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

  • Plant Microbe Interactions
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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.

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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.

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Authors and Affiliations

  1. Mendeleum - Institute of Genetics, Mendel University in Brno, Valtická 334, 691 44, Lednice, Czech Republic

    Ales Eichmeier & Eliska Penazova

  2. Department of Fruit Growing, Mendel University in Brno, Valtická 337, 691 44, Lednice, Czech Republic

    Tomas Kiss & Tomas Necas

  3. Department of Plant Protection, University of Agriculture in Krakow, 29 Listopada 54, 31-425, Krakow, Poland

    Dorota Tekielska

  4. Department of Biology, Faculty of Science, University of Hradec Králové, Rokitanského 62, 500 03, Hradec Králové, Czech Republic

    Marketa Bohunicka

  5. Research and Breeding Institute of Pomology Holovousy Ltd, Holovousy 129, 508 01, Hořice, Czech Republic

    Lucie Valentova & Radek Cmejla

  6. Laboratory of Environmental Microbiology, Institute of Microbiology of the CAS, Vídeňská 1083, 14220, Praha 4, Czech Republic

    Daniel Morais & Petr Baldrian

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  1. Ales Eichmeier
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  2. Tomas Kiss
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Correspondence to Ales Eichmeier.

Electronic supplementary material

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)

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)

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)

High Resolution Image (TIF 349 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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Eichmeier, A., Kiss, T., Necas, T. et al. High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by “Candidatus Phytoplasma prunorum”. Microb Ecol 77, 664–675 (2019). https://doi.org/10.1007/s00248-018-1250-9

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