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. 2016 Jul 15;5(1):1085.
doi: 10.1186/s40064-016-2756-6. eCollection 2016.

Bacteria of the genus Rhodopseudomonas (Bradyrhizobiaceae): obligate symbionts in mycelial cultures of the black truffles Tuber melanosporum and Tuber brumale

Christine Le Roux  1 , Estelle Tournier  1 , Adrien Lies  1 , Hervé Sanguin  1 , Gérard Chevalier  2 , Robin Duponnois  3 , Daniel Mousain  4 , Yves Prin  1
Affiliations

Bacteria of the genus Rhodopseudomonas (Bradyrhizobiaceae): obligate symbionts in mycelial cultures of the black truffles Tuber melanosporum and Tuber brumale

Christine Le Roux et al. Springerplus. .

Abstract

Background: This work aimed at characterizing 12 isolates of the genus Tuber including Tuber melanosporum (11 isolates) and Tuber brumale (one isolate). This was done using internal transcribed spacer (ITS) sequences, confirming their origin.

Results: Analysis of their mating type revealed that both MAT1-1 and MAT1-2 exist within these isolates (with 3 and 8 of each, respectively). We observed that each of these cultures was consistently associated with one bacterium that was intimately linked to fungal growth. These bacterial associates failed to grow in the absence of fungus. We extracted DNA from bacterial colonies in the margin of mycelium and sequenced a nearly complete 16S rDNA gene and a partial ITS fragment. We found they all belonged to the genus Rhodopseudomonas, fitting within different phylogenetic clusters. No relationships were evidenced between bacterial and fungal strains or mating types. Rhodopseudomonas being a sister genus to Bradyrhizobium, we tested the nodulation ability of these bacteria on a promiscuously nodulating legume (Acacia mangium), without success. We failed to identify any nifH genes among these isolates, using two different sets of primers.

Conclusions: While the mechanisms of interaction between Tuber and Rhodopseudomonas remain to be elucidated, their interdependency for in vitro growth seems a novel feature of this fungus.

Keywords: Ascomycete; Cultivability; In vitro production; Procaryote; Truffle.

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Figures

Fig. 1
Fig. 1
Microscopic observation of the bacterial colonization of growing hyphae of Tuber melanosporum isolate Mel18 by Rhodopseudomonas sp., on solid medium. a General view of the peripheral mycelia and the bacterial colonies. Bar is 100 µm. b progressive ensheathment of growing hyphae by Rhodopseudomonas sp. Bar is 50 µm
Fig. 2
Fig. 2
PhyML phylogenetic tree based on nearly complete 16S rRNA (1330 bp) sequences of 12 Tuber spp. associated bacterial strains aligned with Rhodopseudomonas spp. type strains (T) and related strains, including uncultured Bradyrhizobium sp. clones from Tuber borchii and Tuber magnatum ascocarps. Only branch support probabilities (estimated with the approximate likelihood-ratio test) higher than 0.70 are given at the branching points. Gaps were not considered. Scale indicates 2 % sequence divergence. Blastochloris sulfoviridis was chosen as an outgroup
Fig. 3
Fig. 3
BioNJ phylogenetic tree based on 16S-23S rRNA ITS sequences of 11 Tuber spp. associated bacterial strains aligned with Rhodopseudomonas spp. type strains (T) and related strains. Only bootstrap probability values higher than 70 % (100 replicates) are given at the branching points. Gaps were not considered. Scale indicated 10 % sequence divergence. Bradyrhizobium denitrificans was chosen as an outgroup
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