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Microbial Ecology

, Volume 77, Issue 3, pp 664–675 | Cite as

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by “Candidatus Phytoplasma prunorum”

  • Ales EichmeierEmail author
  • Tomas Kiss
  • Tomas Necas
  • Eliska Penazova
  • Dorota Tekielska
  • Marketa Bohunicka
  • Lucie Valentova
  • Radek Cmejla
  • Daniel Morais
  • Petr Baldrian
Plant Microbe Interactions
  • 322 Downloads

Abstract

Candidatus Phytoplasma prunorum” (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.

Keywords

Candidatus Phytoplasma prunorum” Phloem Stone Fruit High-throughput sequencing Bacterial composition 

Notes

Acknowledgements

This research was supported by The Ministry of Agriculture of the Czech Republic, Project no. QJ1510352. Access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum provided under the program “Projects of Large Research, Development, and Innovations Infrastructures” (CESNET LM2015042) is greatly appreciated. The work was supported from EFRR “Multidisciplinary research to increase application potential of nanomaterials in agricultural practice” (No. CZ.02.1.01/0.0/0.0/16_025/0007314). We appreciate the cooperation with Jana Suchá and Jan Wolf regarding the sampling and DNA extraction.

Supplementary material

248_2018_1250_MOESM1_ESM.pdf (149 kb)
Supplementary Figure 1 Heatmap with ascendant hierarchical clustering based on Euclidian distances was created with a dataset of OTUs with abundances >0.5% in >2 samples. (PDF 149 kb)
248_2018_1250_MOESM2_ESM.pdf (128 kb)
Supplementary Figure 2 Alpha diversity of the three groups (A, B+ and B-), expressing number of taxa (S) with bootstrapping 9999. Shannon index (entropy), a diversity index, was calculated, the index taking into account the number of individuals as well as number of taxa. Varies from 0 for communities with only a single taxon to high values for communities with many taxa, each with few individuals. For alpha diversity calculations were used the taxa included in the Table 3. (PDF 127 kb)
248_2018_1250_Fig4_ESM.png (1.1 mb)
Supplementary Figure 3

The box plot graph based on taxa abundancies in % (axis y), the sampling localities, stonefruit species and the individual samples are showed (axis x). (PNG 1084 kb)

248_2018_1250_MOESM3_ESM.tif (349 kb)
High Resolution Image (TIF 349 kb)
248_2018_1250_MOESM4_ESM.odt (16 kb)
Supplementary Table 1 Description of the numbers of reads. Primary analysis provides reads mapped to index ID showing the percentage of the reads per sample, the table contains 60 isolates positive and negative for CPp presence by nested PCR and real-time PCR methods. % Reads Identified (PF) is the total fraction of passing filter reads assigned to an index. Trimming, Merging and QC steps were carried out in CLC Genomics Workbench 6.5.1 (CLC Bio, Denmark) (ODT 16 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ales Eichmeier
    • 1
    Email author
  • Tomas Kiss
    • 2
  • Tomas Necas
    • 2
  • Eliska Penazova
    • 1
  • Dorota Tekielska
    • 3
  • Marketa Bohunicka
    • 4
  • Lucie Valentova
    • 5
  • Radek Cmejla
    • 5
  • Daniel Morais
    • 6
  • Petr Baldrian
    • 6
  1. 1.Mendeleum - Institute of GeneticsMendel University in BrnoLedniceCzech Republic
  2. 2.Department of Fruit GrowingMendel University in BrnoLedniceCzech Republic
  3. 3.Department of Plant ProtectionUniversity of Agriculture in KrakowKrakowPoland
  4. 4.Department of Biology, Faculty of ScienceUniversity of Hradec KrálovéHradec KrálovéCzech Republic
  5. 5.Research and Breeding Institute of Pomology Holovousy LtdHořiceCzech Republic
  6. 6.Laboratory of Environmental MicrobiologyInstitute of Microbiology of the CASPraha 4Czech Republic

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