Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 2;82(10):2943-2949.
doi: 10.1128/AEM.00452-16. Print 2016 May 15.

Isolation of Endohyphal Bacteria from Foliar Ascomycota and In Vitro Establishment of Their Symbiotic Associations

Kayla R Arendt  1 , Kevin L Hockett  1 , Sarah J Araldi-Brondolo  1 , David A Baltrus  1 , A Elizabeth Arnold  2   3
Affiliations

Isolation of Endohyphal Bacteria from Foliar Ascomycota and In Vitro Establishment of Their Symbiotic Associations

Kayla R Arendt et al. Appl Environ Microbiol. .

Abstract

Endohyphal bacteria (EHB) can influence fungal phenotypes and shape the outcomes of plant-fungal interactions. Previous work has suggested that EHB form facultative associations with many foliar fungi in the Ascomycota. These bacteria can be isolated in culture, and fungi can be cured of EHB using antibiotics. Here, we present methods for successfully introducing EHB into axenic mycelia of strains representing two classes of Ascomycota. We first establish in vitro conditions favoring reintroduction of two strains of EHB (Luteibacter sp.) into axenic cultures of their original fungal hosts, focusing on fungi isolated from healthy plant tissue as endophytes: Microdiplodia sp. (Dothideomycetes) and Pestalotiopsis sp. (Sordariomycetes). We then demonstrate that these EHB can be introduced into a novel fungal host under the same conditions, successfully transferring EHB between fungi representing different classes. Finally, we manipulate conditions to optimize reintroduction in a focal EHB-fungal association. We show that EHB infections were initiated and maintained more often under low-nutrient culture conditions and when EHB and fungal hyphae were washed with MgCl2 prior to reassociation. Our study provides new methods for experimental assessment of the effects of EHB on fungal phenotypes and shows how the identity of the fungal host and growth conditions can define the establishment of these widespread and important symbioses.

PubMed Disclaimer

Figures

FIG 1
FIG 1
(a and b) Successful reintroduction of Luteibacter sp. 9143 (tdTomato construct) into hyphae of Pestalotiopsis sp. 9143, illustrated with phase-contrast (a) and dark-field/fluorescence (b) microscopy. (c and d) Absence of Luteibacter sp. 9143 in cured hyphae of Pestalotiopsis sp. 9143, illustrated with phase-contrast (c) and dark-field/fluorescence (d) microscopy. (e) Free-living Luteibacter sp. 9143 (tdTomato construct) in pure culture, seen with dark-field/fluorescence microscopy. Magnification, 400× (a, b, and e) or 1,000× (c and d).
See this image and copyright information in PMC

References

    1. Turner TR, James EK, Poole PS. 2013. The plant microbiome. Genome Biol 14:209. doi: 10.1186/gb-2013-14-6-209. - DOI - PMC - PubMed
    1. Berg G, Grube M, Schloter M, Smalla K. 2014. Unraveling the plant microbiome: looking back and future perspectives. Front Microbiol 5:148. doi: 10.3389/fmicb.2014.00148. - DOI - PMC - PubMed
    1. Lindow SE, Brandl MT. 2003. Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883. doi: 10.1128/AEM.69.4.1875-1883.2003. - DOI - PMC - PubMed
    1. van der Putten WH, Klironomos JN, Wardle DA. 2007. Microbial ecology of biological invasions. ISME J 1:28–37. doi: 10.1038/ismej.2007.9. - DOI - PubMed
    1. Márquez LM, Redman RS, Rodriguez RJ, Roossnick MJ. 2007. A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance. Science 315:513–515. doi: 10.1126/science.1136237. - DOI - PubMed

Publication types