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Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria.

ABSTRACT: Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbiogenesis, to explore and exploit their biotechnological potential and for the description of new taxa. Our objective was to improve the recovery of lichen associated bacteria by developing novel isolation and culture approaches, initially using the lichen Pseudevernia furfuracea. We evaluated the effect of newly developed media enriched with novel lichen extracts, as well as the influence of thalli washing time and different disinfection and processing protocols of thalli. The developed methodology included: i) the use of lichen enriched media to mimic lichen nutrients, supplemented with the fungicide natamycin; ii) an extended washing of thalli to increase the recovery of ectolichenic bacteria, thus allowing the disinfection of thalli to be discarded, hence enhancing endolichenic bacteria recovery; and iii) the use of an antioxidant buffer to prevent or reduce oxidative stress during thalli disruption. The optimized methodology allowed significant increases in the number and diversity of culturable bacteria associated with P. furfuracea, and it was also successfully applied to the lichens Ramalina farinacea and Parmotrema pseudotinctorum. Furthermore, we provide, for the first time, data on the abundance of culturable ecto- and endolichenic bacteria that naturally colonize P. furfuracea, R. farinacea and P. pseudotinctorum, some of which were only able to grow on lichen enriched media. This innovative methodology is also applicable to other microorganisms inhabiting these and other lichen species.

SUBMITTER: Biosca EG

PROVIDER: S-EPMC4975499 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria.

Biosca Elena G EG Flores Raquel R Santander Ricardo D RD Díez-Gil José Luis JL Barreno Eva E

PloS one 20160805 8

Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbi ...[more]

PMID: 27494030

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Project description:Dendrobium plants are members of the family Orchidaceae, many of which are endangered orchids with ornamental and medicinal values. Dendrobium endophytic microbes have attracted attention for the development of strategies for plant protection and utilization of medicinal principles. However, the role of endophytic bacteria is poorly elucidated due to the lack of their successful cultivation. This study obtained a total of 749 endophytic isolates from Dendrobium roots using solid media prepared by simply modified methods (separate sterilization of phosphate and agar [PS] and use of gellan gum as a gelling reagent [GG]) and by a conventional method of autoclaving the phosphate and agar together (PT method). Notably, based on a comparison of 16S rRNA gene sequences between the isolates and the Dendrobium root endophyte community, we successfully retrieved more than 50% (17 out of 30) of the predominant endophytic bacterial operational taxonomic units (OTUs) using PS and GG media, which is a much higher recovery rate than that of PT medium (16.7%). We further found that a number of recalcitrant bacteria, including phylogenetically novel isolates and members of even the rarely cultivated phyla Acidobacteriota and Verrucomicrobiota, were obtained only when using PS and/or GG medium. Intriguingly, the majority of these recalcitrant bacteria formed colonies faster on PS or GG medium than on PT medium, which may have contributed to their successful isolation. Taken together, this study succeeded in isolating a wide variety of Dendrobium endophytic bacteria, including predominant ones using PS and GG media, and enables performance of future studies to clarify their unknown roles associated with the growth of Dendrobium plants. IMPORTANCE Dendrobium endophytic bacteria are of great interest since their functions may contribute to the protection of endangered orchids with ornamental and medicinal values. To understand and reveal the "true roles" of the endophytes, obtaining those axenic cultures is necessary even in the metagenomic era. However, no effective methods for isolating a variety of endophytic bacteria have been established. This study first demonstrated that the use of simply modified medium is quite effective and indeed allows the isolation of more than half of the predominant endophytic bacteria inhabiting Dendrobium roots. Besides, even phylogenetically novel and/or recalcitrant endophytic bacteria were successfully obtained by the same strategy. The obtained endophytic bacteria could serve as "living material" for elucidating their unprecedented functions related to the conservation of endangered orchid plants. Furthermore, the culture method used in this study may enable the isolation of various endophytic bacteria dominating not only in orchid plants but also in other useful plants.

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Dataset Information

Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria.

Publications

Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria.

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