity of Soil Bacterial Communities

Background The development of Tuber melanosporum mycorrhizal symbiosis is associated with the production of an area devoid of vegetation (commonly referred to by the French word ‘brûlé’) around the symbiotic plants and where the fruiting bodies of T. melanosporum are usually collected. The extent of the ecological impact of such an area is still being discovered. While the relationship between T. melanosporum and the other fungi present in the brûlé has been assessed, no data are available on the relationship between this fungus and the bacteria inhabiting the brûlé. Methodology/Principal Findings We used DGGE and DNA microarrays of 16S rRNA gene fragments to compare the bacterial and archaeal communities inside and outside of truffle brûlés. Soil samples were collected in 2008 from four productive T. melanosporum/Quercus pubescens truffle-grounds located in Cahors, France, showing characteristic truffle brûlé. All the samples were analyzed by DGGE and one truffle-ground was analyzed also using phylogenetic microarrays. DGGE profiles showed differences in the bacterial community composition, and the microarrays revealed a few differences in relative richness between the brûlé interior and exterior zones, as well as differences in the relative abundance of several taxa. Conclusions/Significance The different signal intensities we have measured for members of bacteria and archaea inside versus outside the brûlé are the first demonstration, to our knowledge, that not only fungal communities, but also other microorganisms are affected by T. melanosporum. Firmicutes (e.g., Bacillus), several genera of Actinobacteria, and a few Cyanobacteria had greater representation inside the brûlé compared with outside, whereas Pseudomonas and several genera within the class Flavobacteriaceae had higher relative abundances outside the brûlé. The findings from this study may contribute to future searches for microbial bio-indicators of brûlés.