Dataset Information

Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

ABSTRACT: We used a next-generation, Illumina-based sequencing approach to characterize the bacterial community development of apple rhizosphere soil in a replant site (RePlant) and a new planting site (NewPlant) in Beijing. Dwarfing apple nurseries of 'Fuji'/SH6/Pingyitiancha trees were planted in the spring of 2013. Before planting, soil from the apple rhizosphere of the replant site (ReSoil) and from the new planting site (NewSoil) was sampled for analysis on the Illumina MiSeq platform. In late September, the rhizosphere soil from both sites was resampled (RePlant and NewPlant). More than 16,000 valid reads were obtained for each replicate, and the community was composed of five dominant groups (Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria). The bacterial diversity decreased after apple planting. Principal component analyses revealed that the rhizosphere samples were significantly different among treatments. Apple nursery planting showed a large impact on the soil bacterial community, and the community development was significantly different between the replanted and newly planted soils. Verrucomicrobia were less abundant in RePlant soil, while Pseudomonas and Lysobacter were increased in RePlant compared with ReSoil and NewPlant. Both RePlant and ReSoil showed relatively higher invertase and cellulase activities than NewPlant and NewSoil, but only NewPlant soil showed higher urease activity, and this soil also had the higher plant growth. Our experimental results suggest that planting apple nurseries has a significant impact on soil bacterial community development at both replant and new planting sites, and planting on new site resulted in significantly higher soil urease activity and a different bacterial community composition.

SUBMITTER: Sun J

PROVIDER: S-EPMC4216118 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

Sun Jian J Zhang Qiang Q Zhou Jia J Wei Qinping Q

PloS one 20141031 10

We used a next-generation, Illumina-based sequencing approach to characterize the bacterial community development of apple rhizosphere soil in a replant site (RePlant) and a new planting site (NewPlant) in Beijing. Dwarfing apple nurseries of 'Fuji'/SH6/Pingyitiancha trees were planted in the spring of 2013. Before planting, soil from the apple rhizosphere of the replant site (ReSoil) and from the new planting site (NewSoil) was sampled for analysis on the Illumina MiSeq platform. In late Septem ...[more]

PMID: 25360786

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Project description:BackgroundApple replant disease (ARD) is a syndrome that occurs in areas where apple plants or closely related species have been previously cultivated. Even though ARD is a well-known phenomenon, which has been observed in different regions worldwide and occurs independent of the soil type, its causes still remain unclear.ResultsAs expected, the biomass of plants grown in replant soil was significantly lower compared to those grown in control (virgin) soil. A shotgun metagenome analysis showed a clear differentiation between the rhizosphere and bulk soil compartments independent from the soil used. However, significant differences associated with apple replant disease were only observed in the rhizosphere compartment, for which we detected changes in the abundance of major bacterial genera. Interestingly, reads assigned to Actinobacteria were significantly reduced in relative abundance in rhizosphere samples of the soil affected by replant disease. Even though reads assigned to pathogenic fungi were detected, their relative abundance was low and did not differ significantly between the two different soils. Differences in microbiome structure also resulted in shifts in functional pattern. We observed an increase in genes related to stress sensing in the rhizosphere of soils affected by replant disease, whereas genes linked to nutrient sensing and uptake dominated in control soils. Moreover, we observed a lower abundance of genes coding for enzymes which trigger the degradation of aromatic compounds in rhizosphere of soils affected by replant disease, which is probably connected with higher concentration of phenolic compounds, generally associated with disease progression.ConclusionsOur study shows, for the first time, how apple replanting affects soil functioning by altering the soil microbiome. Particularly, the decrease in the abundance of genes which code for enzymes catalyzing the degradation of aromatic compounds, observed in the rhizosphere of plants grown in soil affected by apple replant disease, is of interest. Apple rootstocks are known to synthetize many phenolic compounds, including defense related phytoalexins, which have been considered for long to be connected with the emergence of replant disease. The knowledge gained in this study might help to develop targeted strategies to overcome or at least reduce the effects of ARD symptoms.

Project description:Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized.We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms.The library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost.Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.

Project description:BackgroundBovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences.ResultsLeptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences.ConclusionsThe microbiomes of the cranial lung lobe and mediastinal lymph node from calves which died from BRD and from clinically healthy H-F calves have been characterised. Contigs corresponding to the abundant Leptotrichiaceae OTU were sequenced and found not to be identical to any known bacterial genus. This suggests that we have identified a novel bacterial species associated with BRD.

Project description:Apple replant disease (ARD) is a serious threat to producers of apple trees and fruits worldwide. The ARD etiology is not unraveled and managing options are either economically not applicable or environmentally harmful. Thus, interest is given in biomarkers that allow to indicate ARD situations at early time points in order to classify soils according to ARD severity but also to analyze the effectiveness to potential countermeasures. This study aimed at (i) identifying ARD biomarkers on the transcriptional level in root tissue by analyzing the expression of previously identified candidate genes in ARD soils of different origin and texture and (ii) testing the specificity of these marker genes to ARD. In vitro propagated M26 plantlets were submitted to a bio-test with three ARD soils, either untreated or disinfected by γ-irradiation. Expression of seven candidate genes identified in a previous transcriptomic study was investigated by RT-qPCR in a time course experiment. Already three days after planting, a prominent upregulation of the phytoalexin biosynthesis genes biphenyl synthase 3 (BIS3) and biphenyl 4-hydroxylase (B4Hb) was observed in the untreated ARD variants of all three soils. The phytoalexin composition in roots was comparable for all three soils and the total phytoalexin content correlated with the expression of BIS3 and B4Hb. The third promising candidate gene that was upregulated under ARD conditions was the ethylene-responsive transcription factor 1B-like (ERF1B). In a second experiment M26 plantlets were exposed to different abiotic stressors, namely heat, salt and nutrient starvation, and candidate gene expression was determined in the roots. The expression levels of BIS3 and B4Hb were highly and specifically upregulated in ARD soil, but not upon the abiotic stress conditions, whereas ERF1B also showed higher expression under heat stress. In conclusion, BIS3 and B4Hb are recommended as early ARD biomarkers due to their high expression levels and their high specificity.

Dataset Information

Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

Publications

Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

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