Metagenomics of African Empogona and Tricalysia (Rubiaceae) reveals the presence of leaf endophytes

Background Leaf symbiosis is a phenomenon in which host plants of Rubiaceae interact with bacterial endophytes within their leaves. To date, it has been found in around 650 species belonging to eight genera in four tribes; however, the true extent in Rubiaceae remains unknown. Our aim is to investigate the possible occurrence of leaf endophytes in the African plant genera Empogona and Tricalysia and, if present, to establish their identity. Methods Total DNA was extracted from the leaves of four species of the Coffeeae tribe (Empogona congesta, Tricalysia hensii, T. lasiodelphys, and T. semidecidua) and sequenced. Bacterial reads were filtered out and assembled. Phylogenetic analysis of the endophytes was used to reveal their identity and their relationship with known symbionts. Results All four species have non-nodulated leaf endophytes, which are identified as Caballeronia. The endophytes are distinct from each other but related to other nodulated and non-nodulated endophytes. An apparent phylogenetic or geographic pattern appears to be absent in endophytes or host plants. Caballeronia endophytes are present in the leaves of Empogona and Tricalysia, two genera not previously implicated in leaf symbiosis. This interaction is likely to be more widespread, and future discoveries are inevitable.

[1]  P. Vandamme,et al.  Cyclitol metabolism is a central feature of Burkholderia leaf symbionts. , 2022, Environmental microbiology.

[2]  S. Razafimandimbison,et al.  Target capture data resolve recalcitrant relationships in the coffee family (Rubioideae, Rubiaceae) , 2022, Frontiers in Plant Science.

[3]  K. Gademann,et al.  Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts , 2021, Scientific Reports.

[4]  H. Gross,et al.  Burkholderia in the genomic era: from taxonomy to the discovery of new antimicrobial secondary metabolites , 2021, Critical reviews in microbiology.

[5]  W. Weckwerth,et al.  Dissecting Metabolism of Leaf Nodules in Ardisia crenata and Psychotria punctata , 2021, Frontiers in Molecular Biosciences.

[6]  F. Forest,et al.  Settling a family feud: a high-level phylogenomic framework for the Gentianales based on 353 nuclear genes and partial plastomes. , 2021, American journal of botany.

[7]  S. Janssens,et al.  Patterns of transmission and horizontal gene transfer in the Dioscorea sansibarensis leaf symbiosis revealed by whole-genome sequencing , 2021, Current Biology.

[8]  M. C. Orozco-Mosqueda,et al.  Plant-microbial endophytes interactions: Scrutinizing their beneficial mechanisms from genomic explorations , 2020 .

[9]  H. X. Nguyen,et al.  Identification and characterization of Vietnamese coffee bacterial endophytes displaying in vitro antifungal and nematicidal activities. , 2020, Microbiological research.

[10]  D. Crouzillat,et al.  Complex evolutionary history of coffees revealed by full plastid genomes and 28,800 nuclear SNP analyses, with particular emphasis on Coffea canephora (Robusta coffee). , 2020, Molecular phylogenetics and evolution.

[11]  B. Bremer,et al.  Conflicting phylogenetic signals in genomic data of the coffee family (Rubiaceae) , 2020 .

[12]  C. Guyeux,et al.  Chloroplast genomes of Rubiaceae: Comparative genomics and molecular phylogeny in subfamily Ixoroideae , 2020, PloS one.

[13]  Arne Sinnesael.  Bacterial leaf symbiosis – Origin, function, evolutionary gain, and transmission mode of endophytes in bacteriophilous Rubiaceae , 2020 .

[14]  E. Smets,et al.  Is the bacterial leaf nodule symbiosis obligate for Psychotria umbellata? The development of a Burkholderia-free host plant , 2019, PloS one.

[15]  A. Davis,et al.  Using multiple plastid DNA regions to construct the first phylogenetic tree for Asian genera of Coffeeae (Ixoroideae, Rubiaceae) , 2018, Botanical Journal of the Linnean Society.

[16]  K. Gademann,et al.  Leaf nodule symbiosis: function and transmission of obligate bacterial endophytes. , 2018, Current opinion in plant biology.

[17]  M. Suchard,et al.  Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7 , 2018, Systematic biology.

[18]  P. Poole,et al.  Rhizobia: from saprophytes to endosymbionts , 2018, Nature Reviews Microbiology.

[19]  B. Bremer,et al.  Conflicting results from mitochondrial genomic data challenge current views of Rubiaceae phylogeny. , 2017, American journal of botany.

[20]  B. Bremer,et al.  Historical biogeography and phylogeny of the pantropical Psychotrieae alliance (Rubiaceae), with particular emphasis on the Western Indian Ocean Region. , 2017, American journal of botany.

[21]  S. Janssens,et al.  Non-nodulated bacterial leaf symbiosis promotes the evolutionary success of its host plants in the coffee family (Rubiaceae). , 2017, Molecular phylogenetics and evolution.

[22]  P. Pevzner,et al.  metaSPAdes: a new versatile metagenomic assembler. , 2017, Genome research.

[23]  Justin Chu,et al.  ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter , 2016, bioRxiv.

[24]  L. Eberl,et al.  The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis. , 2016, Environmental microbiology.

[25]  M. Samadpour,et al.  Transfer of eleven species of the genus Burkholderia to the genus Paraburkholderia and proposal of Caballeronia gen. nov. to accommodate twelve species of the genera Burkholderia and Paraburkholderia. , 2016, International journal of systematic and evolutionary microbiology.

[26]  Daniel Mapleson,et al.  KAT: a K-mer analysis toolkit to quality control NGS datasets and genome assemblies , 2016, bioRxiv.

[27]  Jan P. Meier-Kolthoff,et al.  Phylogenomic Study of Burkholderia glathei-like Organisms, Proposal of 13 Novel Burkholderia Species and Emended Descriptions of Burkholderia sordidicola, Burkholderia zhejiangensis, and Burkholderia grimmiae , 2016, Front. Microbiol..

[28]  M. Ducousso,et al.  Burkholderia novacaledonica sp. nov. and B. ultramafica sp. nov. isolated from roots of Costularia spp. pioneer plants of ultramafic soils in New Caledonia. , 2016, Systematic and applied microbiology.

[29]  Anders Krogh,et al.  Fast and sensitive taxonomic classification for metagenomics with Kaiju , 2016, Nature Communications.

[30]  T. Mitchell,et al.  What's Inside That Seed We Brew? A New Approach To Mining the Coffee Microbiome , 2015, Applied and Environmental Microbiology.

[31]  Radhey S. Gupta,et al.  Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species , 2014, Front. Genet..

[32]  Torsten Seemann,et al.  Prokka: rapid prokaryotic genome annotation , 2014, Bioinform..

[33]  B. Bremer,et al.  Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species. , 2014, American journal of botany.

[34]  P. Vandamme,et al.  Intraspecific variation in Burkholderia caledonica: Europe vs. Africa and soil vs. endophytic isolates. , 2014, Systematic and applied microbiology.

[35]  E. Smets,et al.  Phylogenetic lineages in Vanguerieae (Rubiaceae) associated with Burkholderia bacteria in sub-Saharan Africa. , 2013, American journal of botany.

[36]  Michael Roberts,et al.  The MaSuRCA genome assembler , 2013, Bioinform..

[37]  E. Smets,et al.  Symbiotic ß-Proteobacteria beyond Legumes: Burkholderia in Rubiaceae , 2013, PloS one.

[38]  E. Smets,et al.  Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae). , 2012, FEMS microbiology ecology.

[39]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[40]  E. Smets,et al.  Identification, origin, and evolution of leaf nodulating symbionts of Sericanthe (Rubiaceae) , 2011, The Journal of Microbiology.

[41]  P. Vandamme,et al.  Bacterial Leaf Symbiosis in Angiosperms: Host Specificity without Co-Speciation , 2011, PloS one.

[42]  E. Smets,et al.  Endophytic Bacteria in Toxic South African Plants: Identification, Phylogeny and Possible Involvement in Gousiekte , 2011, PloS one.

[43]  A. E. Wyk,et al.  Taxonomy of the genus Keetia (Rubiaceae-subfam. Ixoroideae-tribe Vanguerieae) in southern Africa, with notes on bacterial symbiosis as well as the structure of colleters and the 'stylar head' complex , 2009 .

[44]  M. Fay,et al.  Phylogeny of Tricalysia (Rubiaceae) and its Relationships with Allied Genera Based on Plastid DNA Data: Resurrection of the Genus Empogona1 , 2009 .

[45]  D. Posada.  jModelTest: phylogenetic model averaging. , 2008, Molecular biology and evolution.

[46]  J. Buyer,et al.  Endophytic bacteria in Coffea arabica L. , 2005, Journal of basic microbiology.

[47]  J. Caballero-Mellado,et al.  Burkholderia unamae sp. nov., an N2-fixing rhizospheric and endophytic species. , 2004, International journal of systematic and evolutionary microbiology.

[48]  F. Lutzoni,et al.  Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. , 2003, Molecular biology and evolution.

[49]  R. de Wachter,et al.  Identification of the bacterial endosymbionts in leaf galls of Psychotria (Rubiaceae, angiosperms) and proposal of 'Candidatus Burkholderia kirkii' sp. nov. , 2002, International journal of systematic and evolutionary microbiology.

[50]  K. Katoh,et al.  MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. , 2002, Nucleic acids research.

[51]  F. K. Barker,et al.  The utility of the incongruence length difference test. , 2002, Systematic biology.

[52]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[53]  I. Longden,et al.  EMBOSS: the European Molecular Biology Open Software Suite. , 2000, Trends in genetics : TIG.

[54]  E. Robbrecht.  Sericanthe, a New African Genus of Rubiaceae (Coffeeae) , 1978 .

[55]  N. Lersten,et al.  Bacterial leaf nodule symbiosis in angiosperms with emphasis on Rubiaceae and Myrsinaceae , 1976, The Botanical Review.

[56]  Powo.  Plants of the World Online. , 2020 .

[57]  Mosè Manni,et al.  BUSCO: Assessing Genome Assembly and Annotation Completeness. , 2019, Methods in molecular biology.

[58]  P. Vandamme,et al.  Draft genome and description of Orrella dioscoreae gen. nov. sp. nov., a new species of Alcaligenaceae isolated from leaf acumens of Dioscorea sansibarensis. , 2017, Systematic and applied microbiology.

[59]  E. Smets,et al.  Identification of the bacterial endosymbionts in leaf nodules of Pavetta (Rubiaceae). , 2012, International journal of systematic and evolutionary microbiology.

[60]  R. Wachter,et al.  The taxonomic value of bacterial symbiont identification in African Psychotria (Rubiaceae) , 2001 .

[61]  M. P. Cummings,et al.  PAUP* Phylogenetic analysis using parsimony (*and other methods) Version 4 , 2000 .

[62]  Leigh A. Johnson,et al.  Assessing Congruence: Empirical Examples from Molecular Data , 1998 .

[63]  I. Miller.  Bacterial Leaf Nodule Symbiosis , 1990 .

[64]  A. E. Wyk,et al.  Non-pathological bacterial symbiosis in Pachystigma and Fadogia (Rubiaceae): its evolutionary significance and possible involvement in the aetiology of gousiekte in domestic ruminants. , 1990 .

[65]  C. Bremekamp.  A monograph of the genus Pavetta L.: Additions and Emendations. II , 1939 .

[66]  C. Bremekamp.  A monograph of the genus Pavetta L.: Additions and Emendations , 1939 .

[67]  F. Faber.  Das erbliche Zusammenleben von Bakterien und tropischen Pflanzen , 1912 .