Phylogenetic Characterization and In Situ Detection of a
            <i>Cytophaga-Flexibacter-Bacteroides</i>
            Phylogroup Bacterium in
            <i>Tuber borchii</i>
            Vittad. Ectomycorrhizal Mycelium

114

115

dTruffles (Tuber spp.) are ascomycete subterraneous fungi that form ectomycorrhizas in a symbiotic relationship with plant roots. Their fruiting bodies are appreciated for their distinctive aroma, which might be partially derived from microbes. Indeed, truffle fruiting bodies are colonized by a diverse microbial community made up of bacteria, yeasts, guest filamentous fungi, and viruses. The aim of this minireview is two-fold. First, the current knowledge on the microbial community composition of truffles has been synthesized to highlight similarities and differences among four truffle (Tuber) species (T. magnatum, T. melanosporum, T. aestivum, and T. borchii) at various stages of their life cycle. Second, the potential role of the microbiome in truffle aroma formation has been addressed for the same four species. Our results suggest that on one hand, odorants, which are common to many truffle species, might be of mixed truffle and microbial origin, while on the other hand, less common odorants might be derived from microbes only. They also highlight that bacteria, the dominant group in the microbiome of the truffle, might also be the most important contributors to truffle aroma not only in T. borchii, as already demonstrated, but also in T. magnatum, T. aestivum, and T. melanosporum. Microbes can be found almost everywhere on our planet. They colonize many different types of habitats, among them living organisms, such as plant roots or insect and human guts. Classical microbiological methods have long offered a spotlight view on microbial diversity. Recent high-throughput molecular techniques have revolutionized the field of microbial ecology by unraveling an enormous microbial diversity in numerous organisms and highlighting the deep impact of microbiomes of their host physiology and behavior (1, 2). Truffle fungi are no exception, since they are colonized by a complex microbial community made up of bacteria, yeasts, guest filamentous fungi, and viruses (3-14).Truffles are subterranean ascomycete fungi that form ectomycorrhizas in symbiotic relationship with plant roots (15). Their fruiting bodies are appreciated for their distinctive aroma, which is partially derived from microbes (6,14,16). The aim of this minireview is to synthesize the current knowledge on the composition of the microbial community of truffles and discuss their potential role in truffle aroma formation, specifically focusing on volatiles that are responsible for human-perceived truffle aroma (defined as odorants). TRUFFLE MICROBIOMESTruffles are colonized by microbes at all stages of their life cycle, which include a symbiotic stage in association with a host plant (ectomycorrhiza), a sexual stage (fruiting bodies), and a free-living mycelial stage, which might serve an exploratory purpose in the soil. To date, microbes and microbial communities have been characterized in truffles with culture-dependent and -independent techniques in Ͼ15 papers (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(17)(18)(19)(20)(21). Various life cycle stages of four...

Section: Bacterial Communitiesmentioning

confidence: 99%

Section: Truffle Microbiomesmentioning

confidence: 99%

The Role of the Microbiome of Truffles in Aroma Formation: a Meta-Analysis Approach

Vahdatzadeh

Deveau

Splivallo

2015

114

115

“…These examples, coupled with the discovery of bacteria within hyphae of the ectomycorrhizal Dikarya (Tuber borchii; Ascomycota; Laccaria bicolor and Piriformospora indica; Basidiomycota) (5)(6)(7)(8)58), suggest that the capacity to harbor endohyphal bacteria is widespread among fungi. To date, however, endocellular bacteria have been recovered only from fungi that occur in the soil and rhizosphere (12,31).…”

mentioning

confidence: 99%

Diverse Bacteria Inhabit Living Hyphae of Phylogenetically Diverse Fungal Endophytes

Hoffman

Arnold

2010

221

220

Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, the interplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recently bacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at least one species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylogenetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including representatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photosynthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria using microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophytic Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regions surveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies were incongruent with each other and with taxonomic relationships of host plants. Overall, eight families and 15 distinct genotypes of endohyphal bacteria were recovered; most were members of the Proteobacteria, but a small number of Bacillaceae also were found, including one that appears to occur as an endophyte of plants. Frequent loss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineages of endohyphal bacteria relative to previous studies, is the first to document their occurrence in foliar endophytes representing four of the most species-rich classes of fungi, and highlights for the first time their diversity and phylogenetic relationships with regard both to the endophytes they inhabit and the plants in which these endophyte-bacterium symbiota occur.Traits related to the establishment and outcome of plantfungus symbioses can reflect not only abiotic conditions and the unique interactions of particular fungal and plant genotypes (49,50,56,59,62,67) but also additional microbes that interact intimately with fungal mycelia (4, 12, 42). For example, mycorrhizosphere-associated actinomycetes release volatile compounds that influence spore germination in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita (Glomeromycota) (14). Levy et al. (34) describe Burkholderia spp. that colonize spores and hyphae of the AM fungus Gigaspora decipiens and are associated with decreased spore germination. Diverse "helper" bacteria have been implicated in promoting hyphal growth and the establishment of ectomycorrhizal symbioses (23,26,57,70). Minerdi et al. (43) found that a consortium of ectosymbiotic bacteria limited the ability of the pathogen Fusarium oxysporum to infect and cause vascular wilts in lettuce, with virulence restored to the pathogen when ectosymbionts were removed.In addition to interacting with environmental and ectosymbiotic bacteria, some plant-associated fungi harbor bacteria within their hyphae (first ...

“…revealed the presence of a novel uncultured bacterium associated with the hyphae of this fungus (1). The phylogenetic analysis unequivocally placed the bacterial sequences in a single new rRNA branch in the Sphingobacterium subgroup of the CytophagaFlexibacter-Bacteroides (CFB) phylum (6,18).…”

mentioning

confidence: 87%

“…The phylogenetic analysis unequivocally placed the bacterial sequences in a single new rRNA branch in the Sphingobacterium subgroup of the CytophagaFlexibacter-Bacteroides (CFB) phylum (6,18). A few CFB cells per septum of mycelium were detected by the fluorescent in situ hybridization technique, with both general and specific oligonucleotide probes (1).…”

mentioning

confidence: 99%

Competitive PCR for Quantitation of a Cytophaga-Flexibacter-Bacteroides Phylum Bacterium Associated with the Tuber borchii Vittad. Mycelium

Barbieri¹,

Riccioni²,

Pisano³

et al. 2002

Self Cite

An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10 6 cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.A recent straightforward molecular study aimed at surveying potential endo-and epiphytic bacteria within the mycelium of the ectomycorrhizal fungus Tuber borchii Vittad. revealed the presence of a novel uncultured bacterium associated with the hyphae of this fungus (1). The phylogenetic analysis unequivocally placed the bacterial sequences in a single new rRNA branch in the Sphingobacterium subgroup of the CytophagaFlexibacter-Bacteroides (CFB) phylum (6, 18). A few CFB cells per septum of mycelium were detected by the fluorescent in situ hybridization technique, with both general and specific oligonucleotide probes (1).The occurrence of this novel bacterium associated with the T. borchii fungus will shed light on the relative importance of this bacterial species as a new, significant partner in fungusplant symbiosis. Thus, in order to estimate the abundance of this strain within the fungal host tissue, we used a competitive PCR (cPCR) technique (3,7,10,11,15,22). This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.Mycelia and bacterial strain. Four mycelium strains of T. borchii Vittad. (1BO [ATCC 96540], 10RA, 17BO, and Z43), in which the presence of the CFB bacterium (1) had been revealed, were used in this study. Mycelial cultures were grown in MNN liquid medium (21) and analyzed after 30 days. The 16S rRNA gene (rDNA) from the associated CFB bacterium was amplified by PCR following the procedures described by Barbieri et al. (1). In addition, Sphingobacterium heparinum ATCC 13125 was chosen from among the most closely related culturable strains to determine its rRNA operon copy number.Ergosterol assay. Ergosterol is a component of the fungal membrane and provides a reliable indication of metabolically active fungal biomass (4). The assay for ergosterol was carried out on 30-day-old cultures of the four T. borchii mycelium strains utilized in this study, as described by Zeppa et al. (21). Construction of the QPCR competitor.The 16S rDNA amplification product from the mycelium strain 17BO was cloned into the TA cloning vector pGEM (Promega) and sequenced. The sequence obtained was identical (1,450 of 1,450 nucleotides) to the b-17BO 16S rDNA sequence (GenBank no. AF070444). The recombinant DNA plasmid pb-17BO was cleaved with AgeI (cleavage sites, positions 1243 and 1422, 5Ј-3Ј), generating a 179-bp DNA fragment and the competitor pb-17BO-c, a new plasmid which has a deletion but has the same priming sequences as the DNA target. After li...