PREMISE: Despite the high functional importance of endophytes, we still have limited understanding of the biotic and abiotic factors that influence colonization of plant hosts along major ecological gradients and lack quantitative estimates of their colonization extent. In this study, we hypothesized that the developmental stage of the ecosystem will affect the levels of bacterial and fungal endophytic assemblages in the foliar endosphere. METHODS: We quantified levels of bacterial and fungal endophytes in leaves of four plant hosts at four stages of vegetation succession using an optimized qPCR protocol with bacteria-specific 16S and fungi-targeting primers. RESULTS: (1) The ecosystem developmental stage did not have a significant effect on the colonization levels of bacterial or fungal endophytes. (2) Colonization levels by bacterial and fungal endophytes were governed by different mechanisms. (3) Endophytic colonization levels and their relationship to foliar tissue stoichiometry were highly host specific. CONCLUSIONS: Quantifying colonization levels is important in the study of endophytic ecology, and the fast, relatively low-cost qPCR-based method can supply useful ecological information, which can significantly enhance the interpretation potential of descriptive data generated, for example, by next-generation sequencing.

Biology Centre CAS Institute of Hydrobiology Na Sádkách 702 7 České Budějovice 37005 Czech Republic

Institute for Environmental Studies Faculty of Science Charles University Benátská 2 CZ 12800 Prague Czech Republic

University of South Bohemia Faculty of Science Branišovská 1645 31a České Budějovice 37005 Czech Republic

Zobrazit více v PubMed

Anderson, C. A. , Eser U., Korndorf T., Borsuk M. E., Skotheim J. M., and Gladfelter A. S.. 2013. Nuclear repulsion enables division autonomy in a single cytoplasm. Current Biology 23: 1999–2010. 10.1016/j.cub.2013.07.076 PubMed DOI PMC

Apigo, A. , and Oono R.. 2018. Dimensions of host specificity in foliar fungal endophytes. In: Pirttilä, A. , Frank, A. (eds) Endophytes of Forest Trees. Forestry Sciences, vol 86. Springer, Cham.

Apigo, A. , and Oono R.. 2022. Plant abundance, but not plant evolutionary history, shapes patterns of host specificity in foliar fungal endophytes. Ecosphere 13: e03879.

Barau, J. , Grandis A., de V. M. Carvalho A., Teixeira G. S., Zaparoli G. H. A., do Rio M. C. S., Rincones J., et al. 2015. Apoplastic and intracellular plant sugars regulate developmental transitions in witches’ broom disease of cacao. Journal of Experimental Botany 66: 1325–1337. PubMed PMC

Bazzaz, F. A. 1979. The physiological ecology of plant succession. Annual Review of Ecology, Evolution, and Systematics 10: 351–371.

Bazzaz, F. A. , and Pickett S. T. A.. 1980. Physiological ecology of tropical succession: a comparative review. Annual Review of Ecology, Evolution, and Systematics 11: 287–310.

Bednarek, P. , Kwon C., Schulze‐Lefert P., Parker J. E., and Ellis J. G.. 2010. Not a peripheral issue: secretion in plant–microbe interactions. Current Opinion in Plant Biology 13: 378–387. PubMed

Black, J. , Dean T., Byfield G., Foarde K., and Menetrez M.. 2013. Determining fungi rRNA copy number by PCR. Journal of Biomolecular Techniques 24: 32–38. PubMed PMC

Borniego, M. L. , Molina M. C., Guiamét J. J., and Martinez D. E.. 2020. Physiological and proteomic changes in the apoplast accompany leaf senescence in Arabidopsis . Frontiers in Plant Sciences 10: 1635. PubMed PMC

Borruso, L. , Wellstein C., Bani A., Bacchiocchi S. C., Margoni A., Tonin R., Zerbe S., and Brusetti L.. 2018. Temporal shifts in endophyte bacterial community composition of sessile oak (Quercus petraea) are linked to foliar nitrogen, stomatal length, and herbivory. PeerJ 6: e5769. PubMed PMC

Cantwell, H. , and Nurse P.. 2019. Unraveling nuclear size control. Current Genetics 65: 1281–1285. PubMed PMC

Caporaso, J. G. , Lauber C. L., Walters W. A., Berg‐Lyons D., Lozupone C. A., Turnbaugh P. J., Fierer N., and Knight R.. 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences, USA 108: 4516–4522. PubMed PMC

Carbone, I. , and Kohn L. M.. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553–556.

Dorn‐In, S. , Bassitta R., Schwaiger K., Bauer J., and Hölzel C. S.. 2015. Specific amplification of bacterial DNA by optimized so‐called universal bacterial primers in samples rich in plant DNA. Journal of Microbiological Methods 113: 50–56. PubMed

Ettenauer, J. , Piñar G., Tafer H., and Sterflinger K.. 2014. Quantification of fungal abundance on cultural heritage using real‐time PCR targeting the β‐actin gene. Frontiers in Microbiology 5: 262. PubMed PMC

Gladfelter, A. , and Berman J.. 2009. Dancing genomes: fungal nuclear positioning. Nature Reviews Microbiology 7: 875–886. PubMed PMC

Hammer, Ø. , Harper D. A. T., and Ryan P. D.. 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4(1): 9. http://palaeo-electronica.org/2001_1/past/issue1_01.htm

Harantová, L. , Mudrák O., Kohout P., Elhottová D., Frouz J., and Baldrian P.. 2017. Development of microbial community during primary succession in areas degraded by mining activities. Land Degradation and Development 28: 2574–2584.

Harrison, J. G. , and Griffin E. A.. 2020. The diversity and distribution of endophytes across biomes, plant phylogeny and host tissues: How far have we come and where do we go from here? Environmental Microbiology 22: 2107–2123. PubMed PMC

Heikrujam, J. , Kishor R., and Behari Mazumder P.. 2020. The chemistry behind plant DNA isolation protocols. Biochemical Analysis Tools – Methods for Bio‐Molecules Studies. IntechOpen. 10.5772/intechopen.92206 DOI

Fischer, P. J. , Petrini O., and Sutton B. C.. 1993. A comparative study of fungal endophytes in leaves, xylem, and bark of Eucalyptus nitens in Australia and England. Sydowia. 45: 338–345.

Frouz, J. , and Nováková A.. 2005. Development of soil microbial properties in topsoil layer during spontaneous succession in heaps after brown coal mining in relation to humus microstructure development. Geoderma 129: 54–64.

Frouz, J. , Prach K., Pižl V., Háněl L., Starý J., Tajovský K., Materna J., et al. 2008. Interactions between soil development, vegetation and soil fauna during spontaneous succession in post‐mining sites. European Journal of Soil Biology 44: 109–121.

Guillard, R. R. L. , and Lorenzen C. J.. 1972. Yellow‐green algae with chlorophyllide c . Journal of Phycology 8: 10–14.

Keiblinger, K. M. , Hall E. K., Wanek W., Szukics U., Hämmerle I., Ellersdorfer G., Böck S., et al. 2010. The effect of resource quantity and resource stoichiometry on microbial carbon‐use‐efficiency. FEMS Microbiology Ecology 73: 430–440. PubMed

Lambers, H. , Chapin F. S., and Pons T. L.. 2008. Plant physiological ecology, 2nd edi. Springer, NY, NY, USA.

Lavrinienko, A. , Jernfors T., Koskimäki J. J., Pirttilä A. M., and Watts P. C.. 2021. Does intraspecific variation in rDNA copy number affect analysis of microbial communities? Trends in Microbiology 29: 19–27. PubMed

Liu, M. , Wang Y., Li Q., Xiao W., and Song X.. 2019. Photosynthesis, ecological stoichiometry, and non‐structural carbohydrate response to simulated nitrogen deposition and phosphorus addition in Chinese fir forests. Forests 10: 1068.

Lofgren, L. A. , Uehling J. K., Branco S., Bruns T. D., Martin F., and Kennedy P. G.. 2018. Genome‐based estimates of fungal rDNA copy number variation across phylogenetic scales and ecological lifestyles. Molecular Ecology. 28: 721–730. PubMed

Martínez‐Vilalta, J. , Sala A., Asensio D., Galiano L., Hoch G., Palacio S., Piper F. I., and Lloret F.. 2016. Dynamics of non‐structural carbohydrates in terrestrial plants: a global synthesis. Ecological Monographs 86: 495–516.

Mudrák, O. , Doležal J., and Frouz J.. 2016. Initial species composition predicts the progress in the spontaneous succession on post‐mining sites. Ecological Engineering 95: 665‐670.

Müller, H. , Berg C., Landa B. B., Auerbach A., Moissl‐Eichinger C., and Berg G.. 2015. Plant genotype‐specific archaeal and bacterial endophytes but similar Bacillus antagonists colonize Mediterranean olive trees. Frontiers in Microbiology 6: 138. PubMed PMC

Nakano, M. 2018. 16S rRNA gene primer validation for bacterial diversity analysis of vegetable products. Journal of Food Protection 81: 848–859. PubMed

Nieuwenhuis, B. P. S. , and James T. Y. 2016. The frequency of sex in fungi. Philosophical Transactions of the Royal Society, B, Biological Sciences 371: 20150540. PubMed PMC

Oksanen, J. , Blanchet F. G., Kindt R., Legendre P., Minchin P. R., O'Hara R. B., Simpson G. L., et al. 2014. vegan: Community ecology package. R package version 2.2‐0. Website http://CRAN.Rproject.org/package=vegan.

Olsson, P. A. , Larsson L., Bago B., Wallander H., and Van Aarle I. M.. 2003. Ergosterol and fatty acids for biomass estimation of mycorrhizal fungi. New Phytologist 159(1): 7–10. http://www.jstor.org/stable/1514347 PubMed

R Core Team . 2020. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Website: https://www.R-project.org

Ruijter, J. M. , Ruiz‐Villalba A., van den Hoff A. J. J., Gunst Q. D., Wittwer C. T., and van den Hoff M. J. B.. 2019. Removal of artifact bias from qPCR results using DNA melting curve analysis. Federation of American Societies for Experimental Biology Journal 33: 14542–14555. PubMed

Šimek, K. , and Sirova D.. 2019. Fluorescently labeled bacteria as a tracer to reveal novel pathways of organic carbon flow in aquatic ecosystems. Journal of Visualized Experiments 151: e59903. 10.3791/59903 PubMed DOI

Smith, C. J. , and Osborn A. M.. 2009. Advantages and limitations of quantitative PCR (q‐PCR)‐based approaches in microbial ecology. FEMS Microbiology Ecology 67: 6–20. PubMed

Soong, J. L. , Fuchslueger L., Marañon‐Jimenez S., Torn M. S., Janssens I. A., Penuelas J., and Richter A.. 2020. Microbial carbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling. Global Change Biology 26: 1953–1961. PubMed

Stielow, J. B. , Levesque C. A., Seifert K. A., Meyer W., Irinyi L., Smits D., Renfurm R., et al. 2015. One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes. Persoonia: Molecular Phylogeny and Evolution of Fungi 35: 242–263. PubMed PMC

Taylor, S. C. , Laperriere G., and Germain H.. 2017. Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports 7: 2409. PubMed PMC

Tellez, P. H. , Arnold A. E., Leo A. B., Kitajima K., and Van Bael S. A.. 2022. Traits along the leaf economics spectrum are associated with communities of foliar endophytic symbionts. Frontiers in Microbiology 13: 927780. PubMed PMC

Tyrrell, M. G. , Peabody D. C., Peabody R. B., James‐Pederson M., Hirst R. G., Allan‐Perkins E., Bickford H., et al. 2020. Mosaic fungal individuals have the potential to evolve within a single generation. Scientific Reports 10: 1–13. PubMed PMC

Urbanová, M. , Kopecký J., Valášková V., Ságová‐Marečková M., Elhottová D., Kyselková M., Moënne‐Loccoz Y., and Baldrian P.. 2011. Development of bacterial community during spontaneous succession on spoil heaps after brown coal mining. FEMS Microbiology Ecology 78: 59–69. PubMed

Walters, W. , Hyde E. R., Berg‐Lyons D., Ackermann G., Humphrey G., Parada A., Gilbert J. A., et al. 2016. Improved bacterial 16S rRNA gene (V4 and V4‐5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems 1: e00009. PubMed PMC

Wang, S. , Ventolero M., Hu H., and Li X.. 2022. A revisit to universal single‐copy genes in bacterial genomes. Scientific Reports 12: 14550. PubMed PMC

Wickham, H. 2016. ggplot2: Elegant graphics for data analysis. Springer‐Verlag NY, NY, USA. Website 10.1007/978-0-387-98141-3 DOI

Yu, Y. , Chen Z., Xie H., Feng X., Wang Y., and Xu P.. 2022. Overhauling the effect of surface sterilization on analysis of endophytes in tea plants. Frontiers in Plant Sciences 13: 849658. PubMed PMC