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.
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