f u n g a l b i o l o g y 1 1 4 ( 2 0 1 0 ) 9 3 6 e9 4 2 journal homepage: www.elsevier.com/locate/funbio New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum Elena BARBIERIa,*, Paola CECCAROLIa, Roberta SALTARELLIa, Chiara GUIDIa, Lucia POTENZAa, Marina BASAGLIAb, Federico FONTANAb, Enrico BALDANb, Sergio CASELLAb, Ouafae RYAHIc, Alessandra ZAMBONELLIc, Vilberto STOCCHIa a Dipartimento di Scienze Biomolecolari, University of Urbino “Carlo Bo”, Urbino, Italy Dipartimento di Biotecnologie Agrarie, University of Padova, Agripolis, Legnaro, Italy c Dipartimento di Protezione e Valorizzazione Agroalimentare, University of Bologna, Bologna, Italy b article info abstract Article history: Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Received 3 February 2010 Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate Received in revised form PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata 27 July 2010 at different stages of maturation. Putative amino acid sequences revealed mainly the pres- Accepted 2 September 2010 ence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes Available online 15 September 2010 from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction Corresponding Editor: assay was 0.5e7.5 mmol C2H4 h Andrew N. Miller with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expres- 1 g 1, comparable with early nodules of legumes associated sion gene and activity within truffle. Keywords: ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Nitrogen-fixing bacteria Truffle Tuber magnatum Introduction The functional and taxonomic diversity of soil microbes is influenced by both plant roots that can locally affect the chemistry of the rhizosphere through composition and amount of root exudates (Frey-Klett et al. 2007) and by the presence of symbiotic organisms that colonize plant roots such as ectomycorrhizal fungi (Burke et al. 2006). This effect, known as the “mycorrhizosphere effect” seems to favour the occurrence of bacteria involved in the mycorrhization process, called “Mycorrhizal Helper Bacteria” (MHB), as well as the growth of bacteria called “mycorrhizal associated bacteria” for which different roles, including the nitrogen-fixation ability, were assigned (Frey-Klett et al. 2007). Several studies have found that potential nitrogen-fixing bacteria can be associated with ectomycorrhizal and arbuscular mycorrhizal fungi (Frey-Klett et al. 2007) and that the nifH gene, which encodes for the nitrogenase reductase, is present in Pinus silvestris and Pinus nigra ectomycorrhizae (Timonen & Hurek 2006; Izumi et al. 2006). Recently, the nitrogen-fixing bacteria were also found in the fruiting bodies of Tuber borchii Vittad. and Tuber magnatum Pico (Barbieri et al. 2005, 2007). These species, known as truffles, are ectomycorrhizal fungi belonging to the Tuber genus which form edible fruiting bodies * Corresponding author. Dipartimento di Scienze Biomolecolari, University of Urbino “Carlo Bo”, Via A. Saffi, 2, Urbino (PU) 61029, Italy. Tel.: þ39 0722 303418; fax: þ39 0722 303401. E-mail address: elena.barbieri@uniurb.it 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.09.001 Nitrogen fixation in truffle (Tuber magnatum) appreciated for their peculiar organoleptic properties (Hall et al. 2007). The ability of these bacteria to modify nutrient availability during the biological cycle, is of particular importance in truffles because after the formation of the primordium, the fruiting body seems to become independent from the plant host and needs nutrients in order to complete its maturation process independently (Barry et al. 1994). Nevertheless, no data about nitrogen-fixation activity are available in ectomycorrhizal fungi belonging to the genus Tuber. In this study we performed a combined molecular and biochemical approaches to look into the biodiversity of diazotrophs within Tuber-associated bacteria and to verify whether nitrogenase activity was detectable. 937 cloned using a pGEMR-T vector Systems cloning kit (Promega, Madison, USA). JM109 Escherichia coli cells were transformed and about 50e70 clones of the library from each T. magnatum ascocarp were screened as described in Barbieri et al. (2005). Amplified products were digested with TaqI, AluI and MspI (Fermentas Inc., Glen Burnie MD) according to the manufacturer’s instructions, for restriction fragment length polymorphism (RFLP) analyses that indicate the operational taxonomic units (OTUs). Clones representative of different OTUs were chosen for sequencing and phylogenetic analyses. Sequencing was performed using an ABI 377 DNA sequencer (PerkineElmer, Applied Biosystems Div.). Expression of nifH genes Material and methods Biological samples Nine Tuber magnatum ascomata were freshly collected in North-Central Italy and dried samples of each specimen were preserved in the Herbarium of the Mycology Center of Bologna, University of Bologna (Italy). The ascomata were classified for their maturation stages on the basis of the percentage of asci containing mature spores (Zeppa et al. 2002): immature ascocarps (0e10 %, stage I); mature ascocarps (>40 %, stage II). Acetylene reduction assay (ARA) Nitrogen-fixation activity in Tuber magnatum ascomata, was verified with a direct enzymatic ARA test, as a modification of the method previously described for legume root nodules (Povolo & Casella 2000). The ascomata were washed with sterile water, gently brushed and their surface briefly flamed. Truffles were then incubated in special tubes containing glass beads to reduce the headspace, and the atmosphere was supplemented with 3 % acetylene (V/V). Control samples without acetylene were arranged. At different times, 100 ml of the headspace were collected and injected in a GC-FID (TraceGC 2000 ThermoFinnigan) equipped with a MAPD Varian column (50 m  0.53 mm i.d., 0.50 mm film thickness) with FID detector at 270  C and He as carrier gas. Taking into account the obvious lack of replicates (each truffle is different in weight, size, maturation stage, etc) the data were expressed as “specific ARA”, in mmol C2H4 h 1 g 1, separately for each fruiting body available. NifH diversity Molecular identification of the potential Tuber magnatum associated nitrogen-fixing bacteria was performed by nifH gene analysis. One gram of tissue was removed from the inner part of each ascocarp to avoid soil contaminants and was homogenized in 1 ml of filter-sterilized physiologic solution (0.85 % NaCl) to extract the DNA directly from the ascoma as described by Paolocci et al. (2006). NifH gene was amplified using degenerate primers nifH (forA), nifH (rev) and nifH (forB) by semi-nested PCR as reported in Widmer et al. (1999). The nifH PCR products, resulting of the expected size (371 bp), were Total RNA was extracted following a protocol described by Zeppa et al. (2002) with slight modifications. A portion of the gleba from the ascomata material used for DNA extraction (about 500 mg fresh weight) was homogenized in RLT buffer from Qiagen (RNA Easy Plant Kit Extraction, Qiagen GmbH, Hilden, Germany) by a homogenizer (Kinematica Polytron MR 2100 Littau-Lucerne, Switzerland) and kept at 80  C for expression analyses. All frozen samples were de-frozen on ice for 20 min and thawed for 15 min at 37  C in a water bath to dissolve salts. DNA was removed by DNase I digestion in column during the RNA extraction (Qiagen) and in order to eliminate any residual of genomic DNA, a further DNA digestion was performed using DNase I enzyme (Ambion, Austin, TX, U.S.A.). RNA was quantified spectrophotometrically (UV ¼ 260 nm). Total RNA (500 ng) was reverse transcribed using Omniscript RT (Qiagen) and random hexamers (Promega Corp., Madison, WI, U.S.A.) in a final volume of 20 ml as described in manufacturer’s protocol. Semi-nested PCR was performed in order to improve sensitivity and specificity of expressed nifH sequence recovery (Brown et al. 2003; Knauth et al. 2005). We perform two types of semi-nested PCR by using both specific primers for BradyrhizobiumeTuber-associated bacteria to address direct relationship between the truffle and Bradyrhizobium sp. and universal primers for nitrogenfixer bacteria, as previously applied for nifH gene diversity using DNA as template (Widmer et al. 1999) and described in the above paragraph. In particular, specific primers employed were designed on the variable region of nifH genes sequenced belonged Bradyrhizobium sp. prevalently occurring in Tuber magnatum ascomata: NIFH2F 50 -GAA GGT CGG CTA CCA GAA CA-30 (Azotobacter vinelandii M20568 numbering 231e251) and NIFH5R 50 -AAG TTG ATC GAG GTG ATG ACG-30 (numbering 306e327) (Ueda et al. 1995; Jacobson et al. 1989). To assess primer specificity, control was tested using DNA from nifH plasmid TM4cl2 clone (GenBank GQ464079) closely related to Bradyrhizobium elkanii (GenBank ABG74604). While, universal primers for nitrogen-fixer bacteria nifH (forB) and nifH (rev) were used in the semi-nested PCR for nifH gene diversity as previously applied using DNA as template (Widmer et al. 1999). Aliquots (2 ml) of the initial PCR products were used as the template in the second PCR reaction for both semi-nested PCR approaches. The composition of the reaction mixtures was the same used for DNA approach with slight differences in cycle number, in order to reduce the saturation expression level detectable in the PCR 938 products. The minimum detectable value of PCR product from semi-nested PCR from a known amount of RNA retrotranscribed was reached at 25 cycles (data not shown). Amplification was initiated with a denaturation step of 94  C for 2 min, followed by 25 cycles of 94  C for 30 s, 60  C for 30 s, and 72  C for 30 s, and a final extension at 72  C for 7 min. PCR products were analyzed by electrophoresis using 2 % agarose (BioWhittaker, Rockland, ME, USA) gels. To ensure that the sequences that were recovered were truly from mRNA and not from contaminating DNA, semi-nested PCR was performed directly on an aliquot of each RNA extract without reverse transcription (RT). No amplification products from this control were seen. The nifH PCR products, resulting of the expected size were cloned and analyzed to choose representative of different OTUs for sequencing and phylogenetic analyses as above described. Phylogenetic analysis The amino acid deduced sequences of NifH were used for extensive database searching for both homologous and hortologous sequences. Protein sequence data were taken from SWISS-PROT and EMBL protein databases and the GenBank non-redundant protein database. A progressive multiple protein sequencing alignment was applied using the CLUSTALX package (Thompson et al. 1997). Phylogenetic analyses were performed with the PHYLIP software package, version 3.63 (Felsenstein 2004) and inferred using Neighbour Joining method. Bootstrap analyses were based on 1000 re-samplings of the sequence alignment. Nucleotide sequence accession numbers The NifH sequences of nitrogen-fixing bacteria identified in this study have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers between GQ464066 and GQ464113 for DNA and between HM852954 and HM852969 for mRNA. Results With the aim to verify the occurrence of nitrogen-fixation activity in Tuber magnatum ascoma, a direct enzymatic ARA test was tuned up. All the truffles analyzed showed detectable nitrogenase activity, ranging from 0.5 to 7.5 mmol ethylene produced per hour per gram of truffle (fresh weight), only in the presence of acetylene (Table 1). Additional measurements indicated that for some fruiting bodies ARA activity was 80e90 % in the external part of the ascoma (including the peridium and about 2 mm of external gleba) and only 10e20 % in the inner part of the ascoma (data not shown). Molecular identification of the potential T. magnatum associated nitrogen-fixing bacteria was performed by nifH gene analysis. The nifH PCR products, resulting of the expected size (371 bp) from all samples, were cloned and digested as described (Barbieri et al. 2005). Similarities were determined for the nucleic acid and amino acid sequences, translated in silico, and compared with reference sequences in DDBJ/EMBL/GenBank databases (Table 1). Comparisons of deduced amino acid as well nucleic acid E. Barbieri et al. sequences showed high similarities to those previously described in database (89e100 % and 95e100 %, respectively). Within the amino acid deduced sequences, highly conserved key amino acid residues, previously identified to be potentially important in NifH structure and function, were examined; they included four Cys residues (no. 38, 85, 97, and 132), Arg100, Thr104, Asp125, and Asp129 (Dang et al. 2009). All of these key amino acid residues were conserved in our NifH sequences. Within the sequencing analysis, a similarity threshold up to 98 % was applied to define the OTUs. The homologous coverage indicated that the 50e60 clones analyzed covered more than 92 % of the expected richness in the clone libraries (Loy et al. 2002) (Table 1). As shown in Table 1 and Fig 1, most of the DNA sequences belonged to the Alphaproteobacteria closely related to Bradyrhizobium elkanii (74.5 %, 380 out of the 510 total clones); a small fraction of the clones was related to Sinorhizobium spp. (3.92 %, 20 out of the 510 total clones). Few clones were closely related to the Betaproteobacteria, specifically Azoarcus spp. and Dechloromonas spp. (6.86 %, 35 out of the total clones). No sequence similarities were detected with Burkholderia nifH gene described in spores of arbuscular mycorrhizal fungi (Minerdi et al. 2001). A consistent subgroup includes Gammaproteobacteria (10.8 %, 55 out of the 510 total clones): most of them were described as uncultured nitrogen-fixers closely related to Thioalkalispira spp. and Azotobacter spp. Only few strains were affiliated to Geobacter spp. belonging to Deltaproteobacteria (3.92 %, 20 out of the 510 total clones). Microbial diversity was highest in the immature ascocarps, in which four subdivisions of proteobacteria (alpha, beta, gamma and delta) occurred. By contrast only Alphaproteobacteria, mostly belonging to B. elkanii, were found in the mature ascocarps. Through extraction of mRNA, followed by RT and specific semi-nested PCR for BradyrhizobiumeTuber-associated bacteria, targeting mRNA of nifH, the nifH gene expression of this bacterium was revealed in all T. magnatum samples analyzed (data not shown). For a qualitative nifH gene expression analysis, RT-PCR products using universal primers were applied in four T. magnatum samples, randomly chosen among immature and mature ascomata. We recovered a total of 150 cloned nifH sequences from T. magnatum fruiting body samples. Based on RFLP analysis, the representative OTUs nifH sequences were translated in amino acid sequences for comparative and phylogenetic analysis (Table 2 and Fig 1). The nifH sequence pair wise similarities between expressed nifH sequences and their nearest neighbours ranged from 90 % to 97 %. The expressed T. magnatum associated nifH sequences of Bradyrhizobium sp. represented the major sequences revealed of the Alphaproteobacteria and were highly similar to those recovered from T. magnatum DNA (52 %, 78 out of the 150 total clones). Expressed nifH sequences from Gammaproteobacteria represented only the 10.6 % (16 out of the 150 total clones) closely related to uncultured bacteria (92 % of similarity) described among the dinitrogen-fixing bacteria communities in soil (Giuntini et al. 2006) and formed a new OTU respect the once described from T. magnatum DNA. Nitrogen fixation in truffle (Tuber magnatum) 939 Table 1 e List of Tuber magnatum ascomata, specific ARA test and affiliation of nifH gene clones sequenced in this study. Clone OTU No. of T. magnatum Maturation Fresh Specific ARA clones weight (mmol C2H4 sequenced ascoma degreea h 1 g 1) (g) tBLASTX MATCH Coverageb Seq. identities TM2-2768 0% 1.88 2.58 1, 2, 3, 4 1 55 YP_001207334 [Bradyrhizobium sp. ORS278] AAO48617 [uncultured nitrogen-fixer] 98.1 % 97e98 % TM3-2758 0% 0.83 5.21 1, 2, 3, 4 1 50 YP_001207334 [Bradyrhizobium sp. ORS278] AAO48617 [uncultured nitrogen-fixer] 98 % 95e100 % TM4-2771 0% 0.39 5.40 1, 2, 4, 6 2 10 92.3 % 89e91 % 3, 5, 7, 11, 12, 13 3 25 8, 10 4 10 9 5 10 14 6 10 YP_932042 [Azoarcus sp. BH72] CAD91359 [Dechloromonas sp. SIUL] ABN10977 [Thioalkalispira microaerophila] ABD74409 [uncultured nitrogen-fixer] AAO48655 [uncultured nitrogen-fixer] AAX84830 [Gammaproteobacterium] AAY59348 [uncultured bacterium] 1 2 25 2 1 25 1 7 20 2, 3 1 32 TM5-2757 TM6-2770 5e10 % 5e10 % 0.37 1.50 7.29 3.11 91 e92 % 98 % 98 % 95 % ABD74409 [uncultured nitrogen-fixer] BAG84759 [uncultured bacterium] 96 % YP_001229952 [Geobacter uraniireducens] AAO48617 [uncultured nitrogen-fixer] 96.2 % 92 % 100 % 98 % 100 % TM7-2769 5e10 % 4.11 0.49 1, 2, 3, 4 1 58 AAY59348 [uncultured bacterium] 98.2 % 100 % TM1-2742 40e60 % 4.95 0.44 1, 3, 4, 5, 6, 8, 9, 10 1 55 YP_001207334 [Bradyrhizobium sp. ORS278] AAO48617 97.2 % 95e98 % 2, 7 1 15 1 8 20 2 1 30 1, 2, 3, 4, 5 1 60 TM8-2755 TM9-2767 Total clones 50e60 % 50e90 % 0.92 3.67 2.43 1.65 [uncultured nitrogen-fixer] ACI23531 [Bradyrhizobium canariense] 98 % NP_435695 [Sinorhizobium meliloti] ACI25871 [uncultured soil bacterium] 96 % YP_001207334 [Bradyrhizobium sp. ORS278] AAO48617 [uncultured nitrogen-fixer] 98.3 % 96 % 98 % 97e100 % 510 a Asci containing mature spores. b The coverage for the 16S rRNA gene libraries generated from each T. magnatum ascocarp was determined according to the formula C ¼ [1 (n1  N 1)]  100 %, with n1 being the number of OTUs containing only one sequence and N being the total number of 16S rRNA gene clones analyzed. 940 E. Barbieri et al. 711 956 TM1 cl1, TM1 cl2, TM1 cl3, TM1 cl4, TM1 cl5, TM1 cl6,TM1 cl7, TM1 cl8, TM1 cl9, TM1 cl10 TM1 cl5 mRNA, TM1 cl6 mRNA, TM1 cl8 mRNA, TM 1 cl9 mRNA, TM1 cl10 mRNA TM2 cl1, TM2 cl2, TM2 cl3, TM2 cl4 / TM2 cl3 mRNA, TM2 cl4 mRNA TM3 cl1, TM3 cl2,TM3 cl3, TM3 cl4 TM5 cl2 TM6 cl2, TM6 cl3 TM7 cl1, TM7 cl2, TM7 cl3, TM7 cl4 TM8 cl2 / TM8 cl1 mRNA TM9 cl1,TM9 cl2, TM9 cl3, TM9 cl4, TM9 cl5 (OTU 1) AAO48617 uncultured nitrogen fixing ABG74604 Bradyrhizobium elkanii α-proteobacteria 617 TM8 cl1 (OTU 8) NP_435695 Sinorhizobium meliloti 823 YP_932042 Azoarcus sp. BH72 CAD91359 Dechloromonas sp. SIUL TM4 cl1, TM4 cl2, TM4 cl6, TM5 cl1 (OTU 2) TM4 cl11 mRNA, TM4 cl26 mRNA, TM4 cl18 mRNA (OTU 10) AAS47809 uncultured bacterium 879 420 775 AAX84830 Gammaproteobacterium TM4 cl9 (OTU 5) 977 780 676 β-proteobacteria 978 886 γ-proteobacteria TM4 cl3, TM4 cl5, TM4 cl7, TM4 cl11, TM4 cl12, TM4 cl13 (OTU 3) ABN10977 Thioalkalispira microaerophila TM4 cl14 (OTU 6) 940 YP_002801975 Azotobacter vinelandii 537 TM4 cl8, TM4 cl10 (OTU 4) AAT48908 uncultured bacterium 618 YP_001229952 Geobacter uraniireducens TM6 cl1 (OTU 7) 996 ABC69209 Sulfurospirillum multivorans ABN45924 uncultured bacterium TM2 cl8 mRNA, TM2 cl15 mRNA TM4 cl3 mRNA, TM4 cl16 mRNA (OTU 9) 980 906 406 δ -proteobacteria ε-proteobacteria TM1 cl2 mRNA,TM1 cl4 mRNA, TM1 cl11 mRNA,TM1cl14 mRNA TM8 cl2 mRNA,TM8 cl7 mRNA,TM8 cl16 mRNA (OTU 12) AAP48979 Arcobacter nitrofigilis 886 978 TM4 cl15 mRNA, TM4 cl25 mRNA (OTU 11) AAS47806 uncultured bacterium Firmicutes AAP48978 Desulfotomaculum nigrificans AAU93872 Frankia sp. 0.01 Fig 1 e Neighbour joining phylogenetic analysis of the NifH amino acid deduced sequences extracted from Tuber magnatum DNA and RNA ascomata compared with homologous sequences obtained by tBLASTX algorithm. Bootstrap analyses were based on 1000 re-samplings of the sequence alignment. The sequence of Frankia sp. was included as the out group. Bootstrap values below 50 % are not shown. Sequences determined in this study are shown in bold. Interestingly, the mRNA approach allowed to identify nitrogen-fixing bacteria not closely related to those of the DNA-derived nifH sequences, belonging to the Epsilonproteobacteria and Firmicutes subdivisions (30 %, 46 out of the 150 total clones and 6.6 %, 10 out of the 150 total clones, respectively). Discussion This study reports the first evidence of nitrogen-fixation activity in truffles and offers the first sequences for the nifH gene described in Tuber-associated bacteria. ARA method measures nitrogenase activity, which can be related to the total amount of N that a system or organism is fixing (Staal et al. 2001). The ARA values obtained with Tuber magnatum samples were comparable to those generally obtained in a test tube from legume root nodules. However, while the root nodules maintain the suitable environment for nitrogen fixation once detached from the root and incubated into a test tube (leghemoglobin persists to ensure anaerobic environment), in the case of the fruiting bodies the collection from soil and the transfer to the lab may cause at least a partial loss of the needed environment. Looking at the suitable lab conditions to facilitate the measurement of nitrogen fixation in truffles is matter of further investigation. The nitrogenase activity in the specific association T. magnatum with diazotrophic bacteria, has important implication for the biology of this fungus. T. magnatum is an hypogeous fungus and lives in symbiosis with host plant roots to accomplish its life cycle which involves a first phase of growth as filamentous mycelium, a second phase of symbiotic Nitrogen fixation in truffle (Tuber magnatum) 941 Table 2 e List of Tuber magnatum ascomata, affiliation of nifH gene clones from mRNA nifH sequences analyzed in this study. T. magnatum Clone OTU No. of ascoma sequenced clones TM2-2768 TM4-2771 TM1-2742 TM8-2755 3, 4 1 34 8, 15 9 8 11, 18, 26 10 16 15, 25 11 10 3, 16 9 15 5, 6, 8, 9, 10 1 23 2, 4, 11, 14 12 11 1 21 12 12 1 2, 7, 16 Total clones tBLASTX MATCH Coverage Seq. identities GQ464113 [uncultured nitrogen-fixer TM9cl5 BradyrhizobiumeTuber associated] DQ337206 [Sulfurospirillum multivorans dinitrogenase (nifH ) gene] 98.1 % 97 % 93 % 98 % AY526283 [uncultured bacterium dinitrogenase reductase (nifH ) gene] AY221823 [Desulfotomaculum nigrificans clone CC1095J1 dinitrogenase reductase (nifH ) gene] EF208171 [uncultured nitrogen-fixing bacterium dinitrogenase reductase (nifH ), mRNA] 88.3 % 91 % 80 % 90 % 92.7 % 94 % GQ464103 [uncultured nitrogen-fixer TM7cl1 BradyrhizobiumeTuber associated] AY221824 [Arcobacter nitrofigilis dinitrogenase reductase (nifH ) gene] 93 % 96 % 84 % 95 % GQ464113 [uncultured nitrogen-fixer TM9cl5 BradyrhizobiumeTuber associated] AY221824 [Arcobacter nitrofigilis dinitrogenase reductase (nifH ) gene] 92.8 % 96 % 90 % 94 % 150 association as ectomycorrhizae and finally the organization of hypogeous fruiting body with asci and ascospores (Hall et al. 1998). Although the saprobic strategy of ascocarp development is debated (Zeller et al. 2008), T. magnatum ascomata can grow in soil supported by very low numbers of mycorrhizas (Bertini et al. 2006). Moreover, direct linkages among belowground mycelium, mycorrhizas and fruiting bodies in T. magnatum truffle-ground are absent (Zampieri et al. 2010). Under these conditions, enhanced nutrient availability by nitrogenfixing bacteria could have a beneficial effect on the development and maturation of the fruiting bodies. To establish whether or not the T. magnatum associated bacteria share the nitrogen-fixation genes, the sequences of the nifH genes, which encode the Fe protein subunit of nitrogenase, were determined. The nifH gene is one of the most ancient and conservative structural genes. Its phylogenetic analysis makes it possible to study relationships of nitrogen-fixing microorganisms at different taxonomic levels and provides a good level of estimation of the biodiversity of diazotrophs in various natural habitats (Ueda et al. 1995; Zani et al. 2000; Roesch et al. 2008). RT-PCR for nifH gene expression analysis is also an important approach for studying the nitrogen-fixation activity of bacteria present within environmental samples (Brown et al. 2003). In this study, RT and amplification of nifH cDNA by semi-nested PCR from total RNA extracts of ascocarps of T. magnatum allowed an assessment as to whether nitrogenase genes were expressed on T. magnatum truffles. Although the specific primers for nifH mRNA of BradyrhizobiumeTuber-associated bacteria highlight the expression of this nitrogen-fixing bacteria within ascomata of T. magnatum, the sequences of nifH from DNA and RNA pools showed diverse data in both nucleic and amino acid sequences (Tables 1 and 2). A similar observation has been made for rice endophytic diazotrophs (Knauth et al. 2005) and for ectomycorrhizas of Corsican pine (Izumi et al. 2006). Therefore, PCR based bias may influence the results on microbial functional activities, including the significance of nifH in truffle. This study has shown, through the amplification of nifH mRNA, that bacteria closely related to Bradyrhizobium spp. and bacteria belonging to Epsilonproteobacteria and Firmicutes subdivisions, not previously characterized by DNA based approaches, were active nitrogen-fixing bacteria associated with T. magnatum ascomata. The most representative clones from T. magnatum ascomata DNA and RNA, among the Alphaproteobacteria, were closely related to Bradyrhizobium elkanii and no information is currently available on the specific interaction of this species with Tuber fungi. B. elkanii markedly differs from other Bradyrhizobium species and its morphology, physiology, genetic and symbiotic properties were attributed to adaptation to environmental stress conditions (Hungria & Vargas 2000). T. magnatum ascomata develop in soil exposed to variable temperatures during a period from late summer to autumn/ winter, where drought and moisture alternate and the seasonal fluctuations promote their development. The presence of Bradyrhizobium spp. actively found within the immature and mature fruiting bodies analyzed may also suggest that the conditions these bacteria encounter in the colonized tissue may approach those required for free-living nitrogen fixation (Casella et al. 1988). These were originally described by Agarwal & Keister (1983) for slow growing and by Casella et al. (1988) for some fast growing rhizobia, and later adopted and modified by many authors. T. magnatum Pico is highly regarded by chefs and gourmets, but repeated attempts to cultivate it have been unsuccessful (Hall et al. 2007). Traditional cultivation techniques, which involve planting Tuber colonized plants in suitable sites, do not consider the potential effects of nitrogen-fixing bacteria on truffle development. This is the first demonstration of nitrogenase activity within truffle 942 and this study has provided new insights into the role of functionally active nitrogen-fixing bacteria occurring within fungal tissue during the maturation of T. magnatum ascomata, suggesting new possibilities for improving the cultivation technique of Italian white truffle. Acknowledgement We wish to thank Dr I.R. Hall, Truffles and Mushrooms (Consulting) Ltd, PO Box 268, Dunedin, New Zealand, for a critical reading of the manuscript. references Agarwal AK, Keister DL, 1983. Physiology of ex planta nitrogenase activity in Rhizobium japonicum. Applied and Environmental Microbiology 45: 1592e1601. Barbieri E, Bertini L, Rossi I, Ceccaroli P, Saltarelli R, Guidi C, Zambonelli A, Stocchi V, 2005. New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad. FEMS Microbiology Letters 247: 23e35. Barbieri E, Guidi C, Bertaux J, Frey-Klett P, Garbaye J, Ceccaroli P, Saltarelli R, Zambonelli A, Stocchi V, 2007. Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation. Environmental Microbiology 9: 2234e2246. Barry D, Staunton S, Callot G, 1994. Mode of the absorption of water and nutrients by ascocarps of Tuber melanosporum and Tuber aestivum. A radioactive tracer technique. Canadian Journal of Botany 72: 317e322. Bertini L, Rossi I, Zambonelli A, Amicucci A, Sacchi A, Cecchini M, Gregori G, Stocchi V, 2006. Molecular identification of Tuber magnatum ectomycorrhizae in the field. Microbiological Research 161: 59e64. Brown MM, Friez MJ, Lovell CR, 2003. Expression of nifH genes by diazotrophic bacteria in the rhizosphere of short form Spartina alterniflora. FEMS Microbiology Ecology 43: 411e417. Burke DJ, Kretzer AM, Rygiewicz PT, Topa MA, 2006. Soil bacterial diversity in a loblolly pine plantation: influence of ectomycorrhizas and fertilization. FEMS Microbiology Ecology 57: 409e419. Casella S, Shapleigh JP, Lupi F, Payne WJ, 1988. Nitrite reductase in bacteroids of Rhizobium “hedysari” strain HCNT 1. Archives of Microbiology 149: 384e388. Dang H, Luan X, Zhao J, Li J, 2009. Diverse and novel nifH and nifHlike gene sequences in the deep-sea methane seep sediments of the Okhotsk sea. Environmental Microbiology 75: 2238e2245. Felsenstein J, 2004. PHYLIP (Phylogeny Interference Package) Version 3.63 by Joseph Felsenstein Copyright 1986–2004. Department of Genome Sciences, The University of Washington, Seattle. Frey-Klett P, Garbaye J, Tarkka M, 2007. The mycorrhiza helper bacteria revisited. New Phytologist 176: 22e36. Giuntini E, Bazzicalupo M, Castaldini M, Fabiani A, Miclaus N, Piccolo R, Ranalli G, Santomassimo F, Zanobini S, Mengoni A, 2006. Genetic diversity of dinitrogen-fixing bacterial communities in soil amended with olive husks. Annals of Microbiology 56: 83e88. Hall IR, Zambonelli A, Primavera F, 1998. Ectomycorrhizal fungi with edible fruiting bodies 3. Tuber magnatum, tuberaceae. Economic Botany 52: 192e200. Hall IR, Brown G, Zambonelli A, 2007. Taming the Truffle. The History, Lore, and Science of the Ultimate Mushroom. Timber Press, Portland. Hungria M, Vargas MAT, 2000. Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil. Field Crops Research 65: 151e164. E. Barbieri et al. Izumi H, Anderson IC, Alexander IJ, Edward KK, Moore RB, 2006. Diversity and expression of nitrogenase genes (nifH ) from ectomycorrhizas of Corsican pine (Pinus nigra). Environmental Microbiology 8: 2224e2230. Jacobson MR, Brigle KE, Bennett LT, Setterquist RA, Wilson MS, Cash VL, Beynon J, Newton WE, Dean DR, 1989. Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. Journal of Bacteriology 171: 1017e1027. Knauth S, Hurek T, Brar D, Reinhold-Hurek B, 2005. Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environmental Microbiology 7: 1725e1733. Loy A, Daims H, Wagner M, 2002. Activated sludge e molecular techniques for determining community composition. In: Bitton G (ed), The Encyclopedia of Environmental Microbiology. John Wiley & Sons, Inc., New York, pp. 26e43. Minerdi D, Fani R, Gallo R, Boarino A, Bonfante P, 2001. Nitrogen fixation genes in an endosymbiotic Burkholderia strain. Applied and Environmental Microbiology 67: 725e732. Paolocci F, Rubini A, Riccioni C, Arcioni S, 2006. Reevaluation of the life cycle of Tuber magnatum. Applied and Environmental Microbiology 72: 2390e2393. Povolo S, Casella S, 2000. A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti. Archives of Microbiology 174: 42e49. Roesch LFW, Soares RA, Zanatta GFA, de Camargo O, Passaglia LMP, 2008. Diversity of nitrogen fixing bacterial community assessed by molecular and microbiological techniques. In: Dakora FD, et al. (eds), Biological Nitrogen Fixation: Towards Poverty Alleviation through Sustainable Agriculture Springer Science, pp. 309e311. Staal M, te Lintel-Hekkert S, Harren F, Stal L, 2001. Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on-line monitoring. Environmental Microbiology 3: 343e351. Timonen S, Hurek T, 2006. Characterization of culturable bacterial populations associating with Pinus sylvestriseSuillus bovinus mycorrhizospheres. Canadian Journal of Microbiology 52: 769e778. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876e4882. Ueda T, Suga Y, Yashiro N, Matsuguchi T, 1995. Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. Journal of Bacteriology 177: 1414e1417. Widmer F, Shaffer BT, Porteous LA, Seidler RJ, 1999. Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range. Applied and Environmental Microbiology 65: 374e380. Zampieri E, Murat C, Cagnasso M, Bonfante P, Mello A, 2010. Soil analysis reveals the presence of an extended mycelial network in a Tuber magnatum truffle-ground. FEMS Microbiology Ecology 71: 43e49. Zani S, Mellon MT, Collier JL, Zehr JP, 2000. Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR. Applied and Environmental Microbiology 66: 3119e3124. Zeller B, Brechet C, Maurice JP, Le Tacon F, 2008. Saprotrophic versus symbiotic strategy during truffle ascocarp development under holm oak. A response based on 13C and15N natural abundance. Annals of Forest Science 65: 607e617. Zeppa S, Guidi C, Zambonelli A, Potenza L, Vallorani L, Pierleoni R, Sacconi C, Stocchi V, 2002. Identification of putative genes involved in the development of Tuber borchii fruiting body by mRNA differential display in agarose gel. Current Genetics 42: 161e168.